In depth characterization of diazotroph activity across the Western Tropical South Pacific hot spot of N2 fixation

: Here we report quantification of N2 fixation rates over a ~ 4000 km transect in the western and central tropical South Pacific. Water 160° W, covering contrasting trophic regimes, from oligotrophy in the Melanesian archipelagoes (MA) in the WTSP. In the present study, we report previously undocumented bulk and group-specific N 2 fixation quantification 18 over a ~4000 km transect in the western and central tropical South Pacific. The goals of the study were i) to quantify 19 to horizontal and vertical distribution of N 2 fixation rates in the photic layer in relation with hydrological and biological 20 parameters, ii) to quantify the relative contribution of the dominant diazotrophs ( Trichodesmium and UCYN-B) to N 2 21 fixation at selected stations based on cell-specific measurements, iii) to assess the potential ecological impact of N 2 22 fixation in this region. 0.557 This suggests to fix at high rates and may 20 contribute to revise upward the current N 2 fixation estimates for the Pacific Ocean. Further studies would be required 21 along the annual time scale to assess the seasonal variability of N 2 fixation in this region and perform accurate N 22 budgets. Nonetheless, such high N 2 fixation rates question whether or not these high N inputs can balance the N losses 23 in the ETSP. A recent study based on the N* (the excess of N relative to P) at the South Pacific scale (Fumenia et al., 24 Submitted) reveals a strong positive N* anomaly (indicative of N 2 fixation) in the surface and thermocline waters of 25 the WTSP, which potentially influences the geochemical signature of the thermocline waters further east in the South 26 Pacific through the regional circulation. However, the WTSP is chronically undersampled, and a better description of 27 the mesoscale and general circulation would be necessary to assess how N sources and sinks are coupled at the South 28 Pacific scale.

In the ocean, nitrogen (N) availability in surface waters controls primary production and export of organic matter

17
In the present study, we report previously undocumented bulk and group-specific N2 fixation quantification

27
The west to east zonal transect along ~19°S started in Noumea (New Caledonia) and ended in Papeete (French

6
The sampling and analytical methods used to analyze the parameters reported in the correlation table (Table   7 2) are described in details in related papers in this issue (

38
Briefly, 12 mL were subsampled after incubation into Exetainers ® fixed with HgCl2 (final concentration 20 mg L -1 ) 1 that were preserved upside down in the dark at 4°C until analyzed using a membrane inlet mass spectrometer (MIMS) 2 according to (Kana et al., 1994). Lastly, we collected time zero samples at each station to determine the natural N 3 isotopic signature of ambient particulate nitrogen (PN).

4
Discrete N2 fixation rate measurements were depth integrated over the photic layer using trapezoidal 5 integration procedures assuming that surface N2 fixation rates were identical to those in subsurface (5 m) and 6 considering that rates below the deepest sampled depth were zero (JGOFS, 1988

5
Four other diazotrophic phylotypes were quantified using quantitative PCR (qPCR) as they were too scarce  14 Filters were flash frozen in liquid nitrogen and stored at -80 C until RNA extraction. The RNA extraction and Reverse

15
Transcription were performed as previously described using a Super-Script III first-strand cDNA synthesis kit

30
Trichodesmium filament portions were analyzed to take into account the variability of activity among the population.

31
Data were processed using the LIMAGE software. Briefly, all scans were corrected for any drift of the beam 32 and sample stage during acquisition. Isotope ratio images were created by adding the secondary ion counts for each

18
For nutrients, DFe and Chl a concentrations, the transect was divided into two main characteristic sub-regions:  (Table 1). Those numbers were used as time zero samples to calculate N2 fixation rates.
N2 fixation was detected at all 17 sampled stations and the transect could also be divided into the two main 1 characteristic sub-areas: 1) the MA waters exhibiting average N2 fixation rates of 8.9 ± 10 nmol N L -1 d -1 (range: DL-    (Table 3).

5
The in situ nifH expression for all diazotroph groups targeted by qPCR was estimated using a TaqMAN   6 quantitative reverse transcription PCR (RT-QPCR) ( Table 4). The sampling and filtering time (17-21:00 h) was not 7 optimal for quantifying the nifH gene expression for all diazotrophs, however it is useful evaluation for which

22
The second method was developed because the first had been observed to potentially underestimate N2 fixation rates

8
In the NO3 --depleted MA waters, the DIP concentrations close to the detection limit are indicative of the 9 consumption of DIP by diazotrophs. This is consistent with the negative correlation found between N2 fixation and 10 DIP turn-over time (TDIP, the ratio between DIP concentrations and DIP uptake rates) during the OUTPACE cruise 11 (

5
Our hypothesis to explain the spatial distribution of N2 fixation in this region is the following: when DIP-rich   27 not significantly correlated with N2 fixation rates (Table 2).

28
The relative contribution of different diazotroph phylotypes to bulk N2 fixation has been largely investigated 29 through bulk and size fractionation measurements (usually comparing > and <10 µm size fraction N2 fixation rates),

26
Pacific through the regional circulation. However, the WTSP is chronically undersampled, and a better description of 27 the mesoscale and general circulation would be necessary to assess how N sources and sinks are coupled at the South

11
Picheral are warmly thanked for their efficient help in CTD rosette management and data processing, as well as C.

12
Schmechtig for the LEFE-CyBER database management. Aurelia Lozingot is acknowledged for the administrative 13 work. All data and metadata are available at the following web address: http://www.obs-                                Table 4. Summary of average nifH gene expression data determined by qRT-PCR at selected stations (SD2, SD6, 1 LDB), where the cell-specific N2 fixation rates were measured.