Large-scale meridional and zonal variability in the nitrogen isotopic composition of plankton in the Atlantic Ocean

1 Fernández, A., Marañón, E., Bode, A., 2014. Large-scale meridional and zonal variability 1 in the nitrogen isotopic composition of plankton in the Atlantic Ocean. J. Plankton Res. 2 (2014) 36(4): 1060-1073 doi:10.1093/plankt/fbu041 3 4 Pre-print version 5 6 7 Large-scale meridional and zonal variability in the nitrogen isotopic composition of plankton in the 8 Atlantic Ocean 9 Ana Fernández a, * , Emilio Marañón a , and Antonio Bode b 10 11 a Dpto Ecoloxía e Bioloxía Animal, Universidade de Vigo, E-36310 Vigo, Spain. 12 b Instituto Español de Oceanografía, Centro Oceanográfico de A Coruña, E-15080 A Coruña, Spain. 13 14 Corresponding author: * Tel: +34 986 814087 Fax: +34 986 812556 e-mail: [email protected] 15 16 E-mail addresses: [email protected] (A. Fernández), [email protected] (A. Bode), and 17 [email protected] (E. Marañón). 18 19 Keywords: nitrogen isotopes, δ 15 N, phytoplankton, zooplankton, diazotrophy, Atlantic Ocean. 20 21 2 ABSTRACT 22 23 The zonal (ca. 15º-40ºW along 26-29ºN) and meridional (ca. 30ºN-30ºS along 28º-29ºW) 24 variability of δ 15 N of suspended particles and zooplankton (>40 µm) was studied to assess the 25 influence of nitrogen fixation in the isotopic budget of the tropical and subtropical Atlantic ocean. 26 Two cruises were conducted in October-November 2007 and April-May 2008 comprising a zonal 27 and meridional transect each. In the region between 30º-15ºN, the concurrently measured nitrogen 28 fixation was insufficient to explain the consistent patch of suspended particles with δ 15 N < 2‰ and 29 points to a significant contribution of atmospheric deposition of light nitrogen to the isotopic 30 budget. The equatorial region (15ºN-10ºS) is subject to intense nitrogen fixation, which, according 31 to a two-end-member mixing model, may explain 40-60% of the observed δ 15 N in suspended 32 particles and 3-30% in zooplankton. In the South region between 10ºS-30ºS, low values (<4‰) 33 were measured in suspended particles and zooplankton during 2008. The values of δ 15 N of 34 suspended particles suggest that nitrogen fixation, which is usually low (<10 µmol N m −2 d −1 ), may 35 represent 50-60% of phytoplankton nitrogen in this region. Hence, diazotrophy in the South 36 Atlantic may be more important than previously thought. 37 38 39 40 3 INTRODUCTION 41 In many marine ecosystems, primary production is limited by the availability of nitrogen (Vitousek 42 and Howarth, 1991; Karl et al., 1992; Moore et al., 2013). Reactive nitrogen is supplied to the 43 euphotic zone by different physical, chemical and biological processes such as advective diffusion, 44 atmospheric deposition and biological nitrogen fixation. The latter is mediated by organisms and, in 45 the oligotrophic regions of the oceans, is a relevant source of new nitrogen (Paerl and Zehr, 2000). 46 The ratio of stable isotopes in phytoplankton ( 15 N: 14 N expressed as δ 15 N in ‰) is variable, due to 47 the contrasting preferences of the organisms for each isotope. The metabolic pathways usually 48 discriminate against the heavy isotope ( 15 N), a discrimination that is measured by the isotopic 49 fractionation factor (Montoya, 2008). Besides, the different forms of inorganic nitrogen have 50 distinct signatures of δ 15 N. Deep-nitrate typically ranges between 3-6‰ (Montoya, 2008), 51 atmospheric dinitrogen is, by definition, 0‰, and deep-ammonium lies between 6-8‰ (Miyake and 52 Wada, 1967). Hence, a very different δ 15 N of organic matter is expected, according to the source of 53 nitrogen, if this is completely consumed. The isotopic signature of phytoplankton will depend then 54 on the signature of the source of nitrogen and the degree of fractionation during uptake. Yet, the 55 interpretation of δ 15 N is not so straightforward. In the case of animals (i.e. upper trophic levels), a 56 trophic effect is also observed whereby the tissues of the consumer are usually 2-4‰ heavier than 57 the food, whereas the animal’s excreta, mainly in the form of ammonium, can be 2-4‰ lighter than 58 the food (Montoya, 2008; and references herein). In addition, cultured cyanobacteria growing on 59 excess nitrate showed a strong fractionation factor, yielding δ 15 N values similar to those produced 60 by growth on dinitrogen (Bauersachs et al., 2009). 