Analysis of mechanisms of reproductive isolation and speciation in unicellular conjugatophycean Closterium.

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The Closterium peracerosum-strigosum-littorale complex (C. pslc) is a unicellular alga, and is known to be composed of several reproductively isolated mating groups of heterothallic strains. We have carried out field studies in 8 prefectures such as Tokyo, Ibaraki, Saitama, Nagano, Hiroshima, Yamaguchi, Okinawa, and Shizuoka in Japan, and in five foreign countries such as Germany, Taiwan, New Zealand, Slovakia, and Indonesia, to isolate reproductively isolated C. pslc. Finally, we have established 43 clones from these fields.We have studied phylogenetic relationship using DNA sequences of 1506 group I introns of these clones. Based on the alignment, significant phylogenetic relationships were observed among mating groups II-A and II-B, while mating group I-E was distant from groups II-A and II-B. Indeed, mating group I-E is completely sexually isolated from mating groups II-A and II-B, groups II-A and II-B are partially isolated from each other, and only mating-type plus (mt^+) cells of group II-A and mating-type minus (mt^-) cells of group II-B form zygotes. Sexual cell division in both mating-type cells of group A was stimulated in conditioned media in which cells of group II-B had been cultured. When mt^- cells of group II-B were stimulated in conditioned medium derived from group II-A, mt^+ cells of group II-B did not respond to the conditioned medium. Conditioned media derived from group D did not exhibit SCD-inducing activity against any strain except those within its own group. From the alignment of deduced amino acid sequences from orthologous PR-IP Inducer genes, we detected a significant similarity among groups A and B., and mating group D had low similarity to other mating groups. The existing degree of reproductive isolation can be partially explained by differences in molecular structures and physiological activities of sex pheromones of these heterothallic mating groups.