The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates.

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Paramecium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Paramecium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposition. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL barcode for ciliated protists.

[Comparative description of macronuclear electrophoretic karyotypes of Paramecium primaurelia and Paramecium novaurelia sibling species].

The macronuclear genomes of two sibling species belonging to the Paramecium aurelia complex--P. primaurelia and P. novaurelia--were analyzed by pulsed-field gel electrophoresis (PFGE). Their electrophoretic karyotypes showed a continuous spectrum of different-sized DNA molecules with a species-specific pattern of banding, which was reproducible and did not change with strain senescence. Thus, P. aurelia macronuclear PFGE profiles could be described by an approach analogous to that used for the description of metaphase chromosome banding patterns. At first, well-identifiable regions (size fractions) of a PFGE profile, which can be seen at any resolution, are determined. Then, the bands of the second order of resolution (subfractions) can be defined in some of these regions. The blocks of the first and second orders can be utilized as inner markers of the PFGE profile allowing precise comparison of different PFGE profiles. Such comparative analysis has demonstrated some marking differences in organization of the macronuclear genomes of P. primaurelia and P. novaurelia, and low level of intraspecific polymorphism. Worth noting is that the P. novaurelia strain isolated in USA was different from all other analyzed P. novaurelia strains originating from Europe. The nature of banding of P. aurelia PFGE profiles is discussed. The revealed high order and stability of the macronuclear genome organization makes possible searching for new approaches to study the mechanisms of this non-trivial genome formation and maintenance. Further PFGE analysis of the macronuclear genomes of the other Paramecium species in relation with the Paramecium phylogeny may provide insights into the directions of the evolution of the macronuclear genome in Ciliata.

Species of the Paramecium aurelia complex in Japan.

In Japan, the presence of Paramecium sexaurelia was revealed for the first time, and a new habitat of P. tetraurelia was found there.

Habitat of Paramecium biaurelia in Italy, the Island of Elba.

The new habitat of Paramecium biaurelia was found on the Island Elba.

Zooplankton in the artificial pond of the Botanical Garden of the Jagiellonian University in Kraków.

Paramecium caudatum and the predominant zooplanton organisms were registered in the studied pond of the Botanical Garden of the Jagiellonian University in Kraków. The finding of Stentor coeruleus indicates an improvement in the environmental conditions in Kraków, connected with the partial liquidation of the huge steelworks.

Zooplankton in the ponds with tropical plants in the greenhouses of the Botanical Garden of the Jagiellonian University in Kraków.

The presence of P. tetraurelia was found in the pond in "The Palm-House" greenhouse.

The Paramecium aurelia species complex in Ukraine.

New habitats of Paramecium biaurelia were found in the Gorgany Mts, Eastern Carpathians.

Further investigations on the zooplankton of water bodies in the Botanical Garden of the Jagiellonian University in Kraków.

The aim of this study was to register the zooplanktonic organisms in water bodies in the Botanical Garden of the Jagiellonian University in Kraków, especially from the point of view of the occurrence of species of the Paramecium aurelia complex. In one pond, artificially constructed, the presence of P. tetraurelia was revealed.

Paramecium sexaurelia of the Paramecium aurelia species complex in Thailand.

The clones originating from Thailand, Phuket Island, were identified as Paramecium sexaurelia.

Zooplankton of some water bodies of the Zoological Garden in Kraków.

The aim of this study was to register the zooplanktonic organisms in water bodies in the Zoological Garden in Kraków, especially from a point of view of the occurrence of species of the Paramecium aurelia complex. Only P. caudatum was found in one habitat, in several sampling places. The species seems very resistant and can survive in poor or polluted habitats in which the P. aurelia ssp. are eliminated.

Studies on the Paramecium aurelia species complex in Lake Pamvotis (Ioannina, N.W. Greece).

Paramecium sexaurelia was identified in Lake Pamvotis, Grecce. The occurrence of this species there was confirmed.

Paramecium aurelia species complex in a natural but newly reconstructed pond of the Botanical Garden of the Jagiellonian University in Kraków.

In a natural but newly reconstructed pond the presence of P. biaurelia was revealed. The previous studies conducted in 1992 did not reveal the occurrence of any species of the Paramecium aurelia complex here.

Paramecium triaurelia and other species of the aurelia complex in the Kaczawskie Mts and Plateau (western Sudeten).

The present investigations revealed the occurrence of Paramecium triaurelia on the territory of the Kaczawskie Plateau, while its presence was confirmed in the Kaczawskie Mts. In Poland, the presence of this species was recorded only in the Western Sudeten, in spite of many years of investigation. As the result of the studies also the occurrence of P. biaurelia and P. novaurelia was recorded on the territory of the Kaczawskie Mts and the Kaczawskie Plateau.

Species of the Paramecium aurelia complex in Spain.

The new habitats of P. primaurelia, P. biaurelia, and P. triaurelia of the P. aurelia complex were described in Spain. P. pentaurelia was also found, for the first time in that country.

Studies on the distribution of the Paramecium aurelia species complex of Poland.

Species of the Paramecium aurelia complex occurred in the studied regions of the Carpathians and the Sudetes Mts with varying frequency. P. novaurelia appeared most often, P. biaurelia occurred less frequently but was also very characteristic, both species prevailed over P. primaurelia. P. tetraurelia and P. triaurelia were very rare.

Species of the Paramecium aurelia complex in the middle Sudeten of Czecho-Slovakia.

On the investigated territory three species of the complex were identified, namely Paramecium biaurelia, P. triaurelia, and P. novaurelia. P. novaurelia dominated over the other species with regard to the number of clones established from nature as well as to habitats.