Evaluation of the molecular variability and characteristics of Paramecium polycaryum and Paramecium nephridiatum, within subgenus Cypriostomum (Ciliophora, Protista).

Although some Paramecium species are suitable research objects in many areas of life sciences, the biodiversity structure of other species is almost unknown. In the current survey, we present a molecular analysis of 60 Cypriostomum strains, which for the first time allows for the study of intra- and interspecific relationships within that subgenus, as well as the assessment of the biogeography patterns of its morphospecies. Analysis of COI mtDNA variation revealed three main clades (separated from each other by approximately 130 nucleotide substitutions), each one with internal sub-clusters (differing by 30 to 70 substitutions - a similar range found between P. aurelia cryptic species and P. bursaria syngens). The first clade is represented exclusively by P. polycaryum; the second one includes only four strains identified as P. calkinsi. The third cluster seems to be paraphyletic, as it includes P. nephridiatum, P. woodruffi, and Eucandidatus P. hungarianum. Some strains, previously identified as P. calkinsi, had COI sequences identical or very similar to P. nephridiatum ones. Morphological reinvestigation of several such strains revealed common morphological features with P. nephridiatum. The paper contains new information concerning speciation within particular species, i.e. existence of cryptic species within P. polycaryum (three) and in P. nephridiatum (six).

Molecular Identification of Paramecium bursaria Syngens and Studies on Geographic Distribution using Mitochondrial Cytochrome C Oxidase Subunit I (COI).

Paramecium bursaria is composed of five syngens that are morphologically indistinguishable but sexually isolated. The aim of the present study was to confirm by molecular methods (analyses of mitochondrial COI) the identification of P. bursaria syngens originating from different geographical locations. Phylograms constructed using both the neighbor-joining and maximum-likelihood methods based on a comparison of 34 sequences of P. bursaria strains and P. multimicronucleatum, P. caudatum and P.calkinsi strains used as outgroups revealed five clusters which correspond to results obtained previously by mating reaction. Our analysis shows the existence of 24 haplotypes for the COI gene sequence in the studied strains. The interspecies haplotype diversity was Hd = 0.967. We confirmed genetic differentiation between strains of P. bursaria and the occurrence of a correlation between geographical distribution and the correspondent syngen.

Sampling strategies for improving tree accuracy and phylogenetic analyses: a case study in ciliate protists, with notes on the genus Paramecium.

In order to assess how dataset-selection for multi-gene analyses affects the accuracy of inferred phylogenetic trees in ciliates, we chose five genes and the genus Paramecium, one of the most widely used model protist genera, and compared tree topologies of the single- and multi-gene analyses. Our empirical study shows that: (1) Using multiple genes improves phylogenetic accuracy, even when their one-gene topologies are in conflict with each other. (2) The impact of missing data on phylogenetic accuracy is ambiguous: resolution power and topological similarity, but not number of represented taxa, are the most important criteria of a dataset for inclusion in concatenated analyses. (3) As an example, we tested the three classification models of the genus Paramecium with a multi-gene based approach, and only the monophyly of the subgenus Paramecium is supported.

Paramecium putrinum (Ciliophora, Protozoa): the first insight into the variation of two DNA fragments - molecular support for the existence of cryptic species.

Paramecium putrinum (Claparede & Lachmann 1858) is one of the smallest (80-140 µm long) species of the genus Paramecium. Although it commonly occurs in freshwater reservoirs, no molecular studies of P. putrinum have been conducted to date. Herein we present an assessment of molecular variation in 27 strains collected from widely separated populations by using two selected DNA fragments (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA). Both the trees and haplotype networks reconstructed for both genome fragments show that the studied strains of P. putrinum form five main haplogroups. The mean distance between the studied strains is p-distance=0.007/0.068 (rDNA/COI) and exhibits similar variability as that between P. bursaria syngens. Based on these data, one could hypothesize that the clusters revealed in the present study may correspond to previously reported syngens and that there are at least five cryptic species within P. putrinum.

Multiple lines of evidence shed light on the occurrence of paramecium (ciliophora, oligohymenophorea) in bromeliad tank water.