61 In the Atlantic Ocean, experimental data retrieved during large-scale surveys show that 62 Trichodesmium, the most well-studied diazotroph, is distributed preferentially between 0-20ºN 63 (Tyrrell et al., 2003; Moore et al, 2009; Fernández et al., 2010; Luo et al., 2012). In addition, 64 nitrogen fixation, mostly measured with the method of Montoya et al. (1996), is more significant 65 between 0º-15ºN (Moore et al., 2009; Fernández et al., 2010; Luo et al., 2012). The δ 15 N of 66 diazotrophs usually ranges between −1‰ and −2‰ (Montoya et al., 2002). However, the measured 67 isotopic signature of nitrogen in suspended particles and the biogeochemical estimates of excess 68 nitrogen available in the literature suggest that nitrogen fixation is more relevant in a region further 69 north, between 15º-30ºN (Gruber and Sarmiento, 1997; Mahaffey et al., 2003; Mahaffey et al., 70 2004; Reynolds et al., 2007; Hansell et al., 2004). The time scales reflected by these measurements 71 are different: in situ nitrogen fixation rates generally represent instantaneous rates over a few hours 72 to 1 day, while δ 15 N and excess nitrogen are indicators of the diazotrophic activity over longer 73 periods of days to months. However, the determinants of this disagreement remain undefined. Duce 74 4 et al. (2008) argued that the atmospheric deposition of reactive nitrogen in the oceans has increased 75 due to human activities and is fast approaching the marine N 2 fixation budget. Other studies have 76 also shown an increase of the atmospheric deposition of 15 N-depleted nitrogen in high and 77 temperate latitudes (Hastings et al., 2009; Mara et al., 2009; Morin et al., 2009; Holtgrieve et al., 78 2011), as a result of the increasing anthropogenic production of reactive nitrogen and/or natural 79 speciation processes. In addition, Baker et al. (2007) and Knapp et al. (2010) reported depositional 80 fluxes of low δ 15 N similar to measured N 2 fixation rates in the Atlantic Ocean. 81 As part of a wider project, we have previously described the latitudinal and longitudinal distribution 82 of measured community nitrogen fixation in the tropical and subtropical Atlantic Ocean (Fernández 83 et al., 2010; Fernández et al., 2013) and the relative contribution of nitrogen fixation and nitrate 84 eddy diffusion in supplying new nitrogen to the euphotic layer (Mouriño-Carballido et al., 2011). 85 Here we report on the distribution of δ 15 N in suspended particles and two size-fractions of plankton, 86 with the aim of describing the large-scale latitudinal variability of nitrogen isotopic signatures in the 87 Atlantic Ocean and comparing these inferred patterns of diazotrophy with concurrent, direct 88 measurements of in situ N 2 fixation rates. 89 90 METHODS 91 Sampling, hydrography and chlorophyll a 92 Two research cruises were conducted in the tropical and subtropical Atlantic Ocean during 17 93 November-8 December 2007 and 13 April-2 May 2008 on board the BIO ‘Hespérides’. The tracks 94 followed by the vessel comprised a zonal and a meridional transect in each season (Fig. 1). 95 The vertical distribution of temperature, salinity, dissolved oxygen and fluorescence was measured 96 by a SBE 911plus CTD attached to a rosette equipped with 12-L Niskin bottles which was fired to 97 300m depth, always before dawn. The vertical profiles of fluorescence and oxygen at each station 98 were used to choose the sampling depths for the determination of inorganic nutrients concentration, 99 chlorophyll a concentration, community 15 N 2 fixation and natural abundance of nitrogen isotopes in 100 suspended particles. 