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.

Limited Mutation-Rate Variation Within the Paramecium aurelia Species Complex.

Mutation is one of the most fundamental evolutionary forces. Studying variation in the mutation rate within and among closely-related species can help reveal mechanisms of genome divergence, but such variation is unstudied in the vast majority of organisms. Previous studies on ciliated protozoa have found extremely low mutation rates. In this study, using mutation-accumulation techniques combined with deep whole-genome sequencing, we explore the germline base-substitution mutation-rate variation of three cryptic species in the Paramecium aurelia species complex-P. biaurelia, P. sexaurelia, and P. tetraurelia We find that there is extremely limited variation of the mutation rate and spectrum in the three species and confirm the extremely low mutation rate of ciliates.

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.

Differential retention and divergent resolution of duplicate genes following whole-genome duplication.

The Paramecium aurelia complex is a group of 15 species that share at least three past whole-genome duplications (WGDs). The macronuclear genome sequences of P. biaurelia and P. sexaurelia are presented and compared to the published sequence of P. tetraurelia. Levels of duplicate-gene retention from the recent WGD differ by > 10% across species, with P. sexaurelia losing significantly more genes than P. biaurelia or P. tetraurelia. In addition, historically high rates of gene conversion have homogenized WGD paralogs, probably extending the paralogs' lifetimes. The probability of duplicate retention is positively correlated with GC content and expression level; ribosomal proteins, transcription factors, and intracellular signaling proteins are overrepresented among maintained duplicates. Finally, multiple sources of evidence indicate that P. sexaurelia diverged from the two other lineages immediately following, or perhaps concurrent with, the recent WGD, with approximately half of gene losses between P. tetraurelia and P. sexaurelia representing divergent gene resolutions (i.e., silencing of alternative paralogs), as expected for random duplicate loss between these species. Additionally, though P. biaurelia and P. tetraurelia diverged from each other much later, there are still more than 100 cases of divergent resolution between these two species. Taken together, these results indicate that divergent resolution of duplicate genes between lineages acts to reinforce reproductive isolation between species in the Paramecium aurelia complex.

The first European stand of Paramecium sonneborni (P. aurelia complex), a species known only from North America (Texas, USA).

P. aurelia is currently defined as a complex of 15 sibling species including 14 species designated by Sonneborn (1975) and one, P. sonneborni, by Aufderheide et al. (1983). The latter was known from only one stand (Texas, USA). The main reason for the present study was a new stand of Paramecium in Cyprus, with strains recognized as P. sonneborni based on the results of strain crosses, cytological slides, and molecular analyses of three loci (ITS1-5.8S-ITS2-5'LSU rDNA, COI, CytB). The new stand of P. sonneborni in Europe shows that the species, previously considered endemic, may have a wider range. This demonstrates the impact of under-sampling on the knowledge of the biogeography of microbial eukaryotes. Phylogenetic trees based on all the studied fragments revealed that P. sonneborni forms a separate cluster that is closer to P. jenningsi and P. schewiakoffi than to the other members of the P. aurelia complex.

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).

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.

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 Paramecium quadecaurelia strains (P. aurelia spp. complex, Ciliophora) identified by molecular markers (rDNA and mtDNA).

Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5' rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of the P. aurelia complex, all P. quadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines.

[Analysis of G.F. Gause experimental time-series by means of continuous-time models].

Four population dynamics models, namely Verhulst, Gompertz, Rosenzweig, and Svirezhev ones, have been used to approximate two well-known time-series of Paramecia aurelia and P. caudatum population size (Gause, 1934). The parameters are estimated for each of the models by the least-square method (with global fitting) in two different ways: with and without an additional upper bound for a parameter value. In the latter (traditional) case, when the deviations of theoretical (model) trajectories from experimental time-series have been tested for normality (Kolmogorov-Smirnov test, Shapiro-Wilk test) with zero average, and for the presence/absence of serial correlations (Durbin-Watson criteria), the best results are obtained for the Gompertz and Verhulst models. In the former, more realistic, case (when we impose an additional constraint that the parameter meaning the carrying capacity of the environment has to be greater than any element in the sample), the best results are observed for the Gompertz model. Under this constraint, the canonical technique for deviation analysis can be applied in a restricted version only.

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).

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.

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.

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.

Cytochrome b sequence data suggest rapid speciation within the Paramecium aurelia species complex.

We investigated mitochondrial Cytochrome b sequences from all 15 members of the enigmatic Paramecium aurelia species complex (Ciliophora). The analysis revealed high genetic distances between the different P. aurelia species (6.1-19.8%) and a largely unresolved, star-like phylogenetic tree. This result strongly supports a rapid radiation in the evolutionary history of this species complex and it correlates well with the hypothesis that the extant species diversity may have originated from the neutral consequences of a whole genome duplication in the common ancestor of P. aurelia.

Large global effective population sizes in Paramecium.

The genetic effective population size (N(e)) of a species is an important parameter for understanding evolutionary dynamics because it mediates the relative effects of selection. However, because most N(e) estimates for unicellular organisms are derived either from taxa with poorly understood species boundaries or from host-restricted pathogens and most unicellular species have prominent phases of clonal propagation potentially subject to strong selective sweeps, the hypothesis that N(e) is elevated in single-celled organisms remains controversial. Drawing from observations on well-defined species within the genus Paramecium, we report exceptionally high levels of silent-site polymorphism, which appear to be a reflection of large N(e).

Paramecium aurelia revisited.

The species Paramecium aurelia sensu latu, containing 15 sexually isolated subspecies (syngens), is the classic example of a sibling species complex in the ciliates. Using DNA sequence comparison, it is now possible to see whether this example parallels other studied sibling species complexes. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron for 13 of the syngens plus two other Paramecium species and several Tetrahymena spp. Using available spirotrich sequences of the internal transcribed spacer 2 (ITS2), we established the RNA transcript folding pattern for ciliates. Ciliates exhibit the two highly conserved helices in their RNA transcript folding pattern in common with other eukaryotes, despite their unusual nuclear behavior and their presumed low copy number of micronuclear ribosomal repeats. Consequently, the set of 111-116 ITS2 nucleotide positions that are relatively conserved in evolution can be derived and used for comparative analysis. Mating behavior (i.e. gamete agglutination and fusion) is the character showing greatest correlation with the degree of ITS2 evolution in the P. aurelia complex, as also found in other eukaryotes. The degree of change in the ITS2 relatively conserved sequences found among the sibling species of P. aurelia is the same degree as found among the sibling species of the Drosophila melanogaster-mauritania-sechellia-simulans-yakuba species complex. The relatively conserved subregion of ITS2, determined from transcript secondary structure, is a tool for identifying the level of the biological species in the absence of knowledge of sexual compatibility in both micro- and macro-eukaryote species complexes.