Symbioses between eukaryotes and nitrogen (N₂)-fixing cyanobacteria (or diazotrophs) are quite common in the plankton community. A few genera of diatoms (Bacillariophyceae) such as Rhizosolenia, Hemiaulus and Chaetoceros are well known to form symbioses with the heterocystous diazotrophic cyanobacteria Richelia intracellularis and Calothrix rhizosoleniae. The latter are also called diatom-diazotroph associations, or DDAs. Up to now, the prokaryotic partners have been morphologically and genetically characterized, and the phylogenetic reconstruction of the well conserved nifH gene (encodes for the nitrogenase enzyme) placed the symbionts in 3 clusters based on their host-specificity, i.e. het-1 (Rhizosolenia-R. intracellularis), het-2 (Hemiaulus-R. intracellularis), and het-3 (Chaetoceros-C- rhizosoleniae). Conversely, the diatom-hosts, major representative of the phytoplankton community and crucial contributors to the carbon (C) biogeochemical cycle, have been understudied.

The first aim of this thesis was to genetically and morphologically characterize the diatom-hosts, and to reconstruct the evolutionary background of the partnerships and the symbiont integration in the host. The molecular-clock analysis reconstruction showed the ancient appearance of the DDAs, and the traits characterizing the ancestors. In addition, diatom-hosts bearing internal symbionts (with more eroded draft genomes) appeared earlier than diatom-hosts with external symbionts. Finally a blast survey highlighted a broader distribution of the DDAs than expected.

The second aim of this thesis was to compare genetic and physiological characteristics of the DDAs symbionts with the other eukaryote-diazotroph symbiosis, i.e. prymnesiophyte-UCYN-A (or Candidatus Atelocyanobacterium thalassa). The genome comparison highlighted more genes for transporters in het-3 (external symbiont) and in the UCYN-A based symbiosis, suggesting that symbiont location might be relevant also for metabolic exchanges and interactions with the host and/or environment. Moreover, a summary of methodological biases that brought to an underestimation of the DDAs is reported.

The third aim of this thesis was to determine the distribution of the DDAs in the South Pacific Ocean using a quantitative polymerase chain reaction (qPCR) approach and to outline the environmental drivers of such distribution. Among the het-groups, het-1 was the most abundant/detected and co-occurred with the other 2 symbiotic strains, all responding similarly to the influence of abiotic factors, such as temperature and salinity (positive and negative correlation, respectively). Globally, Trichodesmium dominated the qPCR detections, followed by UCYN-B. UCYN-A phylotypes (A-1, A-2) were detected without their proposed hosts, for which new oligonucleotides were designed. The latter suggested a facultative symbiosis. Finally, microscopy observations of the het-groups highlighted a discrepancy with the qPCR counts (i.e. the former were several order of magnitudes lower), leading to the idea of developing a new approach to quantify the DDAs.  

The fourth aim of this thesis was to develop highly specific in situ hybridization assays (CARD-FISH) to determine the presence of alternative life-stages and/or free-living partners. The new assays were applied to samples collected in the South China Sea and compared with abundance estimates from qPCR assays for the 3 symbiotic strains. Free-living cells were indeed detected along the transect, mainly at deeper depths. Free-living symbionts had two morphotypes: trichomes and single-cells. The latter were interpreted as temporary life-stages. Consistent co-occurrence of the 3 het-groups was also found in the SCS and application of a SEM model predicted positive interactions between the het groups. We interpreted the positive interaction as absence of intra-specific competition, and consistent with the previous study, temperature and salinity were predicted as major drivers of the DDAs distribution.