101 The concentration of chlorophyll a was measured at 6-7 depths distributed through the euphotic 102 layer. At each depth a 250-mL sample was filtered, using low vacuum pressure, through 0.2 µm 103 pore-size polycarbonate filters. The pigments were extracted overnight in 90% acetone at -4ºC. 104 Fluorescence was subsequently measured on board with a Turner Designs 700 fluorometer, 105 calibrated with pure chlorophyll a (Fluka). 106 107 5 Rates of N 2 fixation by the whole planktonic community in a 24-hour incubation period were 108 determined in each station at the surface (5m), an intermediate depth (30-80m) and the depth of the 109 deep chlorophyll maximum (DCM), and are already described in Fernández et al. (2010) and 110 Fernández et al. (2013). Briefly, we incubated triplicate samples following the Montoya et al. 111 (1996) protocol for the 15 N 2 -uptake technique with the modifications of Rees et al. (2009). The 112 equations of Weiss (1970) and Montoya et al. (1996) were used to calculate the initial N 2 113 concentration (assuming equilibrium with atmosphere) and N 2 fixation rates, respectively. The limit 114 of detection, estimated following Montoya et al. (1996), was 0.001 µmol N m −3 d −1 . 115 Natural abundance of nitrogen isotopes in suspended particles 116 For the determination of δ 15 N signature in suspended particles (δ 15 N sp ), 2-L samples were taken at 6 117 depths through the euphotic layer in each pre-dawn station and filtered through a 25-mm diameter 118 GF/F filter (Whatman). All filters were dried at 40ºC during 24 h and then stored until pelletization 119 in tin capsules. The measurement of particulate organic nitrogen (PON) and 15 N atom% was carried 120 out with an elemental analyzer combined with a continuous-flow stable isotope mass-spectrometer 121 (FlashEA112 + Deltaplus, ThermoFinnigan) and using an acetanilide standard as reference. The 122 limit of detection of the equipment was 0.20 µg N. 123 The isotopic signature observed in the suspended particles may be affected by the presence of other 124 types of material in addition to phytoplankton (i.e. bacteria, detritus, zooplankton). The existence of 125 a relationship between the particulate organic nitrogen (PON) to chlorophyll a (chl-a) ratio and the 126 δ 15 N of suspended particles is an indicator of such a trophic effect (Waser et al., 2000). The Pearson 127 product-moment correlation coefficient of PON:chl-a and δ 15 N sp was calculated to test this 128 possibility. 129 The weighted mean of δ 15 N of suspended particles in the euphotic layer was used as an integral of 130 the signature of phytoplankton in the euphotic zone to simplify the comparison with the δ 15 N of the 131 two size-fractions of zooplankton (40-200 µm and >200 µm). It was calculated, following Landrum 132 et al. (2011), as: 133 Weightedmeanδ N   =∑PN  ×∆z  ×δ N     ∑PN  ×∆z    Where [PN] i is the concentration (µM) of particulate nitrogen, δ 15 N sp is the nitrogen isotopic 134 composition of suspended particles ( 15 N: 14 N, ‰), and ∆z i is the depth interval (m). 135 The fraction contribution of diazotroph nitrogen to the bulk suspended particles defined by 136 Montoya et al. (2002) was also calculated as: 137 6 % !"#$%&$'ℎ)=100×, δ N   −δ NO / 0  δ N 12345647  −δ NO / 0  8 Where δ 15 N diazotroph is the nitrogen isotopic composition of diazotrophs ( 15 N: 14 N, ‰) and δ 15 NO 3‒ is 138 the nitrogen isotopic composition of deep-nitrate ( 15 N: 14 N, ‰). As pointed by these authors, this 139 two-end-member mixing model is sensitive to the values of the end members chosen (δ 15 N diazotroph 140 and δ 15 NO 3‒ ). In order to represent only the nitrate in the upper thermocline, and avoid the effect of 141 recently fixed nitrogen recycled between the upper water column and the thermocline in the 142 calculations, the δ 15 NO 3− used was 4.5‰, which is the global average of deep-nitrate (Liu and 143 Kaplan, 1989; Sigman et al., 1997). Due to the fact that most of our stations are oligotrophic, no 144 additional fractionation factor during nitrate uptake was added. As a conservative choice 145 representing the least contribution of nitrogen fixers, and considering the fact that little fractionation 146 occurs during N 2 fixation (Montoya, 2007), the δ 15 N diazotroph used was −2‰ (Montoya et al., 2002). 