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.

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.

The ts111 Mutation of Paramecium tetraurelia Affects a Member of the Protein Palmitoylation Family.

The thermosensitive ts111 mutant of Parameciun tetraurelia carries a recessive mutation which causes cell death after 2-8 divisions at the restrictive temperature of 35 degrees C. Expression at 35 degrees C induces disassembly of the infraciliary lattice (ICL). In this study, we found that the ts111 mutation also results in significant abnormalities in the number and structure of contractile vacuole complexes (CVCs) and in their functioning at the restrictive temperature. In order to characterize the ts111 gene, the complementation cloning was performed by microinjection into the macronucleus of an indexed genomic DNA library. The mutation was complemented by a sequence of 852 bp, which differed from the mutant sequence by a single nucleotide substitution. The deduced protein sequence is 284 amino acids long. It contains a domain referred to as the DHHC domain, associated with 2 trans-membrane helices. The DHHC proteins belong to the Palmitoyl-Acyl Transferases (PATs) protein family, which is implicated in the protein palmitoylation process playing the role in protein addressing. The ts111 mutation induces the amino acid change, localized before the first membrane helix. Transformation of ts111 mutant cells with the TS111-GFP gene fusion showed the expected reparation restoring thermoresistance and also demonstrated a localization of the protein in contractile vacuoles, but not in the ICL. The entire gene silencing in wild type cells at restrictive temperature caused the same effect as the expression of a point mutation in ts111 mutant. The authors propose the following hypotheses: (i) function of CVCs at the restrictive temperature depends in Paramecium on the TS111 protein--a member of the PAT family, and the primary effect of the termosensitive ts111 mutation are morphological abnormalities and dysfunction of CVCs, (ii) disassembly of the ICL is a secondary effect of the ts111 mutation, which results from disturbed regulation of the intracellular concentration of Ca(+2) ions caused by the abnormal functioning of CVC.

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.

Molecular variability of the COI fragment supports the systematic position of Enarmoniini within the subfamily Olethreutinae (Lepidoptera: Tortricidae).

The Tortricidae, a globally distributed family of Lepidoptera, consists of approximately 10,000 described species, of which a large number do not have clearly defined taxonomic positions. In the present paper the systematics of Enarmoniini based on molecular data is compared to systematics based on morphology. Two genera of Enarmoniini were used for analysis: the type-genus Enarmonia (one species examined) and Ancylis (7 species examined). A comparison of a 606 bp homologous fragment of the COI mitochondrial gene revealed that Enarmoniini form a cluster distinct from Olethreutini (3 genera and 7 species examined), Eucosmini (2 genera, 4 species) and Grapholitini (4 genera, 9 species). In our opinion the molecular studies combined with previously obtained morphological data should facilitate a more natural classification system of this relatively poorly explored family of Microlepidoptera. Altogether, 30 species of Tortricidae were examined.

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.

Reassessment of the systematic position of Orthocomotis DOGNIN (Lepidoptera: Tortricidae) based on molecular data with description of new species of Euliini.

The application of molecular analyses for resolving taxonomic problems in the family Torticidae (Lepidoptera) is still uncommon. The majority of papers concern the assessment of population variability of economically important species; reports on the systematic positions ofNeotropical Tortricidae taxa are rare. The Neotropical genus Orthocomotis was classified initially as a member of the tribe Euliini. Then, based on genital morphology, it was moved to the tribe Polyorthini. A comparison of homologous 606 bp fragments of the COI mitochondrial gene revealed that Orthocomotis should be transfered back into the tribe Euliini. Based on an analysis ofphylogenetic relationships the studied genera of Euliini form a monophyletic cluster, clearly separated from tribe Polyorthini in which they were temporarily included. Moreover, in the current paper we describe two new species of the tribe Euliini: Galomecalpa lesta RAZOWSKI & PELZ, sp. n., Gauruncus ischyros RAZOWSKI & PELZ, sp. n.

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.

Interactions between Ciliate Species and Aphanizomenon flos-aquae Vary Depending on the Morphological Form and Biomass of the Diazotrophic Cyanobacterium.

