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.