The mitochondrial genome sequence of the ciliate Paramecium caudatum reveals a shift in nucleotide composition and codon usage within the genus Paramecium.

BACKGROUND: Despite the fact that the organization of the ciliate mitochondrial genome is exceptional, only few ciliate mitochondrial genomes have been sequenced until today. All ciliate mitochondrial genomes are linear. They are 40 kb to 47 kb long and contain some 50 tightly packed genes without introns. Earlier studies documented that the mitochondrial guanine + cytosine contents are very different between Paramecium tetraurelia and all studied Tetrahymena species. This raises the question of whether the high mitochondrial G+C content observed in P. tetraurelia is a characteristic property of Paramecium mtDNA, or whether it is an exception of the ciliate mitochondrial genomes known so far. To test this question, we determined the mitochondrial genome sequence of Paramecium caudatum and compared the gene content and sequence properties to the closely related P. tetraurelia. RESULTS: The guanine + cytosine content of the P. caudatum mitochondrial genome was significantly lower than that of P. tetraurelia (22.4% vs. 41.2%). This difference in the mitochondrial nucleotide composition was accompanied by significantly different codon usage patterns in both species, i.e. within P. caudatum clearly A/T ending codons dominated, whereas for P. tetraurelia the synonymous codons were more balanced with a higher number of G/C ending codons. Further analyses indicated that the nucleotide composition of most members of the genus Paramecium resembles that of P. caudatum and that the shift observed in P. tetraurelia is restricted to the P. aurelia species complex. CONCLUSIONS: Surprisingly, the codon usage bias in the P. caudatum mitochondrial genome, exemplified by the effective number of codons, is more similar to the distantly related T. pyriformis and other single-celled eukaryotes such as Chlamydomonas, than to the closely related P. tetraurelia. These differences in base composition and codon usage bias were, however, not reflected in the amino acid composition. Most probably, the observed picture is best explained by a hitherto unknown (neutral or adaptive) mechanism that increased the guanine + cytosine content in P. tetraurelia mtDNA on the one hand, and strong purifying selection on the ancestral amino acid composition on the other hand. These contradicting forces are counterbalanced by a considerably altered codon usage pattern.

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.

High mitochondrial haplotype diversity of Coleps sp. (Ciliophora: Prostomatida).

To date the awareness of the temporal population structure in eukaryotic microbes is very limited. This is exemplified in the scarce knowledge about the intraspecific genetic variation in ciliates. To elucidate the genetic variation of Coleps (Ciliophora: Prostomatida), we employed the analysis of the mitochondrial apocytochrome b gene of the Coleps community in a young lake in Germany. The analysis of 111 isolates, sampled from April 2005 to September 2006, revealed a high genetic variation for the two dominant Coleps species (11 mitochondrial haplotypes in Coleps spetai, nine in Coleps hirtus hirtus). The study represents one of the largest datasets of intraspecific diversity in a microbial eukaryote and demonstrates for the first time the suitability of a mitochondrial gene for the detection of genetic variation within populations of eukaryotic microbes. However, the results of our study warrant caution in the application of such an approach, as we amplified some non-orthologous cob-like sequences, whose uncritical acceptance would have led to the erroneous discovery of cryptic species.

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.

The Paramecium aurelia species complex, frequency and co-occurrence across Europe.

In Europe, 10 among the 15 known species of the Paramecium aurelia complex have been recorded including P. primaurelia, P. biaurelia, P. triaurelia, P. tetraurelia, P. pentaurelia, P. sexaurelia, P. septaurelia, P. novaurelia, P. dodecaurelia, and P. tredecaurelia. The occurrence of species of this complex in Europe has been estimated on the basis of the number of habitats and the ratio value (r.v.), i.e. the number of habitats for a defined species to the total number of habitats with species of the P. aurelia complex. The r.v. presents the relative frequency of the particular species to others of the complex. In total, 483 habitats were considered. The dominant species is P. novaurelia (r.v. 0.38, found in 186 habitats), followed by P. biaurelia (r.v. 0.26 in 125 habitats) and P. primaurelia (0.23 in 113 habitats). Other species are less frequent, such as P. tetraurelia (r.v. 0.06 found in 29 habitats), P. triaurelia (r.v. 0.04 in 22 habitats), or rare such as P. septaurelia (r.v. 0.025 found in 12 habitats), as well as P. pentaurelia and P. sexaurelia (r.v. 0.02 for both, found in 11 and 10 habitats, respectively). P. dodecaurelia was found in three localities in Europe (r.v. 0.006) and P. tredecaurelia is known from a single habitat (r.v. 0.002). This paper presents new populations, found at present, of species of the P. aurelia complex in Europe (Germany, Italy, Spain, Iceland, Poland, Great Britain) and one new from the USA.

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).

Intraspecific genetic variation in Paramecium revealed by mitochondrial cytochrome C oxidase I sequences.

Studies of intraspecific genetic diversity of ciliates, such as population genetics and biogeography, are particularly hampered by the lack of suitable DNA markers. For example, sequences of the non-coding ribosomal internal transcribed spacer (ITS) regions are often too conserved for intraspecific analyses. We have therefore identified primers for the mitochondrial cytochrome c oxidase I (COI) gene and applied them for intraspecific investigations in Paramecium caudatum and Paramecium multimicronucleatum. Furthermore, we obtained sequences of the ITS regions from the same strains and carried out comparative sequence analyses of both data sets. The mitochondrial sequences revealed substantially higher variation in both Paramecium species, with intraspecific divergences up to 7% in P. caudatum and 9.5% in P. multimicronucleatum. Moreover, an initial survey of the population structure discovered different mitochondrial haplotypes of P. caudatum in one pond, thereby demonstrating the potential of this genetic marker for population genetic analyses. Our primers successfully amplified the COI gene of other Paramecium. This is the first report of intraspecific variation in free-living protozoans based on mitochondrial sequence data. Our results show that the high variation in mitochondrial DNA makes it a suitable marker for intraspecific and population genetic studies.