147 148 Natural abundance of nitrogen isotopes in plankton 149 At each pre-dawn station, zooplankton were collected by vertical tows of a 40µm net of 30cm in 150 diameter through the upper 200m of the water column at a constant towing speed of 60 m min −1 . 151 The content of the collector was suspended in 500 mL of 20 µm-filtered seawater. Two 60 mL sub-152 samples were preserved, one in Lugol's solution and the other in formaldehyde, for the 153 determination of abundance of Trichodesmium and other plankton by microscopical examination. 154 Trichodesmium trichomes were more abundant in the fraction 40-200µm while colonies were 155 present in the >200µm fraction. The rest of the sample was separated into two size fractions by 156 passage through nylon sieves of 40 and 200µm. Each fraction was then re-suspended in 200 mL of 157 20µm-filtered seawater and subsequently filtered in pre-weighted 45-mm diameter GF/F filters by 158 low vacuum pressure. All filters were dried for 24 h at 40ºC and stored until measurement of 159 particulate organic nitrogen and 15 N atom% as previously described. 160 The relative contribution of diazotroph N to zooplankton biomass was estimated following Montoya 161 et al. (2002) two-end-member mixing model for zooplankton: 162 % !"#$%&$'ℎ)=100×,δ N :2;<54;  −δ N 6=>=6=;?=:  δ N 12345647  −δ N 6=>=6=;?=:  8 Where δ 15 N plankton stands for the nitrogen isotopic composition of the plankton size-fraction 163 ( 15 N: 14 N, ‰), and δ 15 N reference pl is the δ 15 N of reference zooplankton. Again, a conservative value of 164 ‒2‰ was used for δ 15 N diazotroph . The δ 15 N of the reference plankton was calculated as the mean of 165 the δ 15 N 40 or δ 15 N 200 measured in the stations where the lowest abundance of Trichodesmium and 166 7 nitrogen fixation were found, i.e., the stations between 0-20ºS in the latitudinal leg of 2007 cruise, 167 where no influence of nitrogen fixation in the samples is expected. The values used were: 4.6‰ for 168 the δ 15 N reference 40 , and 5.9‰ for δ 15 N reference 200. This model is based on the use of reference 169 plankton to account for the trophic effect, i.e., the reference plankton serves as a proxy in both 170 terms of the calculation; therefore, no additional fractionation term for the trophic effect was 171 needed. The assumptions that are implied are: i) the size distribution of grazers in the sample and 172 the reference plankton are similar, ii) the trophic fractionation in the sample and the reference is 173 similar, and iii) in both locations the isotopic composition of the nitrate supporting the food web is 174 the same (J. P. Montoya, Atlanta, personal communication). 175 176 RESULTS 177 Hydrography and fluorescence 178 In the latitudinal transects, the Equatorial upwelling was clearly defined by the rising of the isolines 179 of temperature (T) and salinity (S) in both cruises (Fig. 2a, b, c, d). In order to simplify the analysis 180 of data, we use the changes in the depth of the 16ºC isotherm, above and below 150m, to identify 181 the area affected by the Equatorial upwelling and delimit three main regions along the transects, 182 i.e., North gyre (29º-15ºN), equatorial region (15ºN-10ºS) and South gyre (10º-33ºS). 