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.

a textbook example of an east-west disjunction or a taxonomic misconcept? -Zoologica Scripta , 33 , 213-221. We compare 1036 bp of the mitochondrial cytochrome b gene (cyt b ) from all six Mauremys species with 16 other taxa, representing both currently recognized subfamilies of the Geoemydidae (Geoemydinae and Batagurinae) to contribute a comprehensive dataset towards resolving the conflicting Mauremys taxonomy and phylogeography. Mauremys , a representative of the Geoemydinae, is thought to be an example of a taxon with an east-west disjunction due to Pleistocene glacial extinction, with species occurring in the western Palearctic and species in the eastern Palearctic and Oriental regions. Our results contradict this traditional zoogeographical scheme and the current taxonomy of the Geoemydidae. Mauremys is paraphyletic with respect to two East Asian genera belonging to the Batagurinae: Chinemys and Ocadia . Therefore, Mauremys , as currently understood, clearly represents a taxonomic misconcept. Mauremys + Chinemys + Ocadia contains four well supported clades, two of which -M. japonica + Chinemys + Ocadia and M. annamensis + M. mutica -are confined to eastern Asia. The other two -M. caspica + M. rivulata and M. leprosa -occur in the western Palearctic. Mauremys leprosa may represent an ancient lineage which differentiated before the split between the other western and eastern species occurred. The patchy distribution of the four clades is likely the result of several ancient radiations rather than of a Pleistocene extinction. The sister-group of Mauremys + Chinemys + Ocadia is Cuora , a morphologically highly specialized genus with a complicated shell hinging mechanism.

BackgroundDespite 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.ResultsThe 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.ConclusionsSurprisingly, 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.

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 .

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.