BIOCHEMICAL, PHYSIOLOGICAL, AND MOLECULAR ANALYSIS OF SEXUAL ISOLATION IN THE SPECIES COMPLEX CLOSTERIUM-PERACEROSUM-STRIGOSUM-LITTORALE (CHLOROPHYTA)

Closterium strains obtained from Japan (NIES-64 and -65) and Nepal (NI ES-67 and -68) have been classified as the same taxonomic species; how ever, they are sexually isolated from each other. Wizen NIES-G4 and -6 5 cells were separately incubated in a medium in which both strains ha d previously been cultured together, release of protoplasts from both strains was observed. We suggest that factors responsible for the rele ase of protoplasts from cells of both NIES-64 and -65 are produced in a mixed culture of these cells and function during conjugation. These factors, however, had no effect on the release of protoplasts from cel ls of strains NIES-67 or -68. Alternatively, a protein that is respons ible for the release of protoplasts front cells of NIES-68, called the protoplast-release-inducing protein (PR-IP), had no effect on the rel ease of protoplasts from cells of strains NIES-64 or -65. When the med ia obtained from the culture of NIES-64 and -65 cells at various mixin g ratios were analyzed by western blotting with antiserum to a 42-kDa subunit of PR-IP, no cross reaction was detected. In Southern hyrbridi zation analysis, no hybridizing band was observed when genomic DNAs of NIES-64 and -65 cells were probed with cDNAs encoding the two subunit s of PR-IP. We suggest from these results that the factors responsible for the release of protoplasts from NIES-64 and -65 cells are not str ucturally similar to PR-IF. It is known that the release of PR-IP from NIES-67 cells can be induced by the action of another sex pheromone ( PR-IF inducer) which is released by NIES-68 cells. In contrast, no pro toplast-release-inducing activity was observed from either NIES-64 or -65 in a culture medium conditioned by opposite strains. We suggest th at the conjugation systems employed by strains NIES-64/ NIES-65 and st rains NIES-67/NIES-68 differ, and we propose a possible mechanism of s exual isolation between these biological species.