1. Oceanographic setting
The anticyclonic eddy spanned approximately 150 km in width and was characterised by a warm water core (Fig. 1). Additionally, the eddy embedded two filaments of colder water in its periphery (Fig. 1a), rotating slowly clockwise as it moved south-westward. The filament stretching from the northwest African coastal upwelling (Fig. 1a) was more intense, hence resulting in a more pronounced front in the east. Consequently, temperatures at the sampling depths were below 19°C in the east, gradually increasing to near 23°C in the west (Fig. 1b). At the sampling depths measured inorganic nitrogen (nitrate -NO3−-, ammonium -NH4+-, total inorganic nitrogen -TIN-) and silicate (SiO44−) showed three times higher concentrations in the eastern front (stations 1–2), and remained constant throughout the core and western front (Fig. S1 However, nitrite -NO2−-and phosphate -PO43−- showed a peak at core stations 6–8, and a decrease on the western front (Fig. S1).
2. Environmental variability across the eddy
A principal component analysis (PCA) including the physicochemical parameters revealed significant environmental variability among the 10 sampling stations (Fig. 2a), establishing a spatial gradient across the eddy (east-core-west), predominantly explained by the first axis (39%). The core stations (3–8) showed strong environmental similarity among them (Fig. 2a), mainly driven by the pycnocline depth, temperature, and the depth of the deep chlorophyll maximum (DCM). The second axis (22%) distinguished the core stations containing water more preserved over time from the frontal systems containing newly integrated waters at the edges. Station 1 (eastern edge) and stations 9–10 station (western edge) exhibited the highest dissimilarity in both the first and second PCA axes. The interplay between these environmental parameters explains distinct habitats for coexisting microbial populations, resulting in biological niche differentiation within the eddy.
Measured particulate organic carbon (POC) and nitrogen (PON) showed variations among particles with different sinking rates, collected as fractions (‘suspended’, ‘slow-sinking’ and ‘fast-sinking’) with a marine snow catcher (MSC; see Methods). Considering the sampling volumes and assuming all particles were initially homogenised throughout fractions, at the time of bottle closure there was 45% of total particulate organic matter in the suspended, 45% in the slow-sinking, and 10% in the fast-sinking fraction. After settling on deck for 5 h, there was 38.5% remaining at the top (-7.5%), 35.2% middle (-9.8%), and 26% bottom (+ 16%), with the highest concentrations observed in the fast-sinking fraction (Fig. 2b). However, the C:N ratio was higher in the suspended fraction as compared to the fast-sinking fraction along the eastern frontal edge of the eddy. Towards the eddy core and the western edge, these ratios became more similar (Fig. 2b). The significantly elevated POC concentrations observed at station 1 are likely attributed to the sampling of a more productive water mass closer to the eastern upwelling. This variation does not align with the measured PON concentrations. At stations 1–2 on the eastern front is where a peak of inorganic nitrogen compounds was measured (Fig. S1), likely favouring communities better adapted to consume fixed inorganic nitrogen under high-carbon conditions [4, 27].
3. Diazotroph community distribution
The diazotroph community composition identified using the nifH marker gene revealed a spatial gradient shift across samples (Fig. 3a; Fig. S2), aligning with physicochemical parameters (Fig. 2a). This supports previous research on eddies, indicating that stirring and trapping can influence microbial communities zonally beyond their vertical pumping effects [28].
Alpha diversity analyses revealed no significant differences among MSC fractions (Kruskal-Wallis, p = 0.082), except for a moderate significance between the slow and fast-sinking fractions (Wilcoxon, p < 0.1, Table S1). The similar diversity between the suspended and slow-sinking fractions compared to the fast-sinking fraction could be related to a particular parameter, such as the differences in particulate organic matter concentrations (Fig. 2b) attracting different communities. The Shannon diversity decreased gradually from east to west (Table S2), aligning with previous observations of unique communities in frontal regions [29]. Station 8 presented exceptionally high mean diversity (Kruskal-Wallis, p < 0.05; Table S3; Fig. S3), despite physicochemical similarities with neighbouring stations (Fig. 2a). This is likely influenced by microscale environmental factors (substrate composition) and biotic features (e.g., grazing, colonisation dynamics) not considered in this study, and maybe attributed to water mixing with the west filament and the downwelling pumping near the core [30]. Similar to Alpha diversity, Beta diversity analyses revealed significant diazotroph community variations across the eddy (Adonis PERMANOVA, ANOSIM, p < 0.05; Table S4), primarily driven by station groups (61.24% contribution; Kruskal-Wallis, p < 0.05), while MSC fractions made a minor contribution (4.49%) with no significant differences between them (Adonis p = 0.06; ANOSIM p = 0.7; Table S4).
