Results
Hydrography
Over the 3-year period, hydrographic sampling revealed the expected seasonal cycles of cold season mixing, warm season stratification, the short spring transition, and the vertical distribution of chlorophyll fluorescence (Figure 1). The vertical layer boundaries fluctuated in depth over the observational period, highlighting the ability of this physical framework to transcend the temporal variations in vertical water mass structure associated with winter mixing, mesoscale eddies, location of the nutricline, and varying light penetration (Figure 1, Supp. Figure S1). The depth of the surface mixed layer (Layer 0) varied seasonally from 10 m in the stratified season to 170-212 meters in the mixed. The depth of the DCM (Deep Chlorophyll Maximum; Layer 2) ranged from 70 to 130 meters. The top of Layer 8, corresponding to the deep O2 minimum, varied between 600 and 850 m. Other hydrographic features detected were the uplifts in the deep mesopelagic layers due to the passage large eddies (Figure 1). The most prominent uplifts (of about 200 m) occurred during 2018.
Molecular data
Of the 408 samples (34 sampling months, 12 depths), 369 produced a library. The remaining samples failed at some point of the procedure (sampling at sea or extraction), and did not produce a useable library. It usually affected several samples from the same cast, especially the deeper layers (where DNA concentration was always lower). About ~39 million reads were generated (average ~105,000 reads per sample; 23,000 to 364,000). Of those, the strict QC retained 42% as very high quality (16.5M reads; 44,000 per sample average). There was no link between reads passing QC and months or depths.
Vertical structure of communities
Non-hierarchical clustering based on Bray-Curtis distances showed two local R maxima, at K =2 (ANOSIM R =0.903; p<0.001) and K =9 (R =0.904; p<0.001). In both cases, all pairwise comparisons were significant. The K =2 partitioning roughly divided the epipelagic (photic) and the mesopelagic zones, although when the MLD (Mixed Layer Depth) reached into in the mesopelagic, the upper community followed the MLD depth (Figure 2). TheK =9 clustering aligned closely with the hydrographic layering, with higher numbers of clusters in the epipelagic, and greater homogeneity in mesopelagic. In some cases, single communities spanned several mesopelagic layers. The clusters precisely traced hydrographic events such as the deepening of the MLD in winter, the abrupt succession to a different community following the shoaling of this layer at the spring transition, and the uplift of mesopelagic layers due to the passage of mesoscale eddies.
The epipelagic zone was characterized by a succession of different communities over the annual cycle. Cluster 1 dominated Layer 0 (surface MLD) in the spring and stratified seasons, but was displaced by Cluster 2 during the fall and mixed seasons. Cluster 1 was detected as soon as the MLD shoaled in each year, and remained detectable until the onset of Fall when the MLD (Layer 0) gradually deepened and Cluster 2 became dominant. Cluster 3, found below 1 and 2, corresponded to the lower portion of the epipelagic zone that included the DCM (Figure 2). Clusters 4, 5 and 6 occupied the underlying water mass (upper mesopelagic), corresponding to the local deep winter mixed layer (the Winter Mode Water, WMW; Layers 3 & 4), a very weakly stratified portion of the water column occupying the approximate depth range 150-400m, that did not strictly align with density. Clusters 7, 8 and 9, in the lower mesopelagic (500 – 1000m), aligned with the density fields. Cluster 8 overlapped with Layer 8, the oxygen minimum zone (OMZ, with O2 < 160 µmol kg-1). The mesopelagic layers were uplifted during the passage of the fronts in 2018, detected by the raised density layers (Figures 2). Clusters were more tightly grouped with depth (PERMDISP test for homogeneity of multivariate dispersions; p < 0.0001), indicating a more variable community in the epipelagic compared to the mesopelagic, and in the upper mesopelagic compared to the deep mesopelagic.
The PCoA ordination arranged the samples into an arch, with the first axis roughly corresponding to depth (Figure 3). The BioEnv procedure determined that the best model combined O2, fluorescence, depth and density (ρ=0.729 p < 0.001), but since depth and density are highly correlated, the next best model included O2, fluorescence and density (ρ=0.690, p < 0.001). The proposed hydrographic layering was the best-correlated single environmental variable (ρ=0.651, p < 0.001). Superimposing the non-hierarchical clustering onto the PCoA, theK =2 showed a sharp divide between the clusters, despite the intrusions of the upper cluster into deeper layers. For K =9, separation of clusters 1 & 2 reflected the seasonal shifts in the upper epipelagic layers. The remaining clusters projected consecutively by depth/density in the PCoA, with very little mixing at the boundaries. The PCoA also indicated a larger separation between cluster 7 (occupying depths 500 – 600 m) and the other communities. In contrast, clusters 8 (OMZ) and 9 were very close to each other, with minimal mixing.
