Paramecium Diversity and Phylogeny: Three Loci Molecular Characterization

Abstract

Ciliated protozoan are an important bioindicators for pollution and have long been used for environmental biomonitoring, particularly in water purification plants and in activated sludge processes. Each species of ciliates has its own physical and chemical valences and therefore based on the indicator values of representative ciliates, degree of pollution of a particular water body can be determined. Among ciliates Paramecium has become a privileged model for the study of “species problem” particularly in the case of “Paramecium aurelia complex” that has been intensely investigated. Despite extensive studies taxonomy of Paramecium is still changing. The major problem is uneven sampling of Paramecium with relatively few representatives of each species. Continued discovery of new species from various origins (worldwide) are proof of the fact that the list of Paramecium species is not yet complete. Hence, the addition of new species by more sampling is needed to resolve the phylogeny within the Paramecium genus. For this purpose, present study was aimed at molecular characterization of Paramecium species from water samples originating from various regions of Punjab province of Pakistan. Fragments of 18S rDNA, ITS1-5.8S-ITS2-5’LSU rDNA, COII, Hsp70 and Histone H4 genes were used as molecular markers for the phylogenetic analysis of ten locally isolated strains of Paramecium species including a standing-alone FT8 strain previously isolated by Shakoori et al. (2014). The nucleotide sequences of PCR products of different molecular markers of various isolates (FT2.1, FT3.1, FT4.1, FT5.1, FT6.1, FT7.1, FT9.1, FT10.1 and FT11.1) were compared with the available sequences of these markers in other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all nine strains (FT2.1, FT3.1, FT4.1, FT5.1, FT6.1, FT7.1, FT9.1 FT10.1 and FT11.1) had very close relationship with P. primaurelia except for FT8 strain. FT8 showed its unique position in comparison to all other species in the phylogenetic trees, so became the main focus of our study. Phylogeny of this strain was further carried out with PiggyMac sequence, where it again behaved differently as compared to other species. Sexual behavior, immaturity and maturity periods of FT8 were analyzed by performing daily reisolations that revealed three unique characteristics of this strain; 1) autogamy as only source of the exchange of genetic material 2) no clumps formation before conjugation that is the prerequisite of sexual process, 3) selfing among reactive cells. Second and third characteristics of this species turned our attention towards its mating types (“odd” 0 and “even” E). Why agglutination does not occur in this strain like all other Paramecium species? Or maybe there are circadian rhythms of mating xv types? In order to resolve this mystery, an experiment following circadian rhythms of completely light and completely dark cycles was performed multiple times during a period of one month. However, every time no conjugation upon mixing but selfing in the original cultures was observed. Results of this study could not prove the existence of mating types in FT8 strain but off course detailed analyses at genetic level is required for precise knowledge. Another important aim was to count the number of micronuclei of this unique species with and without Spt5-GFP signals. Four number of MICs on an average were observed, whereas two, three and five MICs were also seen in some karyonides. However, GFP signal was found to be activated till the skein formation of conjugating pairs. No signal in micronuclei of later stages could be observed. Finally, number of events of whole genome duplications (WGDs) of FT8 strain were studied by counting the number of paralogs for highly expressed, highly conserved L8 and S13 ribosomal protein genes. Total three number of genes (L8FT8-1, L8FT8-3 and L8-FT8-4) with L8 amplifications and two genes (S13FT8-1 and S13FT8-2) with S13 after extensive screenings were found. Number of genes obtained from amplifications results supported that this species has not gone through any additional WGD event. However, whole genome sequencing of this species would resolve the complete and precise information of its number of WGDs. Finally, based on the phylogenetic trees of all molecular markers including L8 and S13 genes, it can be concluded that despite occupying unique position in the phylogenetic tree, FT8 was closer to P. multimicronucleatum than to P. caudatum.