Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).     

Centrohelida is still searching for a phylogenetic home: analyses of seven Raphidiophrys contractilis genes

By recent advance in evolutionary biology, the majority of eukaryotes are classified into six eukaryotic assemblages called as "supergroups". However, several eukaryotic groups show no clear evolutionary affinity to any of the six supergroups. Centrohelida, one of major heliozoan groups, are such an unresolved lineage. In this study, we newly determined the genes encoding translation elongation factor 2 (EF2), cytosolic heat shock protein 70 (HSP70), and cytosolic heat shock protein 90 (HSP90) from the centroheliozoan Raphidiophrys contractilis. The three Raphidiophrys genes were then combined with previously determined actin, alpha-tubulin, beta-tubulin, and SSU rRNA sequences to phylogenetically analyze the position of Centrohelida in global eukaryotic phylogeny. Although the multi-gene data sets examined in this study are the largest ones including the centroheliozoan sequences, the relationships between Centrohelida and the eukaryotic groups considered were unresolved. Our careful investigation revealed that the phylogenetic estimates were highly sensitive to genes included in the multi-gene alignment. The signal of SSU rRNA and that of alpha-tubulin appeared to conflict with one another: the former strongly prefers a monophyly of Diplomonadida (e.g., Giardia), Parabasalia (e.g., Trichomonas), Heterolobosea (e.g., Naegleria), and Euglenozoa (e.g., Trypanosoma), while the latter unites Diplomonadida, Parabasalia, Metazoa, and Fungi. In addition, EF2 robustly unites Rhodophyta and Viridiplantae, while the remaining genes considered in this study do not positively support the particular relationship. Thus, it is difficult to identify the phylogenetic relatives of Centrohelida in the present study, since strong (and some are conflicting) gene-specific "signals" are predominant in the current multi-gene data. We concluded that larger scale multi-gene phylogenies are necessary to elucidate the origin and evolution of Centrohelida.     

EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata

Background

Classification of eukaryotes provides a fundamental phylogenetic framework for ecological, medical, and industrial research. In recent years eukaryotes have been classified into six major supergroups: Amoebozoa, Archaeplastida, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. According to this supergroup classification, Archaeplastida and Chromalveolata each arose from a single plastid-generating endosymbiotic event involving a cyanobacterium (Archaeplastida) or red alga (Chromalveolata). Although the plastids within members of the Archaeplastida and Chromalveolata share some features, no nucleocytoplasmic synapomorphies supporting these supergroups are currently known.

Methodology/Principal Findings

This study was designed to test the validity of the Archaeplastida and Chromalveolata through the analysis of nucleus-encoded eukaryotic translation elongation factor 2 (EEF2) and cytosolic heat-shock protein of 70 kDa (HSP70) sequences generated from the glaucophyte Cyanophora paradoxa, the cryptophytes Goniomonas truncata and Guillardia theta, the katablepharid Leucocryptos marina, the rhizarian Thaumatomonas sp. and the green alga Mesostigma viride. The HSP70 phylogeny was largely unresolved except for certain well-established groups. In contrast, EEF2 phylogeny recovered many well-established eukaryotic groups and, most interestingly, revealed a well-supported clade composed of cryptophytes, katablepharids, haptophytes, rhodophytes, and Viridiplantae (green algae and land plants). This clade is further supported by the presence of a two amino acid signature within EEF2, which appears to have arisen from amino acid replacement before the common origin of these eukaryotic groups.

Conclusions/Significance

Our EEF2 analysis strongly refutes the monophyly of the Archaeplastida and the Chromalveolata, adding to a growing body of evidence that limits the utility of these supergroups. In view of EEF2 phylogeny and other morphological evidence, we discuss the possibility of an alternative eukaryotic supergroup.     