183 The hydrographic settings found in these regions were similar in both legs. Surface waters in the 184 equatorial region were always warmer (> 24ºC) and less saline (< 35 psu) than in the gyres in both 185 seasons. In turn, the stability of the water column in the gyres was weaker than that found in the 186 equatorial region, where the average Brunt-Väisäla frequency in the upper 125m was higher 187 (Fernández et al., 2010). The fluorescence profiles showed a well-defined deep chlorophyll 188 maximum (DCM) associated with the thermocline in both transects (Fig. 2e, f). This DCM was 189 shallower and better defined in the equatorial region than in the gyres. By contrast, in the 190 longitudinal sections, waters were warmer and slightly more saline in autumn 2007 than in spring 191 2008 (Fig. 2e), leading to a stronger stability of the water column, as indicated by the higher Brunt-192 Väisäla frequency measured in this cruise (Fernández et al., 2013). The DCM was located at ca. 193 100m in both zonal legs and no apparent trend in depth was observed (Fig. 2e, f). 194 195 Stable nitrogen isotopes in suspended particles (δ 15 N sp ) and particulate organic nitrogen (PON) 196 The Pearson product-moment correlation coefficient of PON:chl-a and δ 15 N sp showed no significant 197 relationship, neither positive nor negative, in our two cruises (Fig. 3). The PON:chl-a ratio 198 represents the contribution of other components of the food web than phytoplankton. If any 199 8 relationship between PON:chl-a ratio and δ 15 N sp is found, this will suggest a significant effect of 200 detrital matter and/or other non-phytoplanktonic organisms on the observed signal of suspended 201 particles. Thus, we can assume that the δ 15 N of suspended particles in our data mainly reflects the 202 composition of phytoplankton (Waser et al., 2000). 203 The zonal distribution of PON showed no apparent trend and the measured concentrations were 204 similar in magnitude in both cruises (Fig. 4b, d), without any significant differences between cruises 205 (t-test, n=60). The mean PON concentrations for the zonal transects were 0.22±0.12 µM in 2007 206 and 0.24±0.06 µM in 2008. In the latitudinal sections, the measured concentrations in the South 207 gyre were similar in both cruises, showing similar values and vertical variability (Fig. 4a, c). In the 208 North gyre and equatorial regions, measured PON differed significantly between cruises (t-test, p < 209 0.01, n=54 and p < 0.05, n=84, respectively). In the North gyre region, the concentrations of PON 210 in 2007 cruise were higher than that of 2008 cruise. In the equatorial region, PON depicted a 211 decreasing pattern in 2007 cruise, but no linear trend was observed in 2008. 212 In the zonal transect of 2007 cruise, the δ 15 N sp increased sharply by 4-6‰ from 80-100m to the base 213 of the euphotic layer, probably reflecting the influence of the African upwelling in the easternmost 214 stations (Fig. 5a). However, the δ 15 N sp distributed uniformly in the euphotic layer in 2008 cruise 215 (Fig. 5b). In the latitudinal transects, the δ 15 N sp was lower in 2007 than in 2008, both in the North 216 gyre (Fig 5c, d) and equatorial region (Fig 5e, f). By contrast, in the South gyre region, the vertical 217 distribution of δ 15 N sp differed between stations in each cruises (Fig 5g, h), with a wide range of 218 values between −4 and 4‰. The difference between cruises were significant in the zonal transect, 219 the North gyre and the equatorial region (t-test, p < 0.01, n=72), but not in the South gyre region. 220 The correlations of δ 15 N of suspended particles with ammonium concentration and with nitrate 221 concentration are shown in Table 1. Considering all the stations in each cruise, δ 15 N sp correlated 222 with ammonium concentration in 2007 (p < 0.05, n=128) and with nitrate in 2008 (p < 0.05, 223 n=119). 