Variations in diazotroph community composition among stations may result from different adaptations to the physicochemical heterogeneity, including their energy acquisition strategies, whether they are cyanobacteria or NCDs. While there was a lack of significant differences in the distribution of these two diazotroph groups (Wilcox, p-value > 0.05), trends were observed. Overall, 52% of the total reads were annotated as cyanobacteria (Fig. 3b), dominating the suspended (60% reads; 80% samples) and the slow-sinking fractions (53.9% reads; 70% samples), but not the fast-sinking fraction (41.6% reads; 50% samples) where NCDs dominated (58.4%). This suggests that diazotrophs associated with fast-sinking particles are mostly non-photosynthetic, while cyanobacterial diazotrophs are more prevalent in the suspended or slow-sinking with smaller particles containing less carbon (Fig. 2b). However, this pattern was not consistently observed across individual stations. Stations 1 and 8, were entirely dominated (> 50% reads) by NCDs in all fractions, whereas stations 3, 4, 6, 9, and 10 were dominated by Cyanobacteria (Fig. 3b). The higher abundance of NCDs in waters influenced by the eastern upwelling (expected to be colder and nutrient rich), can be attributed to the elevated organic carbon concentrations measured in those waters. Cyanobacterial diazotrophs, however, dominated in the core and low-carbon western front as they thrive in anticyclonic eddy conditions, hence exported, and found in sinking particles.
Cyanobacteria
UCYN-A was dominant and ubiquitous in all samples, accounting for 88.5% of the cyanobacteria reads averaging 90.8 ± 19.6% per sample (Fig. 4b) (46% of the overall nifH, averaging 48.6 ± 22.9%). This group is commonly found in anticyclonic eddies in the North Pacific with up to 106 nifH gene copies L− 1 [31], but not in cyclonic eddies [16, 17, 32]. An exception occurred in the suspended particle fraction at station 2, where UCYN-A accounted only for 3% of the reads and Crocosphaera dominated instead (94.5% cyanobacterial, 58% overall reads), becoming the second most abundant cyanobacterial group. While Crocosphaera is typically low or unreported in the northeast compared to the southwest Atlantic [33, 34], high abundances have been linked to high N2 fixation rates measured in anticyclonic eddies in the North Pacific [19, 31]. In this study, 99% of the Crocosphaera sequences were found between stations 1 and 3, seemingly forming a bloom at the eastern edge of the eddy. Considering the sporadic occurrence of this group, it is likely that the appearance of Crocosphaera is a result of the physical accumulation at the eastern front between water bodies, rather than being driven by a response to a new nutrient input, such as PO43− [31]. The third most abundant cyanobacterium was Trichodesmium, found in 19 out of the 30 of the samples with up to 38% of cyanobacteria reads in some samples (Fig. 5b). While Trichodesmium usually thrives in warm waters [35], its distribution here was primarily concentrated along the cold-water eastern edge of the eddy (stations 1–3 comprised 78% of Trichodesmium reads), and present in the western edge, but absent in the core (stations 6–8). Various hypotheses are considered to explain accumulations of this cyanobacterium in response to eddy-wind interactions [36, 37], local nutrient inputs, or advection and trapping from areas where communities are more abundant [38]. In anticyclonic eddies in the North Atlantic, high Trichodesmium abundances are commonly observed [39, 40], particularly at the edges with submesoscale upwelling inputs, which could serve as a nutrient source [41]. Similar to Crocosphaera, the prevalence of Trichodesmium was predominantly driven by the physical accumulation at the frontal systems at the outskirts of the eddy where density contrasts exist [37, 42], specifically in this region [43] rather than by temperature.
UCYN-A and Trichodesmium were indistinctively detected in all MSC fractions, reflecting their distinct vertical movement. While Trichodesmium uses gravitational sinking and buoyancy mechanisms [44, 45, 46], UCYN-A, being a symbiotic organism, benefits from host ballasting and associations with calcium carbonate hosts [47], contributing significantly to carbon export [8].