Protists – A textbook example for a paraphyletic taxon

Protists constitute a paraphyletic taxon since the latter is based on the plesiomorphic character of unicellularity and does not contain all descendants of the stem species. Multicellularity evolved several times independently in metazoans, higher fungi, heterokonts, red and green algae. Various hypotheses have been developed on the evolution and nature of the eukaryotic cell, considering the accumulating data on the chimeric nature of the eukaryote genome. Subsequent evolution of the protists was further complicated by primary, secondary, and even tertiary intertaxonic recombinations. However, multi-gene sequence comparisons and structural data point to a managable number of such events. Several putative monophyletic lineages and a gross picture of eukaryote phylogeny are emerging on the basis of those data. The Chromalveolata comprise Chromista and Alveolata (Dinoflagellata, Apicomplexa, Ciliophora, Perkinsozoa, and Haplospora). Major lineages of the former ‘amoebae’ group within the Heterolobosa, Cercozoa, and Amoebozoa. Cercozoa, including filose testate amoebae, chlorarachnids, and plasmodiophoreans seem to be affiliated with foraminiferans. Amoebozoa consistently form the sister group of the Opisthokonta (including fungi, and with choanoflagellates as sister group of metazoans). A clade of ‘plants’ comprises glaucocystophytes, red algae, green algae, and land vascular plants. The controversial debate on the root of the eukaryote tree has been accelerated by the interpretation of gene fusions as apomorphic characters. In the more traditional view, based on sequence comparisons using archaebacteria as outgroup representatives, parabasaleans and diplomonads branch off first, rendering the biflagellate eukaryotes paraphyletic. In sharp contrast, the root is placed between Bikonta and Opisthokonta plus Amoebozoa on the argument of a single enzyme gene fusion which is postulated to have occurred in the stem species of the Bikonta, and of a double enzyme gene fusion weighed as a synapomorphy of the Opisthokonta and Amoebozoa. We conclude that the paraphyletic taxon ‘protists’ may be maintained for practical reasons. However, introduction of new, clearly recognizable paraphyletic taxa should be avoided.     

Expanding the molecular and morphological diversity of Apusomonadida,a deep-branching group of gliding bacterivorous protists

Apusomonads are cosmopolitan bacterivorous biflagellate protists usually gliding on freshwater and marine sediment or wet soils. These nanoflagellates form a sister lineage to opisthokonts and may have retained ancestral features helpful to understanding the early evolution of this large supergroup. Although molecular environmental analyses indicate that apusomonads are genetically diverse, few species have been described. Here, we morphologically characterize 11 new apusomonad strains. Based on molecular phylogenetic analyses of the rRNA gene operon, we describe four new strains of the known species Multimonas media, Podomonas capensis, Apusomonas proboscidea, and Apusomonas australiensis, and rename Thecamonas oxoniensis as Mylnikovia oxoniensis n. gen., n. comb. Additionally, we describe four new genera and six new species: Catacumbia lutetiensis n. gen. n. sp., Cavaliersmithia chaoae n. gen. n. sp., Singekia montserratensis n. gen. n. sp., Singekia franciliensis n. gen. n. sp., Karpovia croatica n. gen. n. sp., and Chelonemonas dolani n. sp. Our comparative analysis suggests that apusomonad ancestor was a fusiform biflagellate with a dorsal pellicle, a plastic ventral surface, and a sleeve covering the anterior flagellum, that thrived in marine, possibly oxygen-poor, environments. It likely had a complex cell cycle with dormant and multiple fission stages, and sex. Our results extend known apusomonad diversity, allow updating their taxonomy, and provide elements to understand early eukaryotic evolution.     

