Methodology of protistan discovery: from rRNA detection to quality scanning electron microscope images

Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences.     

Phylogenetic relationships of four charophycean green algae inferred from complete nuclear-encoded small subunit RRNA gene sequences

Complete nuclear-encoded (18S) small subunit rRNA gene sequences were determined for four charophycean green algae, Chlorokybus atmophyticus, Coleochaete orbicularis, Klebsormidium flaccidum, and Nitella sp. Chlorokybus atmophyticus and Coleochaete orbicularis have been previously suggested to represent the most basal and most derived taxa within the charophytes, respectively. However, parsimony analysis of our 18S rDNA sequences along with a selection of other complete green algal and land plant 18S rDNA sequences yields a gene tree topology in which Chlorokybus is the most basal taxon, followed by the branching of Coleochaete and Klebsormidium. Two “sister” clades then diverge, one including Nitella and the land plants, and the second, members of the Chlorophyceae and Pleurastrophyceae. Despite producing slightly diiferent gene tree topologies than those inferred from parsimony, distance analyses of the 18S rDNA sequences also do not indicate a strong affinity between the land plants and Coleochaete. Rather, Klebsormidium and Coleochaete are virtually equidistant from the land plant taxa. Other data are needed in order to assess the unexpected findings reported here, particularly the position of Coleochaete.     

Higher plant origins and the phylogeny of green algae

Summary 5S rRNA sequences from six additional green algae lend strong molecular support for the major outlines of higher plant and green algae phylogeny that have been proposed under varying naming conventions by several authors. In particular, the molecular evidence now available unequivocally supports the existence of at least two well-separated divisions of the Chlorobionta: the Chlorophyta and the Streptophyta (i.e., charophytes) (according to the nomenclature of Bremer). The chlamydomonad 5S rRNAs are, however, sufficiently distinct from both clusters that it may ultimately prove preferable to establish a third taxon for them. In support of these conclusions 5S rRNA sequence data now exist for members of four diverse classes of chlorophytes. These sequences all exhibit considerably more phylogenetic affinity to one another than any of them show toward members of the other cluster, the Streptophyta, or the twoChlamydomonas strains. Among the Charophyceae, new 5S rRNA sequences are provided herein for three genera,Spirogyra, Klebsormidium, andColeochate. All of these sequences and the previously publishedNitella sequence show greater resemblance among themselves and to the higher plants than they do to any of the other green algae examined to date. These results demonstrate that an appropriately named taxon that includes these green algae and the higher plants is strongly justified. The 5S rRNA data lack the resolution needed, however, to unequivocally determine which of several subdivisions of the charophytes is the sister group of the land plants. The evolutionary diversity ofChlamydomonas relative to the other green algae was recognized in earlier 5S rRNA studies but was unanticipated by ultrastructural work. These new data provide further evidence for the relative uniqueness of the chlamydomonads and are discussed further.     

The nucleotide sequences of 5S rRNAs from a multicellular green alga, Ulva pertusa, and two brown algae, Eisenia bicyclis and Sargassum fulvellum

The nucleotide sequences of 5S rRNA from a multicellular green alga Ulva pertusa, and multicellular brown algae Eisenia bicyclis and Sargassum fulvellum, have been determined. The 5S rRNA from Ulva is composed of 120 nucleotides, and those from Eisenia and Sargassum have 118 nucleotides. The nucleotide sequence of Ulva 5S rRNA is rather similar to 5S rRNAs from unicellular green algae and higher plants, while those of Eisenia and Sargassum 5S rRNAs are unique.     

Identification of the green alga, Chlorella vulgaris (SDC1) using cyanobacteria derived 16S rDNA primers: targeting the chloroplast

We have tested a set of oligonucleotide primers originally developed for the specific amplification of 16S rRNA gene segments from cyanobacteria, in order to determine their versatility as an identification tool for phototrophic eucaryotes. Using web-based bioinformatics tools we determined that these primers not only targeted cyanobacterium sequences as previously described, but also 87% of sequences derived from phototrophic eucaryotes. In order to qualify our finding, a type culture and environmental strain from the freshwater unicellular, green algae genus Chlorella Beijerinck, were selected for further study. Subsequently, we sequenced a 578-bp fragment of the 16S rRNA gene, which proved to be present within the chloroplast genome, performed sequence analysis and positively identified our solvent-degrading environmental strain (SDC1) as Chlorella vulgaris.     

