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).

The Evolutionary Relationships between Endosymbiotic Green Algae of Paramecium bursaria Syngens Originating from Different Geographical Locations.

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

New Stands of Species of the Paramecium aurelia Complex in Yakutia, Russia.

Paramecium is one of the most studied genera among ciliates. In particular, it is a model organism for investigation of the sibling species problem (also known as the cryptic species problem), spatial distribution, and its role in speciation. The global distribution of Paramecium species and of sibling species belonging to the P. aurelia species complex (Ciliophora, Protista) still need study, e.g. sampling in some territories has been quite limited, while Europe has been investigated for years with the majority of the P. aurelia species isolated from here. The large territory of Yakutia (republic Sakha in the Russian Federation), known for its climate extremes and continuous permafrost that extended over several glacial and interglacial cycles of the Pleistocene, has not been studied before. In the present study we collected paramecia in the central part of Yakutia. Newly established strains were identified to species according to morphology and, in case of the P. aurelia complex, by crossing with the test strains (the reference strains for the particular species). New stands of P. primaurelia, P. biaurelia and P. novaurelia were described from the territory of Yakutia.

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.

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.

Redescription of an early-derivative mite, Pentasetacus araucariae (Eriophyoidea, Phytoptidae), and new hypotheses on the eriophyoid reproductive anatomy.

A unique set of plesiomorphic characters, and its association with an ancient gymnosperm, Araucaria araucana, have made Pentasetacus araucariae a putative relict of a lineage of gymnosperm-associated mites, itself possibly basal to all extant eriophyoids. However, the suboptimal description of this species is impeding morphological comparisons with other species, which are fundamental to eriophyoid systematics. Herein, we designate a female lectotype from syntype specimens and use additional non-type material to redescribe P. araucariae based on external and internal anatomy using different microscopic and 3D reconstruction techniques. Contrarily to statements in the literature, P. araucariae has undivided empodia in all instars, short spermathecal tubes, and large, globose spermathecae in females, as well as rudimentary genital fovea in immatures. In addition, males of P. araucariae were shown to have genitalic attributes similar to a species of Trisetacus studied in parallel, including two reservoir-like structures, which may represent parts of the genital chamber and of the ductus ejaculatorius, respectively, as well as paired testes and ducti deferentes. This is contrary to previous, limited knowledge on eriophyoids indicating that they possess a single testis. Although their short spermathecal tubes weaken the cladistic relationship between P. araucariae (Pentasetacinae) and conifer-associated Nalepellinae (e.g. Trisetacus) having long tubes, the structural similarities in male genitalia may reinforce it.

New stands of species of the Paramecium aurelia complex (Ciliophora, Protozoa) in Europe and South America (Ecuador).

The occurrence of species of the P. aurelia complex has been studied at a large scale in Europe and the majority of known species of the complex have been found there. However, a different number of habitats were studied in particular zones of Europe, the greatest number in the central zone. Herein new stands of several species of the Paramecium aurelia complex are presented from Europe including P. primaurelia, P. biaurelia, P. triaurelia, P. octaurelia, P. novaurelia, and P. dodecaurelia. In South America, studies concerning the distribution of the P. aurelia species complex were carried out only occasionally and the presence of some cosmopolitan species of the complex has been recorded, i.e. P. primaurelia, P. biaurelia, and P. tetraurelia. Recently, new stands of P. primaurelia and P. septaurelia were found in Ecuador. Ciliate biogeography and distribution is also discussed.

New stands of species of the Paramecium aurelia complex in Africa and Europe.

The relevance of geographical distribution and the roles of dispersal and spatial isolation during the speciation of microorganisms are nowadays of great interest. The Paramecium aurelia species complex is a perfect model system to explore these questions given its long history as a study subject and broad distribution. However, the world-wide distribution of the Paramecium aurelia complex (Ciliophora, Protista) still needs study, e.g., sampling in the southern hemisphere has been quite limited, while Europe has been investigated for years, with the majority of aurelia species isolated from here. Recently, new stands of species of the P. aurelia complex were found in southern Europe (Malta, Bulgaria, Cyprus) and in the Czech Republic (P. primaurelia, P. triaurelia, P. octaurelia). In Africa (Republic of South Africa), new stands of P. primaurelia, P. triaurelia, and P. octaurelia were found. Interestingly, the rare species P. triaurelia, and P. octaurelia were found to co-occur both in South Africa (SA 13) and the Czech Republic (CKV 8). Newly established strains were identified to species by crossing with the test strains (the reference strains for the particular species).

Phylogenetic placement of two previously described intranuclear bacteria from the ciliate Paramecium bursaria (Protozoa, Ciliophora): 'Holospora acuminata' and 'Holospora curviuscula'.

'Holospora acuminata' infects micronuclei of Paramecium bursaria (Protozoa, Ciliophora), whereas 'Holospora curviuscula' infects the macronucleus in other clones of the same host species. Because these micro-organisms have not been cultivated, their description has been based only on some morphological properties and host and nuclear specificities. One16S rRNA gene sequence of 'H. curviuscula' is present in databases. The systematic position of the representative strain of 'H. curviuscula', strain MC-3, was determined in this study. Moreover, for the first time, two strains of 'H. acuminata', KBN10-1 and AC61-10, were investigated. Phylogenetic analysis indicated that all three strains belonged to the genus Holospora, family Holosporaceae, order Rickettsiales within the Alphaproteobacteria.

New stands of species of the Paramecium aurelia complex (Ciliophora, Protista) in Russia (Siberia, Kamchatka).