The protistan origins of animals and fungi

Recent molecular studies suggest that Opisthokonta, the eukaryotic supergroup including animals and fungi, should be expanded to include a diverse collection of primitively single-celled eukaryotes previously classified as Protozoa. These taxa include corallochytreans, nucleariids, ministeriids, choanoflagellates, and ichthyosporeans. Assignment of many of these taxa to Opisthokonta remains uncorroborated as it is based solely on small subunit ribosomal RNA trees lacking resolution and significant bootstrap support for critical nodes. Therefore, important details of the phylogenetic relationships of these putative opisthokonts with each other and with animals and fungi remain unclear. We have sequenced elongation factor 1-alpha (EF-1alpha), actin, beta-tubulin, and HSP70, and/or alpha-tubulin from representatives of each of the proposed protistan opisthokont lineages, constituting the first protein-coding gene data for some of them. Our results show that members of all opisthokont protist groups encode a approximately 12-amino acid insertion in EF-1alpha, previously found exclusively in animals and fungi. Phylogenetic analyses of combined multigene data sets including a diverse set of opisthokont and nonopisthokont taxa place all of the proposed opisthokont protists unequivocally in an exclusive clade with animals and fungi. Within this clade, the nucleariid appears as the closest sister taxon to fungi, while the corallochytrean and ichthyosporean form a group which, together with the ministeriid and choanoflagellates, form two to three separate sister lineages to animals. These results further establish Opisthokonta as a bona fide taxonomic group and suggest that any further testing of the legitimacy of this taxon should, at the least, include data from opisthokont protists. Our results also underline the critical position of these "animal-fungal allies" with respect to the origin and early evolution of animals and fungi.     

Phylogeny of the Centrohelida Inferred from SSU rRNA, Tubulins, and Actin Genes

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, α-tubulin, and β-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an α- + β-tubulin + actin data set, and a separate model for SSU rRNA, α- and β-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira–Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree. [Reviewing Editor: Dr. Martin Kreitman]     

PHYLOGENETIC IMPLICATIONS OF THE CAD COMPLEX FROM THE PRIMITIVE RED ALGA CYANIDIOSCHYZON MEROLAE (CYANIDIALES,RHODOPHYTA)1

The de novo pyrimidine biosynthetic pathway consists of six enzymes: carbamoyl‐phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine‐5′‐monophosphate decarboxylase. The origin and organization of the first three enzymes differ markedly between Opisthokonta (Metazoa and Fungi) and the Amoebozoa and green plants. However, no information has been available regarding the characteristics of such genes in other photosynthetic eukaryotes. In this study, we examined the pyrimidine biosynthetic cluster in the primitive red alga Cyanidioschyzon merolae P. DeLuca et al. isolate 10D. Unlike the situation in green plants, the CPS II, ACT, and DHO of C. merolae were fused to form a single open reading frame (the CAD complex), as in the Opisthokonta and Amoebozoa. Phylogenetic analysis of the CPS domain sequences suggested that this red algal CAD complex did not result from a recent lateral gene transfer from Metazoa or Fungi but that the fusion of the three genes occurred before the divergence of Opisthokonta, Amoebozoa, and the red algae. These results cast doubt on the recent hypothesis that the Opisthokonta and Amoebozoa form a monophyletic group, based on the presence in both of the CAD complex.     

18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa

Cochliopodium is a very distinctive genus of discoid amoebae covered by a dorsal tectum of carbohydrate microscales. Its phylogenetic position is unclear, since although sharing many features with naked "gymnamoebae", the tectum sets it apart. We sequenced 18S ribosomal RNA genes from three Cochliopodium species (minus, spiniferum and Cochliopodium sp., a new species resembling C. minutum). Phylogenetic analysis shows Cochliopodium as robustly holophyletic and within Amoebozoa, in full accord with morphological data. Cochliopodium is always one of the basal branches within Amoebozoa but its precise position is unstable. In Bayesian analysis it is sister to holophyletic Glycostylida, but distance trees mostly place it between Dermamoeba and a possibly artifactual long-branch cluster including Thecamoeba. These positions are poorly supported and basal amoebozoan branching ill-resolved, making it unclear whether Discosea (Glycostylida, Himatismenida, Dermamoebida) is holophyletic; however, Thecamoeba seems not specifically related to Dermamoeba. We also sequenced the small-subunit rRNA gene of Vannella persistens, which constantly grouped with other Vannella species, and two Hartmannella strains. Our trees suggest that Vexilliferidae, Variosea and Hartmannella are polyphyletic, confirming the existence of two very distinct Hartmannella clades: that comprising H. cantabrigiensis and another divergent species is sister to Glaeseria, whilst Hartmannella vermiformis branches more deeply.     