224 225 Nitrogen isotopic signature in the euphotic layer 226 In order to compare the δ 15 N of suspended particles and plankton net tows, we calculated the 227 weighted mean of δ 15 N of suspended particles to obtain an integrative δ 15 N sp signature for the whole 228 euphotic layer. The patterns described by the δ 15 N of the planktonic 40-200 µm (δ 15 N 40 ) and >200 229 µm (δ 15 N 200 ) size-fractions were very similar in all stations, with a few exceptions in the equatorial 230 region and the zonal legs, and closely matched that of δ 15 N sp (Fig. 6). The average differences 231 between δ 15 N sp and the two plankton size fractions were in the range previously described 232 9 (Minagawa and Wada, 1984): between the δ 15 N sp and the δ 15 N 40 that difference was 3.2‰ in 2007 233 and 2.6‰ in 2008; between the δ 15 N sp and the δ 15 N 200 was 4.3‰ in 2007 and 1.9‰ in 2008. 234 In the autumn 2007 meridional transect, the isotopic signature of suspended particles showed two 235 minima (< ‒2‰) in the North gyre and South gyre regions. In the equatorial region, δ 15 N sp 236 oscillated around 0‰ (Fig. 6). The δ 15 N 40 and δ 15 N 200 roughly followed these patterns. By contrast, 237 the distributions were dome-shaped in spring 2008, reaching peak values in the equatorial region. In 238 both cruises, the gyres presented low δ 15 N values in most of the stations. Besides, a positive 239 statistical correlation between δ 15 N in the three fractions suggests a regular impact of light nitrogen 240 across trophic levels (Table 2). 241 A two-way factorial ANOVA indicated significant differences between regions and cruises, and for 242 δ 15 N 40 and δ 15 N 200 , a significant interaction region-cruise, which enhances the difference (Table 3). 243 The differences between regions appeared to be significant only for the North gyre-equatorial 244 region (post-hoc Tukey HSD test), as can be also seen in figure 6. 245 We tried to estimate if Trichodesmium could be the major influence on the patterns observed but no 246 significant correlation (Pearson’s r) was found between the measured filament abundance 247 (Fernández et al., 2010; Fernández et al., 2013) and the δ 15 N of suspended particles (p=n.s., n=42), 248 the 40-200 µm (p=n.s., n=41) or the >200 µm plankton size-fractions (p=n.s., n=42). 249 Nitrogen fixation rates (Fig. 6) were previously reported in Fernández et al. (2010) and Fernández 250 et al. (2013). Briefly, in the longitudinal transects no apparent trend was depicted in 2007, while a 251 clear increasing pattern to the East appeared in 2008 (Fig. 6b, d). In the spring 2008 zonal leg, the 252 average vertically integrated N 2 fixation was 7-fold higher than that of autumn 2007 (8.3±3.3 µmol 253 N m −2 d −1 vs. 1.2±0.5 µmol N m −2 d −1 ). In both meridional transects, the highest integrated rates (ca. 254 250 and 150 µmol N m −2 d −1 in 2007 and 2008, respectively) were measured at stations located 255 within the equatorial region (Fig. 6a, c). Besides, the North gyre showed higher diazotrophic 256 activities than the South gyre. But, while N 2 fixation south of the Equator was almost undetectable 257 during the 2007 cruise, substantial rates were measured in the Southern Hemisphere in 2008 (Fig. 258 6a, c). 259 260 Diazotroph nitrogen contribution to δ 15 N in the euphotic layer 261 The contribution of diazotrophs to the observed δ 15 N of suspended particles, 40-200µm and >200 262 µm plankton size-fractions, estimated by the two-end-member mixing models, decreased to the 263 South in 2007 cruise (Table 4). In 2008, the minimum was observed in the equatorial region (Table 264 4). The importance of this contribution is higher in 2007, except in the South gyre, where the 265 16 have addressed the depositional and diazotrophic fluxes together (Baker et al., 2007; Knapp et al., 469 2011) and further studies are needed to accurately define the strength, frequency and isotopic 470 composition of the atmospheric depositional flux against the flux of nitrogen fixation in the North 471 Atlantic. The equatorial region (15ºN-10ºS) is subject to relatively intense nitrogen fixation 472 throughout the year (Moore