Possible Third Step Preventing Conjugation between Different Species of Blepharisma

In the genus Blepharisma, reproductive isolation between different species appears to be established at least by two barriers: (1) a mating pheromone, i.e., gamone 1, and (2) a factor involved in pair formation. Using four species, we experimentally investigated other potential barriers to interspecific conjugation in Blepharisma, as well as the first and second barriers. Cell-free fluid from type I cells (CFF1) of B. americanum had no effect on B. undulans, B. japonicum, or B. stoltei. Type II cells of B. americanum responded to CFF1 from B. americanum but not to CFF1 from B. undulans, B. japonicum, or B. stoltei. Gamone 1, therefore, seems to be the first reproductive barrier (with the inclusion of B. americanum species [megakaryotype 3]) as reported previously. In pretreated cells with complementary gamones in B. undulans and B. americanum, inter-species pair formation was rare, but pair formation between B. americanum and B. japonicum and between B. americanum and B. stoltei occurred at relatively high frequency. Most of the inter-species B. americanum−B. stoltei pairs underwent nuclear changes specific to conjugation. No significant difference was observed between the intra- and inter-species pairs over the time course of the nuclear changes, but the percentage of abnormal cells was higher in inter-species pairs than in intra-species pairs, and no progenies were produced by inter-species pairs. These results suggest a third barrier or step, in addition to the first and second ones, in nuclear changes after pair formation that prevents interspecific conjugation in Blepharisma.