New stands of P. primaurelia, P. biaurelia, and P. dodecaurelia were found in Russia. P. primaurelia was recorded in Tulun (Siberia, Irkutsk region) and in three stands situated on the Kamchatka peninsula: in Lake Chalaktyrskoye, in the Valley of Geysers, and Petropavlovsk Kamchatski. P. biaurelia was also found in Tulun and in two stands in the vicinity of Lake Baikal and the Buriatia region. P. dodecaurelia was recorded in Cheboksary in European Russia and in other stands situated in Asian Russia: Novosibirsk, the vicinity of Lake Baikal, Buriatia, Kamchatka (Petropavlovsk Kamchatski, Lake Chalaktyrskoye, and Nalychevo). These data extend the ranges of species of the P. aurelia complex in Russia, however, this large territory remains understudied.

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.

New stands of species of the Paramecium aurelia complex (Ciliophora, Protozoa) in the Mediterranean region (Italy, Greece, Morocco).

New stands of species of the Paramecium aurelia complex are presented in the paper, P. primaurelia recorded in Italy (Pisa) and in Morocco (Marrakesh), P. biaurelia in Italy (Calabria), P. triaurelia in Morocco (Ifrane), P. pentaurelia in Greece (Kastorya), and P. dodecaurelia in Italy (Padua).

Genetic Diversity of Symbiotic Green Algae of Paramecium bursaria Syngens Originating from Distant Geographical Locations.

Paramecium bursaria (Ehrenberg 1831) is a ciliate species living in a symbiotic relationship with green algae. The aim of the study was to identify green algal symbionts of P. bursaria originating from distant geographical locations and to answer the question of whether the occurrence of endosymbiont taxa was correlated with a specific ciliate syngen (sexually separated sibling group). In a comparative analysis, we investigated 43 P. bursaria symbiont strains based on molecular features. Three DNA fragments were sequenced: two from the nuclear genomes-a fragment of the ITS1-5.8S rDNA-ITS2 region and a fragment of the gene encoding large subunit ribosomal RNA (28S rDNA), as well as a fragment of the plastid genome comprising the 3'rpl36-5'infA genes. The analysis of two ribosomal sequences showed the presence of 29 haplotypes (haplotype diversity Hd = 0.98736 for ITS1-5.8S rDNA-ITS2 and Hd = 0.908 for 28S rDNA) in the former two regions, and 36 haplotypes in the 3'rpl36-5'infA gene fragment (Hd = 0.984). The following symbiotic strains were identified: Chlorella vulgaris, Chlorella variabilis, Chlorella sorokiniana and Micractinium conductrix. We rejected the hypotheses concerning (i) the correlation between P. bursaria syngen and symbiotic species, and (ii) the relationship between symbiotic species and geographic distribution.

Electrophoretic karyotype polymorphism of sibling species of the Paramecium aurelia complex.

Variability of karyotypes is one of the main mechanisms of speciation in organisms. Electrophoretic karyotypes of the macronucleus (MAC) obtained by pulsed-field gel electrophoresis were compared for 86 strains of all 15 sibling species of the Paramecium aurelia complex in order to determine if karyotype differences corresponded to biological species boundaries. Because the electrophoretic karyotype of the MAC reflects indirectly the frequency and distribution of fragmentation sites in the micronuclear (MIC) chromosomes, any change in MAC electrophoretic karyotype may be a marker of certain chromosomal mutations in the MIC. Thirteen main variants of electrophoretic MAC karyotypes were observed in this species complex. Ten of them appeared to correspond to biological species, while the three other variants characterized several species each. Intraspecific polymorphism was observed for several species: in some cases a certain variant of MAC karyotype was specific for all strains from the same part of the world. Distribution of the MAC karyotype variants along molecular phylogenetic trees of the P. aurelia complex shows that isolation of each species or group of species of this complex was accompanied by divergence in the molecular organization of the genome.

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.

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).

Molecular studies on intraspecific differentiation of Paramecium dodecaurelia, with description of new stands of the species (Protozoa, Ciliophora).

New stands of Paramecium dodecaurelia were found in Russia, Ukraine, Kazahstan, Poland, and Tenerife. Molecular studies (RAPD and sequencing of LSU rRNA and CO1 mtDNA gene fragments) of P. dodecaurelia strains depicted intraspecific polymorphism within the species, revealed as distinct differences between strains from the USA in comparison with strains from different regions of the Palearctic.

Comparative Genomic Analysis of Holospora spp., Intranuclear Symbionts of Paramecia.

While most endosymbiotic bacteria are transmitted only vertically, Holospora spp., an alphaproteobacterium from the Rickettsiales order, can desert its host and invade a new one. All bacteria from the genus Holospora are intranuclear symbionts of ciliates Paramecium spp. with strict species and nuclear specificity. Comparative metabolic reconstruction based on the newly sequenced genome of Holospora curviuscula, a macronuclear symbiont of Paramecium bursaria, and known genomes of other Holospora species shows that even though all Holospora spp. can persist outside the host, they cannot synthesize most of the essential small molecules, such as amino acids, and lack some central energy metabolic pathways, including glycolysis and the citric acid cycle. As the main energy source, Holospora spp. likely rely on nucleotides pirated from the host. Holospora-specific genes absent from other Rickettsiales are possibly involved in the lifestyle switch from the infectious to the reproductive form and in cell invasion.

Polymorphism within Paramecium sexaurelia (Ciliophora, Oligohymenophorea) and description of a new stand of the species in China.

The presence of Paramecium sexaurelia from the Paramecium aurelia complex was recorded for the first time in China (Beijing). RAPD fingerprints (band patterns) of P. sexaurelia strains, the new strain from China and others from Asia, as well as from Europe and Puerto Rico, showed polymorphism within the species as several groups of genotypes characterized by different band patterns.

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.