Aphanizomenon flos-aquae can form extensive blooms from freshwater to the brackish environment and, being a diazotrophic species, contribute significantly to the nitrogen and carbon cycle. It occurs as single filaments or aggregates and could be used as an alternative nutrients source for bacteria and ciliates. Ciliates are a group of organisms playing a crucial role in the transfer of nitrogen from primary producers to higher trophic levels in aquatic food webs. The aim of the experiment was to study the effects of the cyanobacterium A. flos-aquae on the community of five ciliate species (Spirostomum minus, Euplotes aediculatus, Strobilidium sp., Vorticella sp. and Paramecium tetraurelia). The response of each species to the presence of a low/high cyanobacterial biomass and to the different morphological forms of A. flos-aquae (single filaments or aggregates) was demonstrated. The results of the experiment showed the variability of interactions between the cyanobacterium A. flos-aquae and ciliates and pointed out the possible benefits that A. flos-aquae provides to the ciliates (e.g., a substrate for the development of bacteria as food for ciliates or as a source of nitrogen and carbon).

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.

Evolutionary Plasticity of Mating-Type Determination Mechanisms in Paramecium aurelia Sibling Species.

The Paramecium aurelia complex, a group of morphologically similar but sexually incompatible sibling species, is a unique example of the evolutionary plasticity of mating-type systems. Each species has two mating types, O (Odd) and E (Even). Although O and E types are homologous in all species, three different modes of determination and inheritance have been described: genetic determination by Mendelian alleles, stochastic developmental determination, and maternally inherited developmental determination. Previous work in three species of the latter kind has revealed the key roles of the E-specific transmembrane protein mtA and its highly specific transcription factor mtB: type O clones are produced by maternally inherited genome rearrangements that inactivate either mtA or mtB during development. Here we show, through transcriptome analyses in five additional species representing the three determination systems, that mtA expression specifies type E in all cases. We further show that the Mendelian system depends on functional and nonfunctional mtA alleles, and identify novel developmental rearrangements in mtA and mtB which now explain all cases of maternally inherited mating-type determination. Epistasis between these genes likely evolved from less specific interactions between paralogs in the P. aurelia common ancestor, after a whole-genome duplication, but the mtB gene was subsequently lost in three P. aurelia species which appear to have returned to an ancestral regulation mechanism. These results suggest a model accounting for evolutionary transitions between determination systems, and highlight the diversity of molecular solutions explored among sibling species to maintain an essential mating-type polymorphism in cell populations.

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 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.

Molecular approach to the systematics of European Tortricini (Lepidoptera: Tortricidae).

Tortricini is a cosmopolitan tribe of the subfamily Tortricinae of the lepidopteran family Tortricidae. The most recent systematic of Tortricini are based on the external morphology of imagines and the structure of their genital organs. The present paper is the first comparative molecular study of the representatives of this tribe. We examined DNA variation in a 606 bp fragment of COI mtDNA obtained from 23 species of Tortricini and two representatives of other tribes (Archips podanus of Archipini and Aethes hartmanniana of Cochylini). The position of Spatalistis, Tortrix, Aleimma and Acleris, and some groupings of species within Acleris were confirmed by molecular data, including the synonymization of Croesia and Phylacophora with Acleris. The positions of a few groupings of the Acleris species remain unresolved.

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.

First stand of Paramecium octaurelia in Europe and molecular characteristics of other known strains of this species.

The first stand of Paramecium octaurelia in Europe (Germany) is described and interesting intra-specific polymorphism is compared within the species using strains originating from different continents (Europe, N. America and Asia). Sequenced fragments of 5' LSU rDNA and COI mtDNA revealed that the studied strains form two groups, one with strains from Germany and USA, and a second group from Israel.

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.

First American stand of Paramecium novaurelia and intra-specific differentiation of the species.

A stand of Paramecium novaurelia was found in Boston Massachusetts, USA, the first on this continent. Molecular studies (RAPD and sequencing ofrRNA [3'SSUrRNA-ITS1, 5' LSU rRNA] and COI mtDNA fragments) of P. novaurelia strains show intra-specific polymorphism within the species as strain clusters characterized by variable relationships.