Phytotelmata are vegetal structures that hold water from the rain, and organic matter from the forest and the soil, resulting in small, compartmentalized bodies of water, which provide an essential environment for the establishment and development of many organisms. These microenvironments generally harbor endemic species, but many organisms that are found in lakes and rivers, are also present. Here, we report, for the first time, the occurrence of the ciliate genus Paramecium in the tank of the bromeliad species Aechmaea distichantha. The identification of the Paramecium species was performed based on live observations, protargol impregnation, scanning electronic microscopy, and sequencing of the 18s rRNA. The absence of Paramecium from bromeliad tank water was highlighted in several earlier investigations, and may be due to the fact that this species is unable to make cysts. The occurrence of Paramecium multimicronucleatum in our samples may be explained by the proximity between the bromeliads and the river, a potential source of the species. Further, we also believe that the counting methodology used in our study provides a more accurate analysis of the species diversity, since we investigated all samples within a maximum period of 6 h after sampling, allowing minimum loss of specimens.

Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family.

BACKGROUND: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. RESULTS: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. CONCLUSIONS: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

Variation in ribosomal and mitochondrial DNA sequences demonstrates the existence of intraspecific groups in Paramecium multimicronucleatum (Ciliophora, Oligohymenophorea).

This is the first phylogenetic study of the intraspecific variability within Paramecium multimicronucleatum with the application of two-loci analysis (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) carried out on numerous strains originated from different continents. The species has been shown to have a complex structure of several sibling species within taxonomic species. Our analysis revealed the existence of 10 haplotypes for the rDNA fragment and 15 haplotypes for the COI fragment in the studied material. The mean distance for all of the studied P. multimicronucleatum sequence pairs was p=0.025/0.082 (rDNA/COI). Despite the greater variation of the COI fragment, the COI-derived tree topology is similar to the tree topology constructed on the basis of the rDNA fragment. P. multimicronucleatum strains are divided into three main clades. The tree based on COI fragment analysis presents a greater resolution of the studied P. multimicronucleatum strains. Our results indicate that the strains of P. multimicronucleatum that appear in different clades on the trees could belong to different syngens.

Survey of Paramecium duboscqui using three markers and assessment of the molecular variability in the genus Paramecium.

The genus Paramecium (phylum Ciliophora) is one of the best-known among protozoa. Nevertheless, the knowledge on the diversity and distribution of species within this genus was remarkably scarce until recent times. In the last years a constantly growing amount of data has formed, especially on the distribution of species and the characterization of molecular markers. Much effort has been made on detecting clades inside each morphospecies, which could suggest the presence of sibling species complexes as in the famous case of Paramecium aurelia. In this work we present new data on Paramecium duboscqui, one of the morphospecies that have not yet been surveyed employing DNA sequences as markers. We obtained data from nine strains sampled around the world, using the three most commonly employed markers (18S rRNA gene, ITS1-5.8S-ITS2 and COI gene sequences). Moreover, we compared our results with those already available for other Paramecium species, and performed phylogenetic analyses for the entire genus. We also expanded the knowledge on the ITS2 secondary structure and its usefulness in studies on Paramecium. Our approach, that considers the data of all the species together, highlighted some characteristic patterns as well as some ambiguities that should be further investigated.

Morphological and molecular characterization of Paramecium (Viridoparamecium nov. subgen.) chlorelligerum Kahl (Ciliophora).

We redescribe Paramecium chlorelligerum, a forgotten species, which Kahl (Tierwelt Dtl., 1935, 30:651) briefly but precisely described in the addendum to his ciliate monographs as a Paramecium with symbiotic green algae. The redescription is based on classical morphological methods and the analysis of the small subunit (SSU) rDNA. Morphologically, P. chlorelligerum differs from P. (C.) bursaria, the second green species in the genus, by having a special swimming shape, the length of the caudal cilia, the size of the micronucleus, the size of the symbiotic algae, the contractile vacuoles (with collecting vesicles vs. collecting canals), and the number of excretory pores/contractile vacuole (1 vs. 2-3). The molecular investigations show that P. chlorelligerum forms a distinct branch distant from the P. (Chloroparamecium) bursaria clade. Thus, we classify P. chlorelligerum in a new subgenus: Paramecium (Viridoparamecium) chlorelligerum. The symbiotic alga belongs to the little-known genus Meyerella, as yet recorded only from the plankton of a North American lake.

Intra-specific differentiation of Paramecium bursaria strains by molecular methods--preliminary studies.

