Genome-defence small RNAs exapted for epigenetic mating-type inheritance.

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

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).

Seasonal Variability of the Paramecium aurelia Complex in the Botanical Garden of the Jagiellonian University, Krak6w - in the Light of Species Composition and COI Haplotype Variation.

The temporal occurrence of some Paramecium aurelia species is still an intriguing problem as cysts were never reported to exist in the Paramecium genus. A sequence of species occurrence was studied (by strain crosses and molecular identification) in five water-bodies of the Jagiellonian University Botanical Garden in Krak6w in different sampling sites and different seasons of the year. In the current study 20 P. aurelia strains were isolated from collected water samples and identified as P. biaurelia, P. tetraurelia, P. sexaurelia (the first record in Poland), P. novaurelia (the first record in the Botanical Garden). Generally only one species was found in the particular water body in a single sampling point in a given season - an exception was observed in the case of some strains of P. tetraurelia and P. sexaurelia. The latter species were mostly isolated from two water bodies situated in the Palm Houses (higher temperature preference) and P. biaurelia with P. novaurelia from water bodies located outside (lower temperature preference). Sequencing of the COImtDNA fragment revealed 9 haplotypes in the studied area which were characteristic for particular species. The most variable species was P. sexaurelia - 8 strains studied and 3 haplotypes identified. In contrast, P. novaurelia has only one haplotype for 6 strains collected in different seasons. The present study supports the hypothesis that botanical garden water bodies may be a hot-spot for microbial eukaryotic species-such as Paramecium.

New Stands of Species of the Paramecium aurelia Complex; is the Occurrence of Particular Species Limited by Temperature Barriers?

The occurrence of ciliates, especially the Paramecium aurelia complex, has not yet been studied in many parts of the world, or sampling was done only occasionally. Generally, the southern hemisphere still awaits investigation. In North America only the USA was studied in greater detail; the majority of species of the complex were there recorded. In Asia, more frequent sampling was performed only in Japan and Asiatic Russia. Europe was studied more carefully, however, a different number of habitats was studied in particular zones of Europe, the least in the southern zone. New stands of P. tetraurelia , P. sexaurelia, P. octaurelia, and P. novaurelia were revealed as a result of the present investigations carried out in Africa (Mozambique--P. tetraurelia, P. sexaurelia), Asia (Indonesia--P. sexaurelia), borderland of Asia and Europe (Georgia--P. octaurelia), and Europe (Macedonia--P. tetraurelia and Romania--P. novaurelia). Are climatic zones the main factor limiting the occurrence of species of the P. aurelia complex? Analysis of data on the distribution of the P. aurelia species complex in warm "tropical" zones on different continents may suggest such preferences for some species, including P. sexaurelia, P. octaurelia, P. tredecaurelia, P. quadecaurelia. The first two of these species were recorded herein in warm or "tropical" zone.

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.

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.

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).

An assessment of haplotype variation in ribosomal and mitochondrial DNA fragments suggests incomplete lineage sorting in some species of the Paramecium aurelia complex (Ciliophora, Protozoa).

The Paramecium aurelia complex (Ciliophora, Protozoa) Sonneborn (1975) is composed of 15 sibling species, which are morphologically indistinguishable but sexually isolated. Therefore, the P. aurelia complex seems to be an ideal model for testing hypotheses about recent speciation events. Here we present two-locus (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA) analyses using over 120 strains collected from around the world and representing all currently known species of the complex. According to our findings, the studied species show different levels of haplotype variability. Some of them appear on the obtained trees as polyphyletic (e.g., P. dodecaurelia), while others as monophyletic (e.g., P. quadecaurelia), clusters. The revealed discrepancies, which are manifested by different mating behavior and haplotypes not characteristic of particular species, may be explained by incomplete lineage sorting. Furthermore, the phenomena of hybridization and introgression are considered as another explanation for our results. Despite the above discrepancies, "polyphyletic taxa" should be considered true biological species based on the results of genetic crosses. Using a combination of both strain crosses (the biological species concept) and molecular methods (the phylogenetic species concept) seems to be the appropriate way of delimiting species in closely related eukaryotic microorganisms such as the P. aurelia complex.

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 in the Paramecium aurelia species complex.

Current understanding of the population genetics of free-living unicellular eukaryotes is limited, and the amount of genetic variability in these organisms is still a matter of debate. We characterized-reproductively and genetically-worldwide samples of multiple Paramecium species belonging to a cryptic species complex, Paramecium aurelia, whose species have been shown to be reproductively isolated. We found that levels of genetic diversity both in the nucleus and in the mitochondrion are substantial within groups of reproductively compatible P. aurelia strains but drop considerably when strains are partitioned according to their phylogenetic groupings. Our study reveals the existence of discrepancies between the mating behavior of a number of P. aurelia strains and their multilocus genetic profile, a controversial finding that has major consequences for both the current methods of species assignment and the species problem in the P. aurelia complex.

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.

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-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.

Paramecium sexaurelia--intra-specific polymorphism and relationships with other Paramecium aurelia spp., revealed by cytochrome b sequence data.

Genetic distances among strains of P. sexaurelia were compared by sequencing the mitochondrial cytochrome b gene. The relationships of P. sexaurelia with representative strains of other species of the P. aurelia complex and related species, i.e., P. schewiakoffi, P. jenningsi and P. multimicronucleatum were evaluated. All investigated P. sexaurelia and P. dodecaurelia strains grouped together with high support. This P. sexaurelia/P. dodecaurelia cluster was furthermore composed of three distinct, strongly supported subgroups. Two of these groups contained both P. sexaurelia and P. dodecaurelia strains, suggesting that these species are not monophyletic. The third branch was composed of strains from Sevilla, Spain and was deeply separated (12-14% p-distance) from the other P. sexaurelia/P. dodecaurelia clades. This illustrates the urgent need for further work and more intense sampling of these "rare" species, in order to understand the status and the relationships of P. sexaurelia and P. dodecaurelia within the P. aurelia species complex. We recommend that general investigations on the speciation process be conducted within and between species of the P. aurelia complex due to the high genetic variation combined with observations that for some of the species possible cryptic speciation may have occurred. This is emphasized by data showing that for some species within this complex, species status may be less static and more dynamic than originally thought.

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.

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.

Molecular polymorphism of Paramecium tetraurelia (Ciliophora, Protozoa) in strains originating from different continents.

Fragments of LSU rDNA and COI mtDNA genes were sequenced in Paramecium tetraurelia strains originating from different continents, i.e. from Australia (Sydney), Europe (Spain, Poland), Asia (Israel, India, Japan) and North America (Indiana) in order to investigate intra-specific polymorphism in this species. Phylogenetic trees (based on analyses using the NJ, MP and BI methods) revealed that P. tetraurelia strains from Australia, Europe, North America and Asia (Israel, Japan) belong to one group divided into two main clusters, while a strain from India is separate and belongs to a different group. The Indian strain groups together with strains representing different species of the P. aurelia complex: P. septaurelia, P. octaurelia, and P. dodecaurelia. Polymorphism within P. tetraurelia was confirmed, however, it seems that the applied markers did not explain the ways of divergence of strains within species (Indian strain and others), and also did not show correlations between geographic origin of strains and their genetic diversity. Some species of the P. aurelia complex seem closely related.