Two distinct green algal symbionts cohabiting in the Japanese black salamander Hynobius nigrescens

Green algae in the genus Oophila are associated with eggs of amphibians, e.g. salamanders and frogs, and are the only known photosynthetic endosymbionts in vertebrates. However, phylogenetic placements of "Oophila" algae associated with amphibians into two distinct clades have caused significant confusion and hindered our understanding of the nature and diversity of this symbiotic system. To gain insights into this unexplored symbiotic model, we carried out a sampling of the Japanese black salamander Hynobius nigrescens eggs, an endemic species in Japan, covering five distinct locations. Egg masses from H. nigrescens all contained green algae, some of which were sampled, isolated, and cultured. In total, 32 algal isolates could grow as free-living cultures and were used for phylogenetic and morphological analyses. Our results supported the co-existence of two Oophila subgroups with H. nigrescens embryos . One is associated with the known subclades of O. amblystomatis found in North America, and the other belongs to an Oophila /Chlorococcum group in the Stephanosphaerinia. The isolates in each subgroup showed identical morphology across sampling sites, consistent with our phylogenetic findings. These new, unique cultures of amphibian-associated green algae will help to understand photosymbiosis between vertebrates and algae.