Identifying and Distinguishing Sibling Species in the Tetrahymena pyriformis Complex (Ciliophora, Oligohymenophorea) Using PCR/RFLP Analysis of Nuclear Ribosomal DNA

ABSTRACT We describe a riboprinting strategy for identifying and distinguishing among sibling species in the Tetrahymena pyriformis complex. It involves use of the polymerase chain reaction to amplify a large segment of the nuclear ribosomal DNA and internal transcribed spacers, and digestion of this DNA with restriction enzymes. Unique restriction fragment length patterns or haplotypes were then used to distinguish species into: (1) six taxa that were identifiable to the species level, (2) eight taxa that were separated into four pairs, and (3) a group of eight taxa that were identical to each other. The latter result indicates that a more variable molecule is needed to distinguish the most closely related species in the complex. There was no intraspecific variation between two strains from one species ( Tetrahymena thermophila ) nor among multiple isoiates from another species ( Tetrahymena empidokyrea ). This approach provides an alternative to traditional techniques for identifying T. pyriformis species that require living reference specimens and/or that reveal high levels of intraspecific variation.