Ten strains of Paramecium bursaria and also P. caudatum, P. multimicronucleatum, P. tetraurelia strains (as outgroups) were characterized by using Random Amplified Polymorphic DNA (RAPD), Amplified Ribosomal DNA Restriction Analysis (ARDRA) and sequencing of the non-coding ribosomal internal transcribed spacer (ITS) regions. RAPD analysis revealed that all Paramecium bursaria strains possessed characteristic band patterns; there was a correlation between the degree of differentiation of DNA revealed by RAPD-fingerprinting and the geographic origin of a particular strain. ARDRA riboprinting (using a fragment of SSU-LSU rDNA, about 3085 bp) with restriction enzymes DraI, EcoRV, HhaI, HindIII, MspI, PstI distinguished groups of P. bursaria strains with characteristic band patterns originating from different sites. Comparison of the 550 bp ITS 1-5.8S-ITS2 fragment showed differentiation (0.9%) of the P. bursaria strains as three main groups of strains connected by site of origin in the constructed tree.

Species of the Paramecium aurelia complex in Russia: new stands and overall distribution.

New stands of Paramecium biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, P. novaurelia, and P. dodecaurelia were recorded in Russia. Especially interesting is the record of P. novaurelia in Vladivostok, Russian Far East, as it is a very rare species outside of Europe. The distribution of species of the Paramecium aurelia complex in Eurasia with emphasis on findings in Russia is discussed.

Species of the Paramecium aurelia complex in Italy.

New stands of Paramecium pentaurelia were recorded in Valmarana, Veneto region in Italy and one stand of P. primaurelia was found at the same locality.

New, world-wide data on the distribution of species of the Paramecium aurelia complex (Ciliophora, Protozoa).

This is the first report on the presence of P. biaurelia in Tasmania, an island that has probably never been investigated before for the occurrence of the P. aurelia species. P. tetraurelia was recorded in Brazil, another very poorly investigated country in terms of this species complex. New stands of P. biaurelia and P. tetraurelia were also recorded in Japan. We present data concerning the occurrence and distribution of the P. aurelia species on different continents as a background for the newly described stands of P. aurelia spp.

Intra-population genetic diversity and its effects on outlining genetic diversity of ciliate populations: Using Paramecium multimicronucleatum as an example.

Questions regarding ciliate distribution (endemism vs. cosmopolitanism) and degree of genetic diversity (high vs. low) remain unsettled, even when the same organism is under investigation. Presence of genes with high copy number and amplification of non-dominant haplotypes might account for the observed discordance in these studies. Herein, we used direct PCR and cloning sequencing to examine intra-population sequence diversity and its effect on assessments of phylogeography of Paramecium multimicronucleatum. Totally, 381 ITS1-5.8S rDNA-ITS2-28S rDNA and 304 mitochondrial cytochrome oxidase subunit I (COI) gene sequences were generated for 18 populations of P. multimicronucleatum. The following results were obtained: (1) Direct sequencing of PCR products captured the dominant ITS and LSU haplotypes, indicating that it is an appropriate strategy for constructing phylogeography of large-scale spatial populations. (2) Deep cloning was deemed more appropriate for the COI gene for population level studies, as direct sequencing could not easily capture the dominant haplotypes. (3) No endemic populations of P. multinucleatum were noted, indicating origin from a single founder population. (4) Nuclear genetic diversity within temporal populations was high, but only the dominant haplotypes seemed to be passed on to subsequent generations.

Multiple origins of the symbioses in Paramecium bursaria.

Many organisms have symbioses with photosynthetic algae as typified by corals, clams, lichens, and some protozoa. Paramecium bursaria contains green algal symbionts and this unicellular ciliate is a textbook example used for microscopic observation in junior high school science projects. We have determined molecular phylogenies for the green algal symbionts. The symbiotic algae are the main constituent of the Paramecium cytoplasm, and we have recognized a total of four species, of which two were newly discovered in the present study. One should be regarded genetically as Chlorella vulgaris, and it belongs phylogenetically to the Chlorella clade (Chlorellaceae, Trebouxiophyceae) as well as "American" and "European" groups, which we previously introduced. Their genetic dissimilarities are 0.50-0.83% in 18S rDNA comparisons, but those of the internal transcribed spacer 2 (ITS2) reach an unambiguous level (22.6-26.6%). These dissimilarities suggest that they are equivalent to discrete species derived from multiple origins as paramecian symbionts. Another newcomer was clearly separated from the Chlorellaceae, and this alga clustered with Coccomyxa spp. in ITS2 analyses. These symbiotic relations indicate multiple origins of symbionts.