Mesostigmatophyceae, a new class of streptophyte green algae revealed by SSU rRNA sequence comparisons

Complete nuclear-encoded SSU rRNA sequences have been obtained from three taxa of streptophyte green algae (Klebsormidium nitens, Nitella capillaris, Chaetosphaeridium globosum) and two strains of the scaly green flagellate Mesostigma viride. Phylogenetic analyses of 70 taxa of Viridiplantae (Chlorophyta and Streptophyta) and 57 taxa of streptophyte green algae and embryophyte plants using distance, parsimony and likelihood methods revealed a novel monophyletic lineage among the Streptophyta comprising the genera Mesostigma and Chaetosphaeridium. This lineage is described here as the Mesostigmatophyceae classis nova. Our analyses demonstrate that (1) scaly green flagellates (prasinophytes) are polyphyletic, (2) a scaly green flagellate is a member of the Streptophyta and forms a clade with the oogamous, filamentous Chaetosphaeridium to the exclusion of all other known streptophyte green algae, (3) a previously published SSU rRNA sequence of Chaetosphaeridium (AF113506) is chimeric and contains part of a fungal SSU rRNA, and (4) the phylogenetic relationships between the Mesostigmatophyceae and other streptophyte green algae remain unresolved by SSU rRNA sequence comparisons.     

The nucleotide sequences of 5S rRNAs from two red algae, Gracilaria compressa and Porphyra tenera

The nucleotide sequences of 5S rRNA from two red algae, Gracilaria compressa and Porphyra tenera have been determined. The two 5S rRNAs are fairly dissimilar to each other in their sequences (65% identity), although they are both composed of 121 nucleotides. Their secondary structures are generally of the eukaryotic with a prokaryotic characteristic. Judged from the 5S rRNA sequence data, the red algae are phylogenically distinct from green and brown algae, and they, Porphyra in particular, are evolutionally most ancient among the eukaryotes of which 5S rRNA sequence has been determined.     

Eukaryogenesis: a model derivated from ribosomal RNA molecular phylogenise]

We have undertaken the construction of a broad molecular phylogeny of protists through the comparison of 28S rRNA molecules. The sequences from several major protistan phyla were aligned and combined with a broad database of metazoans, metaphytes, fungi and bacteria and we have derived dendrograms from both distance matrix and parsimony methods. In agreement with classical systematics, a number of monophyletic groups separated by large evolutionary distances were observed (those of the ciliates, the chlorophytes, etc.). From this analysis, several inferences on the eukaryogenesis can be made among which the ancient origin of the cytoskeleton, the late occurrence of the chloroplastic endosymbiosis and the simultaneous emergence of the triploblastic and diploblastic metazoan patterns.     

Estimating Protistan Diversity Using High‐Throughput Sequencing

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full‐length sequences. A total of 7,432 full‐length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full‐length 18S rRNA gene sequences. Present high‐throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.     

Evolutionary change in 5S rRNA secondary structure and a phylogenic tree of 352 5S rRNA species

The secondary structure models of 5S rRNA have been constructed from the primary structure of 352 5S rRNA species available at present. All the 5S rRNAs examined can take essentially the same secondary structure, however they reveal characteristic differences between eukaryotes, metabacteria (= archaebacteria) and eubacteria. These three types of models can be further subgrouped by minor but characteristic differences. A phylogenic tree of organisms has been constructed using these 5S rRNA sequences by the weighted pairing method (WPG method). The tree reveals that there exist several major groups of eubacteria which seem to have diverged into different directions in the early stages of bacterial evolution. After emergence of eubacteria, metabacteria and eukaryotes separated from each other from their common ancestor. In the eukaryotic evolution, red algae (Rhodophyta) emerged first, and thereafter, thraustocytrids-Proctista, Ascomycota, green plants (green algae and land plants), Basidiomycota, Chromophyta (brown algae, diatoms and golden-yellow algae), slime- and water molds, various protozoans, and animals emerged in this order.     

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O₂/H₂S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O₂/H₂S gradient.     

Phylogenetic relationships of bryophytes inferred from nuclear-encoded rRNA gene sequences

We investigate phylogenetic relationships among hornworts, liverworts and mosses, and their relationships to other green plant groups, by analysis of nucleotide variation in complete 18s rRNA gene sequences of three green algae, two hornworts, seven liverworts, nine mosses, and six tracheophytes. Parsimony and maximum-likelihood analyses yield a single optimal tree in which the hornworts are resolved as the basal group among land plants, and the liverworts and mosses are sister taxa that together form the sister clade to the tracheophytes. This phylogeny is internally robust as indicated by decay indices and by comparison (using both parsimony and likelihood criteria) to topologies representing five alternative hypotheses of bryophyte relationships. We discuss some possible reasons for differences between the phylogeny inferred from the rRNA data and those inferred from other character sets.     