et al., 2009; Fernández et al., 2010; Grosskopf et al., 2012) which may 473 explain 40 to 60% of the observed δ 15 N sp signal. However, this nitrogen of diazotrophic origin 474 seems to be inefficiently transferred to upper trophic levels. In the South gyre, the low δ 15 N sp and 475 the daily estimated contribution of nitrogen fixation to the supply of new nitrogen (Mouriño-476 Carballido et al., 2011) suggest that diazotrophs can contribute up to half of the nitrogen in 477 phytoplankton at different time scales (Fig. 4). Even though the measured nitrogen fixation rates are 478 low (Moore et al., 2009; Fernández et al., 2010; Grosskopf et al., 2012), their impact in the nitrogen 479 isotopic budget of this region may be large. Hence, a re-evaluation of the importance of diazotrophy 480 in the South Atlantic Ocean is needed through new studies that should address the annual variability 481 in nitrogen fixation rates as well as the distribution and relative importance of the different groups 482 of diazotrophs. 483 484 ACKNOWLEDGEMENTS 485 486 The authors appreciate the careful revision and thourough comments of two anonymous reviewers 487 who helped to improve the quality of the manuscript. We thank N. Lluch and P. Chouciño for 488 technical assistance. Stable isotopes were analyzed at SXAI-Universidade da Coruña. We also 489 thank the officers and crew of the BIO Hespérides and the staff of the Marine Technology Unit 490 (UTM), for their support during the work at sea. A. F. was supported by grant 491 PGIDIT05PXIC31201PN of the Xunta de Galicia. This is a contribution of the project 492 TRYNITROP (Trichodesmium and N 2 fixation in the tropical Atlantic Ocean) funded by the 493 Spanish Ministry of Science and Technology through grants CTM2004-05174-C01 and CTM2004-494 05174-C02 to A. B. and E. M., respectively. 495 496 497 17 REFERENCES 498 Baker, A. R., Weston, K., Kelly, S. D et al. (2007) Dry and wet deposition of nutrients from the 499 tropical Atlantic atmosphere: Links to primary productivity and nitrogen fixation. Deep-Sea 500 Research Part I-Oceanographic Research Papers, 54, 1704-1720. 501 Bauersachs, T., Schouten, S., Compaore, J. et al. (2009) Nitrogen isotopic fractionation associated 502 with growth on dinitrogen gas and nitrate by cyanobacteria. Limnology and Oceanography, 503 54, 1403-1411. 504 Checkley, D. M. and Miller, C. A. (1989) Nitrogen isotope fractionation by oceanic zooplankton. 505 Deep-Sea Research Part a-Oceanographic Research Papers, 36, 1449-1456. 506 Duce, R. A., Laroche, J., Altieri, K. et al. 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(2000) Geographic variations in the nitrogen 625 isotope composition of surface particulate nitrogen and new production across the North 626 Atlantic Ocean. Deep-Sea Research Part I-Oceanographic Research Papers, 47, 1207-627 1226. 628 629 630 20 Table and figure legends 631 Table 1. Pearson product-moment correlation coefficient between δ 15 N of suspended particles 632 (δ 15 N sp ) and nutrient concentration: ammonium (NH 4 ) and nitrate (NO 3 ), in 2007 and 2008 cruises. 633 Numbers in brackets represent the total number of samples used for the analysis. 634 Table 2. Pearson product-moment correlation coefficient between δ 15 N of suspended particles 635 (δ 15 N sp ), δ 15 N of 40-200µm plankton size-fraction (δ 15 N 40 ), and δ 15 N of >200µm plankton size-636 fraction (δ 15 N 200 ) in the latitudinal transects of 2007 and 2008 cruises. ** p< 0.01, n=17. 637 Table 3. Two-way factorial ANOVA (region, cruise) of δ 15 N of suspended particles (δ 15 N sp ), δ 15 N 638 of 40-200µm plankton size-fraction (δ 15 N 40 ), and δ 15 N of >200µm plankton size-fraction (δ 15 N 200 ) 639 in the 2007 and 2008 cruises. DF, degrees of freedom; SS, sums of squares; MS, mean of squares; 640 F, F statistic; p, probability. 