Paramecium species of the upper and lower Volga River basin, Russia.

The Volga, which is the largest river in Europe (3690 km long), flows from the north (Tver' region) to the south (Caspian Sea), and its extensive basin (1380 km2) includes very different biotopes. Thus, analysis ofthe occurrence of Paramecium species along this large river basin may significantly enhance our understanding of species distribution according to temperature regime, food richness and other possible factors. The present paper concerns the occurrence of species of the P. aurelia complex in the sampling areas of the Upper Volga River, and a comparison with the occurrence of species of the P. aurelia complex in the Lower Volga region. In the Upper Volga basin, P. biaurelia was the most abundant among species of the complex recorded (among P. triaurelia, P. decaurelia, P. dodecaurelia), in the Lower Volga region eight species of the complex were recorded (P. primaurelia, P. biaurelia, P. triaurelia, P. pentaurelia, P. sexaurelia, P. septaurelia, P. novaurelia, P. decaurelia).

[Comparative description of macronuclear electrophoretic karyotypes of Paramecium primaurelia and Paramecium novaurelia sibling species].

The macronuclear genomes of two sibling species belonging to the Paramecium aurelia complex--P. primaurelia and P. novaurelia--were analyzed by pulsed-field gel electrophoresis (PFGE). Their electrophoretic karyotypes showed a continuous spectrum of different-sized DNA molecules with a species-specific pattern of banding, which was reproducible and did not change with strain senescence. Thus, P. aurelia macronuclear PFGE profiles could be described by an approach analogous to that used for the description of metaphase chromosome banding patterns. At first, well-identifiable regions (size fractions) of a PFGE profile, which can be seen at any resolution, are determined. Then, the bands of the second order of resolution (subfractions) can be defined in some of these regions. The blocks of the first and second orders can be utilized as inner markers of the PFGE profile allowing precise comparison of different PFGE profiles. Such comparative analysis has demonstrated some marking differences in organization of the macronuclear genomes of P. primaurelia and P. novaurelia, and low level of intraspecific polymorphism. Worth noting is that the P. novaurelia strain isolated in USA was different from all other analyzed P. novaurelia strains originating from Europe. The nature of banding of P. aurelia PFGE profiles is discussed. The revealed high order and stability of the macronuclear genome organization makes possible searching for new approaches to study the mechanisms of this non-trivial genome formation and maintenance. Further PFGE analysis of the macronuclear genomes of the other Paramecium species in relation with the Paramecium phylogeny may provide insights into the directions of the evolution of the macronuclear genome in Ciliata.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Relationships of new sibling species of Paramecium jenningsi based on sequences of the histone H4 gene fragment.

Paramecium jenningsi Diller & Earl, 1958 belongs to the "aurelia" subgroup of the genus, together with Paramecium caudatum, Paramecium multimicronucleatum, Paramecium schewiakoffi and species of the Paramecium aurelia complex. The original assumption that the morphospecies P. jenningsi was a single genetic species was questioned because a comparison of genome analyses suggested the possibility that this morphospecies contained two sibling species. To refine understanding of relationships between the strains of P. jenningsi, a molecular phylogenetic analysis was conducted using H4 gene sequences. Some polymorphic sites were found among the compared sequences, and specific patterns of single nucleotide polymorphism (SNP) markers characterize two groups of strains of P. jenningsi. Phylogenetic trees constructed by different methods identified two clearly different groups (from Japan and mainland Asia) whatever the method used. The sequences of the H4 gene analyzed in the present study are closely related, and provide a good subject for phylogenetic analysis. The presence of two isolated groups of strains in the P. jenningsi group can reveal the evolutionary relationship between them; it confirms the presence of two sibling species among the known strains of P. jenningsi, and the close relationships between them and species of the P. aurelia complex.

Strains of Paramecium decaurelia (Ciliophora, Protozoa) from Russia with molecular characteristics of other known strains of the species.

The presence of Paramecium decaurelia from the Paramecium aurelia species complex was demonstrated in Yaroslavl, Russia, (European part, northwestern Russia) and in the Altai Mts (Asiatic part of Russia, western Siberia). RAPD-PCR fingerprints of the newly identified strains of P. decaurelia, rare throughout the world, were compared to those characteristic for the other known strains ofthe species. P. decaurelia strains show some polymorphism within species, strains from Russia have 60% similarity of band patterns, and strains from USA and Japan about 70% similarity of band patterns.