Putative N2-fixing heterotrophic bacteria associated with dinoflagellate–Cyanobacteria consortia in the low-nitrogen Indian Ocean

Heterotrophic dinoflagellates bearing unicellular cyanobacterial symbionts are common within the order Dinophysiales. However, the ecological role of these symbionts is unclear. Due to the occurrence of such consortia in oceanic waters characterized by low nitrogen concentrations, we hypothesized that the symbionts fix gaseous nitrogen (N 2 ). Individual heterotrophic dinoflagellates containing cyanobacterial symbionts were isolated from the open Indian Ocean and off Western Australia, and characterized using light microscopy, transmission electron microscopy (TEM), and nitrogenase (nifH) gene amplification, cloning, and sequencing. Cyanobacteria, heterotrophic bacteria and eukaryotic algae were recognized as symbionts of the heterotrophic dinoflagellates. nifH gene sequences were obtained from 23 of 37 (62%) specimens of dinoflagellates (Ornithocercus spp. and Amphisolenia spp.). Interestingly, only 2 specimens contained cyanobacterial nifH sequences, while 21 specimens contained nifH genes related to heterotrophic bacteria. Of the 137 nifH sequences obtained 68% were most similar to Alpha-, Beta-, and Gammaproteobacteria, 8% clustered with anaerobic bacteria, and 5% were related to second alternative nitrogenases (anfH). Twelve sequences from 5 host cells formed a discrete cluster which may represent a not yet classified nifH cluster. Eight dinoflagellates contained only 1 type of nifH sequence (> 99% sequence identity) but overall the putative N 2 -fixing symbionts did not appear host specific and mixed assemblages were often found in single host cells. This study provides the first insights into the nifH diversity of dinoflagellate symbionts and suggests a symbiotic co-existence of non-diazotrophic cyanobacteria and N 2 -fixing heterotrophic bacteria in heterotrophic dinoflagellates.