Root of the Eukaryota tree as inferred from combined maximum likelihood analyses of multiple molecular sequence data

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.     

Evolutionary history of "early-diverging" eukaryotes: the excavate taxon Carpediemonas is a close relative of Giardia

Diplomonads, such as Giardia, and their close relatives retortamonads have been proposed as early-branching eukaryotes that diverged before the acquisition-retention of mitochondria, and they have become key organisms in attempts to understand the evolution of eukaryotic cells. In this phylogenetic study we focus on a series of eukaryotes suggested to be relatives of diplomonads on morphological grounds, the "excavate taxa". Phylogenies of small subunit ribosomal RNA (SSU rRNA) genes, alpha-tubulin, beta-tubulin, and combined alpha- + beta-tubulin all scatter the various excavate taxa across the diversity of eukaryotes. But all phylogenies place the excavate taxon Carpediemonas as the closest relative of diplomonads (and, where data are available, retortamonads). This novel relationship is recovered across phylogenetic methods and across various taxon-deletion experiments. Statistical support is strongest under maximum-likelihood (ML) (when among-site rate variation is modeled) and when the most divergent diplomonad sequences are excluded, suggesting a true relationship rather than an artifact of long-branch attraction. When all diplomonads are excluded, our ML SSU rRNA tree actually places retortamonads and Carpediemonas away from the base of the eukaryotes. The branches separating excavate taxa are mostly not well supported (especially in analyses of SSU rRNA data). Statistical tests of the SSU rRNA data, including an "expected likelihood weights" approach, do not reject trees where excavate taxa are constrained to be a clade (with or without parabasalids and Euglenozoa). Although diplomonads and retortamonads lack any mitochondria-like organelle, Carpediemonas contains double membrane-bounded structures physically resembling hydrogenosomes. The phylogenetic position of Carpediemonas suggests that it will be valuable in interpreting the evolutionary significance of many molecular and cellular peculiarities of diplomonads.     

Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.     

A new scenario of plastid evolution: plastid primary endosymbiosis before the divergence of the “Plantae,” emended

A recent hypothesis on the origin of eukaryotic phototrophs proposes that red algae, green plants (land plants plus green algae), and glaucophytes constitute the primary photosynthetic eukaryotes, whose plastids may have originated directly from a cyanobacterium-like prokaryote via primary endosymbiosis, whereas the plastids of other lineages of eukaryotic phototrophs appear to be the result of secondary endosymbiotic events involving a phototrophic eukaryote and a host cell. However, the phylogenetic relationships among the three lineages of primary photosynthetic eukaryotes remained unresolved because previous nuclear multigene phylogenies used incomplete red algal gene sequences derived mainly from Porphyra (Rhodophyceae, one of the two lineages of the Rhodophyta), and lacked sequences from the Cyanidiophyceae (the other red algal lineage). Recently, the complete nuclear genome sequences from the red alga Cyanidioschyzon merolae 10D of the Cyanidiophyceae were determined. Using this genomic information, nuclear multigene phylogenetic analyses of various lineages of mitochondrion-containing eukaryotes were conducted. Since bacterial and amitochondrial eukaryotic genes present serious problems to eukaryotic phylogenies, basal eukaryotes were deduced based on the paralogous comparison of the concatenated - and -tubulin. The comparison demonstrated that cellular slime molds (Amoebozoa) represent the most basal position within the mitochondrion-containing organisms. With the cellular slime molds as the outgroup, phylogenetic analyses based on a 1,525-amino acid sequence of four concatenated nuclear genes [actin, elongation factor-1( EF-1), -tubulin, and -tubulin] resolved the presence of two large, robust monophyletic groups and the basal eukaryotic lineages (Amoebozoa). One of the two groups corresponded to the Opisthokonta (Metazoa and Fungi), whereas the other included various lineages containing primary and secondary plastids (red algae, green plants, glaucophytes, euglenoids, heterokonts, and apicomplexans), Ciliophora, Kinetoplastida, dinoflagellates, and Heterolobosea, for which the red algae represented the most basal lineage. Therefore, the plastid primary endosymbiosis likely occurred once in the common ancestor of the latter group, and the primary plastids were subsequently lost in the ancestor(s) of organisms within the group that now lacks primary plastids. A new concept of Plantae was proposed for phototrophic and nonphototrophic organisms belonging to this group on the basis of their common history of plastid primary endosymbiosis. This new scenario of plastid evolution is discussed here, and is compared with recent genome information and findings on the secondary endosymbiosis of the Euglena plastid.     

