Distribution of rickettsial endosymbionts and their possible transmission within the Pleodorina japonica (Volvocales, Chlorophyceae) population

The green alga Pleodorina japonica is an interesting volvocine species that harbors abundant rickettsial endosymbionts ("MIDORIKO") within its cytoplasm. However, the diversity and transmission of these endosymbionts within the species remain unclear. In this study, we examined the presence or absence of "MIDORIKO" and the genetic diversity in 21 culture strains of the host P. japonica population from various localities in Japan. Genomic polymerase chain reactions using "MIDORIKO"-specific primers and 4',6-diamidino-2-phenylindole-staining demonstrated that only five of the 21 strains harbored "MIDORIKO." The 16S ribosomal DNA sequences of "MIDORIKO" from these five strains (1148 bp) were identical to each other and distinct from the sequences of the rickettsial endosymbionts harbored by other algal species and protists, suggesting that "MIDORIKO" from P. japonica is specific to P. japonica. The phylogenetic results for the 21 host strains, which were resolved based on three nuclear genes encoding oxygen-evolving enhancer protein 1, F1F0 ATP synthase subunit beta and actin disagreed significantly. None of the three gene phylogenies supported the close relationship of the five "MIDORIKO"-harboring strains. A recombination test using the three concatenated genes provided strong evidence of recombination. Therefore, gene flow by sexual reproduction has likely occurred in the natural habitats of P. japonica. The transmission of "MIDORIKO" among different P. japonica genotypes could also be considered to occur via sexual reproduction, although it is likely infrequent via that method given the sporadic nature of "MIDORIKO" within the P. japonica population. Although P. japonica exhibits homothallic sexual reproduction, the present genetic data demonstrate that it is undoubtedly a biological species.