The taxonomic composition showed 40-45% of the reads corresponding to Syndiniales, a parasitic group (Figure 4); the proportion originated from free dispersal states versus the parasitic state is unknown. AtK =2, the taxonomic composition showed a shift from a more diverse community in the epipelagic, with many different clades including autotrophs, heterotrophs and mixotrophs, to a heterotrophic, Rhizaria-dominated, community at depth (Figure 5). This result was more pronounced if the Syndiniales were not considered in the analyses. SIMPER analyses indicated that the epipelagic cluster was characterized by a large presence of autotrophs (mostly PelagophyceaePelagomonas calceolata ) and mixotrophs (from Ochrophyta, Stramenopiles and Dinophyta) although some of the latter clades include all trophic possibilities. In contrast, heterotrophs overwhelmingly dominated the deep cluster, principally Radiolarians (Polycystinea and Acantharea), and representatives of Stramenopiles (Labyrinthulea). The dissimilarity between groups was then driven by the mixture of autotrophs (especially P. calceolata and the ChlorophytaOstreococcus sp.) and heterotrophs/mixotrophs (Stramenopiles MAST-4A, and several Dinophyceae lineages) in the epipelagic, compared to their virtual absence in the mesopelagic, where depth-specific heterotrophic Radiolaria dominated.
The taxonomic composition at K =9 reflected a more detailed partitioning (Figure 4). Alveolata dominated the near-surface community during the spring and stratified seasons even if excluding Syndiniales (cluster 1), together with a mixture of Hacrobia and Stramenopiles. In contrast, the near-surface taxa during the mixed periods (cluster 2) exhibited greater abundances of Stramenopiles and Rhizaria. Cluster 3, below clusters 1 & 2, contained the highest concentrations of Archaeplastida, although Rhizaria was the dominant free-living group. A gradual decrease of all the non-Rhizaria groups was associated with increasing depth: Hacrobia and Stramenopiles disappeared almost completely by cluster 7, with only Alveolata maintaining a significant presence. Syndiniales abundances slightly decreased with depth, but had a greater presence in Cluster 7 (500-600 m). SIMPER analyses complimented these findings, indicating that taxa showed a clear affinity with cluster. There was no single ASV/OTU with a significant presence in all clusters, and most were significant only in one or two clusters. A few Rhizaria, however, showed high numbers in several mesopelagic layers (Figure 5; Table 1). Cluster 1 (surface stratified) was characterized by mixotrophs and heterotrophs of Alveolata Dinophyta (e.g., Warnowia sp., Karlodinium sp. orLepidodinium spp.), Hacrobia Prymnesiophyceae (Chrysochromulina sp.) and several Stramenopiles. The only autotroph was a clade of Phaeocystis sp., although this group is known to appear as free living or as a symbiont autotroph in Rhizaria colonies. Cluster 2 (near-surface, mixed periods) shows large abundances of P. calceolata (autotroph), but most of the other clades belong to mixotrophs (e.g. several Dinophyceae, and MOCH-2), or heterotrophs (e.g., MAST lineages 25, 4A, 4C and 1D, Hacrobia Pterocystida). Cluster 3 (DCM) showed several mixotrophs among the dominant lineages, but the main groups were autotrophs (P. calceolata ,Ostreococcus sp., Bathycoccus prasinos , and the HaptophytaPhaeocystis globosa ). Heterotrophs characteristic from this clade included the Hacrobia Leucocryptos marina and several Rhizaria (although these might have autotrophic symbionts such asPhaeocytis ). Clade 5, below the Chlorophyll maximum, showed significant abundances of the autotroph P. calceolata , however the shift towards a heterotrophic Rhizaria-dominated community was noticeable, together with heterotrophic (Telonemia sp.,Leucocryptos marina ) and likely mixotrophic (Prymnesiophyceae Clade E ) Hacrobia. Different Rhizaria dominated all remaining clusters, while some heterotrophic Hacrobia and Stramenopiles were still among the dominant clades in clusters 5 and 6.