Rooting the eukaryotic tree with mitochondrial and bacterial proteins

By exploiting the large body of genome data and the considerable progress in phylogenetic methodology, recent phylogenomic studies have provided new insights into the relationships among major eukaryotic groups. However, confident placement of the eukaryotic root remains a major challenge. This is due to the large evolutionary distance separating eukaryotes from their closest relatives, the Archaea, implying a weak phylogenetic signal and strong long-branch attraction artifacts. Here, we apply a new approach to the rooting of the eukaryotic tree by using a subset of genomic information with more recent evolutionary origin-mitochondrial sequences, whose closest relatives are α-Proteobacteria. For this, we identified and assembled a data set of 42 mitochondrial proteins (mainly encoded by the nuclear genome) and performed Bayesian and maximum likelihood analyses. Taxon sampling includes the recently sequenced Thecamonas trahens, a member of the phylogenetically elusive Apusozoa. This data set confirms the relationships of several eukaryotic supergroups seen before and places the eukaryotic root between the monophyletic "unikonts" and "bikonts." We further show that T. trahens branches sister to Opisthokonta with significant statistical support and question the bikont/excavate affiliation of Malawimonas species. The mitochondrial data set developed here (to be expanded in the future) constitutes a unique alternative means in resolving deep eukaryotic relationships.     

The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)

Mantamonasis a novel genus of marine gliding zooflagellates probably related to apusomonad and planomonad Apusozoa. Using phase and differential interference contrast microscopy we describe the type species Mantamonas plasticasp. n. from coastal sediment in Cumbria, England. Cells are ～5μm long, ～5μm wide, asymmetric, flattened, biciliate, and somewhat plastic. The posterior cilium, on which they glide smoothly over the substratum, is long and highly acronematic. The much thinner, shorter, and almost immobile anterior cilium points forward to the cell's left. These morphological and behavioural traits suggest thatMantamonasis a member of the protozoan phylum Apusozoa. Analyses of 18S and 28S rRNA gene sequences of Mantamonas plasticaand a second genetically very different marine species from coastal sediment in Tanzania show Mantamonasas a robustly monophyletic clade, that is very divergent from all other eukaryotes. 18S rRNA trees mostly placeMantamonaswithin unikonts (opisthokonts, Apusozoa, and Amoebozoa) but its precise position varies with phylogenetic algorithm and/or taxon and nucleotide position sampling; it may group equally weakly as sister to Planomonadida, Apusomonadida or Breviata. On 28S rRNA and joint 18/28S rRNA phylogenies (including 11 other newly obtained apusozoan/amoebozoan 28S rRNA sequences) it consistently strongly groups with Apusomonadida (Apusozoa).     

Evolution and diversity of dictyostelid social amoebae

Dictyostelid social amoebae are a large and ancient group of soil microbes with an unusual multicellular stage in their life cycle. Taxonomically, they belong to the eukaryotic supergroup Amoebozoa, the sister group to Opisthokonta (animals + fungi). Roughly half of the ~150 known dictyostelid species were discovered during the last five years and probably many more remain to be found. The traditional classification system of Dictyostelia was completely overturned by cladistic analyses and molecular phylogenies of the past six years. As a result, it now appears that, instead of three major divisions there are eight, none of which correspond to traditional higher-level taxa. In addition to the widely studied Dictyostelium discoideum, there are now efforts to develop model organisms and complete genome sequences for each major group. Thus Dictyostelia is becoming an excellent model for both practical, medically related research and for studying basic principles in cell-cell communication and developmental evolution. In this review we summarize the latest information about their life cycle, taxonomy, evolutionary history, genome projects and practical importance.     

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).