Microeukaryote community patterns along an O2/H2S gradient in a supersulfidic anoxic fjord (Framvaren, Norway)

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O(2)/H(2)S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O(2)/H(2)S gradient.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.      

Comparative study on the evolution of chloroplast ribosomal 5S RNA of a living fossil plant, Cycas revoluta Thumb

The complete nucleotide sequence of Cycas revoluta Thunb chloroplast 5 S rRNA was determined. It consists of 122 nucleotides. This is the only known 5 S rRNA sequence in Gymnospermae. It is highly homologous with chloroplast 5 S rRNA of higher plants (92-97%), but less homologous (about 54%) with those of lower plants. There is however 67% homology between Cycas and a procaryote a. nidulans. The chloroplast 5 S rRNAs of Angiospermae are nearly identical with each other (95-97%). S. oligorhize and L. minor have 100% homology among themselves. We have constructed a phylogenic tree of 5 S rRNA sequences from fifteen plant chloroplasts. The result suggests that the emergence of algae occurred at an early stage of plant chloroplast evolution and that green plants originated from green algae. This is in agreement with the classical view and other theories of molecular evolution. However there is no common ancestor in the case of Bryophyta and ferns. Among the Angiospermae, a precise evolutionary process cannot be deduced because the Knuc values among the species are very close to each other.     

Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and cytochrome oxidase subunit I genes

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA andcoxI from 14 silk-producing species were used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis weighted towards transversions relative to transitions (ts, tv4) forcoxI resulted in more robust bootstrap support over unweighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and agrees with morphological and cytological data.     

Environmental determinants of the distribution of planktonic diplonemids and kinetoplastids in the oceans

We analysed a widely used barcode, the V9 region of the 18S rRNA gene, to study the effect of environmental conditions on the distribution of two related heterotrophic protistan lineages in marine plankton, kinetoplastids and diplonemids. We relied on a major published dataset (Tara Oceans) where samples from the mesopelagic zone were available from just 32 of 123 locations, and both groups are most abundant in this zone. To close sampling gaps and obtain more information from the deeper ocean, we collected 57 new samples targeting especially the mesopelagic zone. We sampled in three geographic regions: the Arctic, two depth transects in the Adriatic Sea, and the anoxic Cariaco Basin. In agreement with previous studies, both protist groups are most abundant and diverse in the mesopelagic zone. In addition to that, we found that their abundance, richness, and community structure also depend on geography, oxygen concentration, salinity, temperature, and other environmental variables reflecting the abundance of algae and nutrients. Both groups studied here demonstrated similar patterns, although some differences were also observed. Kinetoplastids and diplonemids prefer tropical regions and nutrient-rich conditions and avoid high oxygen concentration, high salinity, and high density of algae.     

An early origin of plastids within the cyanobacterial divergence is suggested by evolutionary trees based on complete 16S rRNA sequences

It is generally accepted that the plastids arose from a cyanobacterial ancestor, but the exact phylogenetic relationships between cyanobacteria and plastids are still controversial. Most studies based on partial 16S rRNA sequences suggested a relatively late origin of plastids within the cyanobacterial divergence. In order to clarify the exact relationship and divergence order of cyanobacteria and plastids, we studied their phylogeny on the basis of nearly complete 16S rRNA gene sequences. The data set comprised 15 strains of cyanobacteria from different morphological groups, 1 prochlorophyte, and plastids belonging to 8 species of plants and 12 species of diverse algae. This set included three cyanobacterial sequences determined in this study. This is the most comprehensive set of complete cyanobacterial and plastidial 16S rRNA sequences used so far. Phylogenetic trees were constructed using neighbor joining and maximum parsimony, and the reliability of the tree topologies was tested by different methods. Our results suggest an early origin of plastids within the cyanobacterial divergence, preceded only by the divergence of two cyanobacterial genera, Gloeobacter and Pseudanabaena.      

Nucleotide sequences of 5s rRNAs from sponge Halichondria japonica and tunicate Halocynthia roretzi and their phylogenetic positions

The nucleotide sequences of 5S rRNAs from sponge Halichondria japonica and tunicate Halocynthia roretzi were determined by chemical and enzymatic gel methods. Their phylogenetic positions among metazoans were derived from the 5S rRNA sequences by a computer analysis based on the maximum parsimony principle. It was suggested that the sponge is closely related to several invertebrates and the tunicate has affinity to vertebrates rather than invertebrates.