Non-cyanobacterial diazotrophs
NCDs accounted for the remaining 48% of reads, emphasising that in dynamic systems like anticyclonic eddies, these can be as abundant and potentially as active, as cyanobacterial diazotrophs. Their prevalence in eddies has been explained by organic matter accumulating at the core of anticyclonic eddies [18] or alleviation of phosphorus stress in cyclonic eddies [48]. Overall, Gammaproteobacteria (mostly Marinobacter) dominated the NCDs reads (39%), followed by Betaproteobacteria (27%) and Alphaproteobacteria (15%). The wide biogeography and depth range of Gammaproteobacteria has been explained by their capability of associating with different particle sizes [8, 11]. While Alphaproteobacteria and minor NCDs groups were predominant in only 3 and 2 samples out of 30 respectively, Betaproteobacteria (13) and Gammaproteobacteria (12) exhibited similar dominance across samples, indicating significant contributions from both groups to the NCDs community dynamics in the studied environment.
In all three MSC fractions a similar spatial distribution pattern was observed. In the suspended and slow fraction Gammaproteobacteria dominated on the east and Betaproteobacteria on the west, while in the fast fraction Gammaproteobacteria was most abundant in the east. However, Alphaproteobacteria was notoriously present, dominating the NCDs community in both eddy fronts (Fig. 5c). Interestingly, Betaproteobacteria showed a statistically significant but moderate negative relationship with Gammaproteobacteria (R=-0.47, p-value < 0.05, Table S5), establishing community niche differentiations across the eddy substructure. This suggests that while Gammaproteobacteria may be more competitive in particle accumulation frontal zones of environments with colder-water and high iron to phosphorus ratios [49, 50], close to the African coast, Betaproteobacteria may be more competitive in warmer-waters with residual nutrients at surface. Despite the wide diversity of metabolic lifestyles within Gammaproteobacteria, it is likely that their distribution is driven by the availability of nitrate and phosphate dragged from the eastern front, contrary to Betaproteobacteria which lack nitrate transporters [4].
Alphaproteobacteria (mainly Hyphomicrobiales, formerly Rhizobiales) dominated exclusively in the fast-sinking fraction, particularly at the eddy edges (stations 1, 9, 10; Fig. 4c). Although not typically considered a major player in marine ecosystems, Hyphomicrobiales is a metabolically and morphologically heterogeneous group [51]. This group has been documented in studies focused on deep environments, such as hydrothermal vents displaying quorum sensing activities [52], or marine sediments, where it may be involved in organic matter degradation. Genera within Hyphomicrobiales include aerobic chemoheterotrophs and facultatively methylotrophs, whereas some can grow anaerobically by denitrification or fermentation [53]. Additionally, Alphaproteobacteria showed a significant negative correlation with UCYN-A (R=-0.43, p-value < 0.05, Table S5). The slight tendency to decrease in abundance when UCYN-A is more prevalent, could be attributed to a habitat preference of UCYN-A near the surface [54] and Alphaproteobacteria in deep environments as explained above, rather than a resource competition.
Two samples from the eastern stations showed an exceptional diversity in community composition. The suspended fraction on station 2 where Crocosphaera dominated, was the only station not dominated by Proteobacteria, but by a merged group of unknown NCDs. In addition, high relative abundance of Desulfuromonadia (previously Deltaproteobacteria, [55]) was found in the slow-sinking fraction of the carbon-rich station 1 (67% of NCDs reads and 58% overall nifH reads), often seen deeper down the water column or marine sediments [56]). Deltaproteobacteria MAGs have been reported as lacking nitrate, nitrite and urea transporters and have a dissimilatory sulphate reduction metabolism [4], potentially favoured in low-nitrogen environments which differentiates them from Alphaproteobacteria and Gammaproteobacteria.
The biological distinctive station 8 (Fig. 3a), characterised by the highest Alpha diversity (Fig. S2) and NCDs dominance in all fractions (Fig. 3b), was explained by its unique NCDs community. Betaproteobacteria accounted for > 50% of the overall diazotroph reads in the fast-sinking (Fig. 4b) with noticeable peaks of the genera Aquabacterium or Gammaproteobacteria Xanthomonadales.