The dissimilarity between consecutive clusters reflected transitions in community function. The mixotroph-dominated cluster one (surface stratified) was replaced by the autotroph-dominated community occupying the ML during the periods of deep mixing (cluster 2). In cluster 3 there was a larger increase of the autotrophs at the cost of mixotrophs from cluster 2. Deeper, differences were due to the progressive decrease in autotrophs, increase of heterotrophs, and layer to layer replacements between different heterotrophs (mostly Rhizaria).
Seasonality
When considering all samples together, there was no discernible seasonal signal. Seasonality, however, appeared when analyzing single depths/clusters. Seasonality was evident in the top layers (surface and 40 m especially; ANOSIM R =0.66 and R =0.62 respectively; p<0.001), but the signal faded with depth, with ANOSIMR progressively decaying, and becoming non-significant below 300m. There was no significant seasonality in the mesopelagic. In the lower mesopelagic the passage of eddies was, however, detectable as a group of dissimilar samples (Figure 6).
Near the surface, the highly diverse community was composed of many taxa with low relative abundances (mostly < 2% average), dominated by mixotrophs and heterotroph clades. Only winter and spring had an autotroph (P. calceolata ) among the most abundant clades (only representing ~ 2% of the reads), while a single heterotroph identified as Warnowia sp. represented 8% of reads in spring. By summer, mixotrophs, and a diverse community of heterotrophs, including Telonemia , Warnowia , several Hacrobia Centroheliozoa and the Rhizaria Minorisa minutadominated the community. In the fall, mixotrophs gained more prominence compared to heterotrophs.
Cluster 3 was generally associated with the Layer 2 (broadly defined DCM) but, since the DCM varied substantially, sometimes this layer did not capture the feature. Cluster 3 community exhibited depth dependence, but no apparent seasonal cycle. When DCM samples were restricted to those acquired within 90% of the actual chlorophyll maximum, a strong and significant seasonal pattern was detected (ANOSIM R = 0.0762, p < 0.001; Figure 6). The stratified and fall periods, despite mixing in the 2PCos representation, were statistically different (R = 0.5, p < 0.05) and were separated in the 3-PCoA (Figure S2). SIMPER analyses on this narrow Chl-a max showed that the main difference is the spring season, during which a few autotrophic clades dominate the community (Ostreococcus sp., P. calceolata and B. prasinos ). Three different clades ofMicromonas , and P. globosa , were also among the most abundant clades. Together, autotrophs represented over 30% of the reads of the non-parasitic community. Other main groups included Dinophyceae (likely mixotrophs) and heterotrophs such as Telonemia sp. and Radiolaria (although these might have autotrophic endosymbionts). After transitioning to summer, only P. calceolata , B. prasinosand P. globosa showed relatively high abundances in the DCM; however, their prevalence was much lower (just above 5%). In contrast, mixotrophs and heterotrophs increased in relative abundance. In the fall transition, only P. calceolata and P. globosa were the autotrophs among the main taxa (with slightly higher relative abundances compared to summer), while the proportion of heterotrophs (Rhizaria andTelonemia ) increased. During winter, there was again a strong shift to autotrophs (especially P. calceolata , B. prasinusand Ostreococcus. sp.; about 10% of the total reads combined) and mixotrophs, with no free heterotrophs among the main lineages in the community.
Diversity
Diversity indices indicated a mismatch between species-based and phylogenetic-based diversity indices (Figure 7). The data showed a higher number of species (S), species diversity (H’) and evenness (J’) in the upper layers of the water column, gradually decreasing with depth, reaching a minimum in the cluster corresponding to the oxygen minimum zone. These profiles also revealed an interesting feature: superimposed upon the general trend of decreasing diversity and evenness with increasing depth throughout the upper 1000m, the indices identified three subgroups (clusters 1-3, 4-6 and 7-9) each of which exhibited its own decreasing trend. The subgroups correspond respectively to the epipelagic, upper mesopelagic, and lower mesopelagic portions of the water column. The profile for Faith’s phylogenetic diversity was distinctly different from these: it showed rising diversity from clusters 1 to 4, followed by a steady decrease with depth to a minimum in the OMZ.