Genetic analysis of the Paramecium aurelia species complex (Protozoa: Ciliophora) by classical and molecular methods

The Paramecium aurelia complex includes 15 species (sibling species) and is characterised by inbreeding (to varying degrees in different species), causing an increase in intra-specific differentiation. Investigations into inter- and intraspecific differentiation of strains originating from remote habitats within species of the com- plex were carried out by classical inter-strain crosses and molecular analyses (RAPD- PCR fingerprints, ARDRA riboprints, RFLP-PCR analysis). RAPD analysis showed that all species in the complex possessed characteristic band patterns and the majority were also polymorphic intra-specifically. A correlation exists between the degree of inbreeding characteristic for a species with differentiation of DNA genotypes revealed by RAPD analysis within species, where inbreeders showed substantial variability of band patterns and moderate inbreeders were highly similar. RFLP analysis (a 480bp fragment of the gene coding the Hsp 70 protein) with the application of restriction enzyme TruII distinguished among species, while digestion with restriction enzyme AluI distinguished groups of species (clusters) and both enzymes revealed intra- species polymorphism within P. dodecaurelia. ARDRA riboprinting (using a fragment of SSU-LSU rDNA, about 2400 bp) with restriction enzymes HhaI, AluI, HinfI, TaqI distinguished groups of species with different band patterns. The majority of en- zymes also demonstrated intra-specific differentiation within P. dodecaurelia. TaqI also revealed intraspecific differences in P. biaurelia and P. tetraurelia. All species in the P. aurelia complex showed a high percentage of surviving hybrid clones in F1 obtained by conjugation and F2 obtained by autogamy in inter-strain crosses. A low percentage was observed only in F2 inter-strain hybrids of P. tredecaurelia, however no cytological changes in the nuclear apparatuses were detected and similar band patterns existed in the studied strains. Future studies, including sequencing of rDNA fragments, may disclose deeper relationships of the species.