Biological n2 fixation in the upwelling region off nw iberian peninsulamagnitude, relevance and players

Resumo

The classical paradigm about marine N2 fixation establishes that this process is mainly constrained to nitrogen-poor tropical and (sub)tropical regions, and sustained by the colonial cyanobacterium Trichodesmium spp. and diatom-diazotroph associations. Therefore, knowledge of the diversity, activity and ecology of N2-fixing (diazotrophic) plankton is mainly limited to these oligotrophic regions. However, the application of molecular techniques allowed determining a high phylogenic diversity and wide distribution of marine diazotrophs, which extends the range of ocean environments where biological N2 fixation may be relevant. Between February 2014 and December 2015, we carried out 10 one-day samplings in the upwelling system off NW Iberia in order to: 1) describe the seasonal variability in the magnitude of N2 fixation, and to compare its biogeochemical role as a mechanism of new nitrogen supply versus nitrate turbulent diffusion, and 2) investigate the variability, in connection with the hydrodynamic forcing, of diazotroph abundance and community composition. Furthermore, considering the contamination of some commercial 15N2 gas stocks with 15N–labeled nitrate and ammonium used for nitrogen fixation measurements, we carried out laboratory and field experiments in productive waters of the temperate northwestern Iberian upwelling system to 3) test the susceptibility of 15N-contaminants to assimilation by non-diazotroph organisms, and to determine the potential overestimation of N2 fixation rates in the field. Our results indicate that the magnitude of N2 fixation in this region was relatively low (0.001±0.002 – 0.095±0.024 µmol N m-3 d-1), comparable to the lower-end of rates described for the subtropical NE Atlantic. Maximum rates were observed at the surface during both upwelling and relaxation conditions. The comparison with nitrate diffusive fluxes revealed the minor role of N2 fixation (<2%) as a mechanism of new nitrogen supply into the euphotic layer. Small diazotrophs (<10 µm) were responsible for all N2 fixation activity detected in the region. In downwelling conditions, characterized by deeper mixed layers and a homogeneous water column, non-cyanobacterial diazotrophs belonging mainly to nifH clusters 1G (Gammaproteobacteria) and 3 (putative anaerobes) dominated the community. In contrast, in upwelling and relaxation conditions, affected by enhanced vertical stratification and hydrographic variability, the diazotrophic community was more heterogeneous and less diverse, with prevalence of Candidatus Atelocyanobacterium thalassa or UCYN-A (unicellular cyanobacterial diazotroph of subcluster 1B) and non-cyanobacterial diazotrophs from cluster 1G and 3. Based on oligotyping analysis of UCYN-A phylotype, UCYN-A2 sublineage was the most abundant (74%), followed by UCYN-A1 (23%) and UCYN-A4 (2%). These sublineages were mainly represented by the oligotypes oligo3 (67%), oligo1 (18%) and oligo 4 (2%), respectively. UCYN-A1 oligotypes were detected at relatively low frequencies during the three hydrographic conditions, whereas UCYN-A2 exhibited higher abundances during upwelling and relaxation. Quantitative PCR targeting the nifH gene revealed the highest abundances of UCYN-A1 and UCYN-A2 mainly at well-lit surface waters under upwelling and relaxation conditions, and of Gammaproteobacteria γ-24774A11 at deep waters during downwelling. Maximum abundance for the three groups were up to 6.7 × 10^2, 1.5 × 10^3 and 2.4 × 10^4 nifH copies L-1, respectively. Our findings demonstrate measurable N2 fixation activity and presence of a diverse and temporally variable diazotroph community throughout the year in a nitrogen-rich temperate region, and outline the environmental conditions that define the ecological niches of UCYN-A and Gammaproteobacteria.