Using nuclear-encoded LSU and SSU rDNA sequences to identify the eukaryotic endosymbiont in Amphisolenia bidentata...

The marine dinoflagellate Amphisolenia bidentata possesses complete intracellular symbionts of prokaryotic and eukaryotic origin. This was confirmed ultrastructurally little over 20 years ago when it was showed that the eukaryotic endosymbiont had a nucleus, a chloroplast and mitochondria. We collected Amphisolenia bidentata cells in the Indian Ocean and the identity of the eukaryotic endosymbionts was investigated using both microscopical and molecular methods. Individual specimens of Amphisolenia bidentata were identified by light microscopy and selected for single-cell PCR. Host and endosymbiont nuclear-encoded LSU and SSU rDNA sequences were determined by PCR cloning. Blast searches showed the endosymbiont LSU sequence to have affinity to Pelagophyceae, an algal class within Chromalveolata that also includes dinoflagellates. Since more SSU rDNA sequences from pelagophytes are available we performed a SSU based phylogeny of chromalveolates. The eukaryotic endosymbiont clustered within a clade comprising flagellated and coccoid pelagophytes whereas Amphisolenia bidentata formed a sister taxon to other dinophysioids. Molecular data therefore resolved the endosymbiont in A. bidentata being a pelagophyte and thus identified the ninth novel chloroplast type in dinoflagellates and a new species association. Based on sequence divergence estimates and phylogenetic inference the endosymbiont in A. bidentata likely represents an undescribed genus of pelagophytes.