641 Table 4. Mean±Standard deviation of the contribution of diazotroph nitrogen to δ 15 N of suspended 642 particles, 40-200µm plankton size-fraction (δ 15 N 40 ) and >200µm plankton size-fraction (δ 15 N 200 ) 643 according to the two-end-member mixing model proposed by Montoya et al. (2002). The reference 644 zooplankton used in each fraction corresponded to the average of the stations sampled in the South 645 gyre during 2007, where Trichodesmium abundance was < 1 trichome L −1 , δ 15 N 40 = 4.6‰, δ 15 N 200 = 646 5.9‰. The values of % of diazotroph N above 100 and below 0 were discarded in the calculation of 647 the regions average. Number of samples is indicated in parentheses. 648 Figure 1. Sampling stations during the TRYNITROP cruises on board the BIO ‘Hespérides’. White 649 circles represent the autumn 2007 cruise (17 November - 8 December 2007), and grey triangles the 650 spring 2008 cruise (13 April - 2 May 2008). 651 Figure 2. Zonal and meridional vertical distribution of temperature (ºC), salinity and fluorescence in 652 autumn 2007 and spring 2008 cruises. Dashed lines in the temperature panels define the limits of 653 the three major regions identified by the depth of 16ºC isotherm: North gyre, equatorial region and 654 South gyre. 655 Figure 3. Relationship between the δ 15 N of suspended particles (δ 15 N sp ) and the particulate organic 656 nitrogen (PON) to chlorophyll a (chl-a) ratio during the autumn 2007 (a) and the spring 2008 (b) 657 cruises. 658 Figure 4. Zonal and meridional distributions of particulate organic nitrogen of suspended particles 659 (PON) during the autumn 2007 (a, b) and spring 2008 (c, d) cruises. Dashed lines define the limits 660 of the three major regions identified by the depth of 16ºC isotherm in the meridional transect: North 661 gyre, equatorial region and South gyre. In the legend z1 to z6 represent the sampled depths from 662 deeper depth, z1 (DCM), to shallower depth, z6 (5m). 663 21 Figure 5. Vertical distribution of δ 15 N of suspended particles (δ 15 N sp ) in autumn 2007 and spring 664 2008 cruises grouped by region: a, b) zonal transect (15º-38º W), c, d) North gyre region (30º-665 15ºN), e, f) equatorial region (15ºN-10ºS), and g, h) South gyre region (10º-30ºS). 666 Figure 6. Zonal and meridional distributions of the weighted mean of δ 15 N of suspended particles 667 (δ 15 N sp ), the δ 15 N of 40-200µm plankton size-fraction (δ 15 N 40 ), the δ 15 N of >200µm plankton size-668 fraction (δ 15 N 200 ), and concurrent measured N 2 fixation (Fernández et al., 2010; Fernández et al., 669 2013) in autumn 2007 (a, b) and spring 2008 (c, d).Dashed lines define the limits of the three major 670 regions identified by the depth of 16ºC isotherm in the meridional transect: North gyre, equatorial 671 region and South gyre. 672 673 22 Table 1. 674 2007 cruise 2008 cruise All stations North gyre a Equatorial region South gyre All stations North gyre a Equatorial region South gyre NH 4 0.22* [128] 0.55* [54] 0.43** [38] n.s. n.s. n.s. 0.47** [42] −0.38* [36] NO 3 n.s. 0.30* [54] n.s. n.s. 0.21* [120] n.s. n.s. 0.51** [36] ** p < 0.01, * p < 0.05, n.s. no significance 675 a includes the zonal and meridional legs in the North gyre. 676 677 23 Table 2. 678 δ 15 N sp δ 15 N 40 2007 2008 2007 2008 δ 15 N 40 0.66 ** 0.82 ** − − δ 15 N 200 0.66 ** 0.74 ** 0.90 ** 0.78 ** 679 24 Table 3. 680 681 δ 15 N sp δ 15 N 40 δ 15 N 200 DF SS MS F p SS MS F p SS MS F p region 2 23.6 11.8 6.3 0.004 26.6 13.3 11.5 0.000 25.7 12.8 15.4 0.000 cruise 1 16.0 16.0 8.6 0.006 4.8 4.8 4.2 0.048 15.9 15.9 19.2 0.000 region vs.cruise 2 6.0 3.0 1.6 0.213 20.9 10.5 9.0 0.000 21.7 10.9 13.1 0.000 682 25 Table 4. 683 684 Region Suspended particles 40-200µm zooplankton >200µm zooplankton 2007 2008 2007 2008 2007 2008 Longitudinal transect 81 (29) 59 (3) 52 (14) 16 (9) 43 (4) 31 (6) North gyre region 85 (14) 61 (10) 48 (22) 21 (11) 41 (12) 36 (8) Equatorial region 62 (27) 39 (8) 25 (18) 3 (2) 11 (3) 29 (12) South gyre region 49 (12) 58 (18) 15 (20) 36 (16) 13 (15) 40 (17) 685 686