Evolutionary relationship and species separation of four morphologically similar stichotrichous ciliates (Protozoa, Ciliophora)

Phylogenetic and taxonomic studies on ciliate protists using molecular approaches have been demonstrated to be very reliable to form strong conclusions and results. In the present work, species separation of some morphologically similar stichotrichous ciliates, two species of Pseudokeronopsis and two species of Apokeronopsis, was reexamined using amplified ribosomal DNA restriction analysis (PCR-RFLP). Five of 10 restriction enzymes revealed species-specific polymorphic patterns, of which four similar stichotrichs could be significantly separated and identified. Among them, EcoR I offered almost no significantly different restriction fragment patterns, but the four species could be separated from one another and identified with Hae III. Distinctly different restriction digestion haplotypes and similarity indices separated the species, and were used to construct a phylogeny. Phylogenies based on ITS2 nucleotide sequences and ITS2 secondary structures supported the separation of Pseudokeronopsis and Apokeronopsis using RFLP analysis, although three Pseudokeronopsis carnea populations did not cluster together. In addition, phylogenetic analyses using multiple algorithms confirmed that these two genera formed two distinct groups within the urostylids.