The study area is characterized by a seasonal cycle with high values of solar irradiance and upwelling index, and low rainfall and river flow during spring and summer (March to September, Fig. 2). In 2011, there was a relatively long rainfall period from mid October–December in addition to the episodic rains recorded in previous winter and spring (January–May). Several upwelling events occurred along the year but more persistently during spring and summer, while downwelling prevailed in winter and also in autumn. The river flow showed high values during the winter–spring period but was between 0.5 and 3 m3 s−1 for most of the summer and autumn (Fig. 2c). River flow was uncorrelated with rainfall when data from the same calendar day were compared but showed a positive correlation at lags of up to 7 days, with a maximum correlation value with the rainfall recorded 5 days earlier (r = 0.212, N = 365, P < 0.05). This latter correlation, along with the low flow measured during spring and summer points out to the major role of the reservoir in regulating the freshwater flow to the estuary, while attending the demands for urban freshwater and flood control during periods of heavy rain. When accumulated in periods of 15 days, values of upwelling index were also negatively correlated with rainfall and positively with irradiance (Table 1S in the Supplement). Either precipitation, irradiance or upwelling index were significantly correlated when accumulated in periods of 7, 15 or 30 days (values not shown); therefore, values accumulated for 15-day periods prior to each sampling date were used in subsequent analysis.
Variability in physical and chemical properties of water
The thermal cycle of progressive warming of surface water during spring and summer, and cooling during autumn and winter was more pronounced in the river and reservoir compared to the bay and estuary (Fig. 3a). The seasonal variability in salinity was reduced when compared with the large spatial variability, with a marked saline front delineating the influence of the saline waters in the estuary near the location of St. 5 (Fig. 3b). The range of salinity values observed was 3.0, 28.3 and 0.0 at stations 1 (bay), 5 (estuary) and 10 (reservoir), respectively. These values are considerably smaller than the range of salinity observed across the 15 km separating the bay station from the reservoir at all sampling times (salinity range >35). Similarly, all water variables mainly showed spatial gradients, while temporal (i.e., seasonal) variability was comparatively smaller (Table 2S in the Supplement). For instance, nitrate had higher concentrations in freshwater than in marine water (Fig. 3c), and phosphate displayed maximal values in the estuary (Fig. 3d). However, relative increases in nitrate concentrations in the bay and decreases in the estuary and river waters during summer must be noticed. Phosphate concentrations were more variable near the saline front in the estuary. Silicate and ammonium concentrations (not shown) displayed similar variability to either nitrate or phosphate, respectively, as indicated by their correlations (Table 2S in the Supplement).
Maximum values of particulate organic matter concentrations were found in the estuary in spring, in the river and reservoir in late summer and in autumn in the bay (Fig. 3e). In general, POC was significantly correlated with chlorophyll a (Table 1S in the Supplement), which always showed the highest values in the reservoir (Fig. 3f). However, concentrations exceeding 5 µg L−1 indicated blooms during spring and late summer in the estuary and in the bay. In turn, dissolved organic matter was always higher in freshwater, with concentrations increasing during spring and summer, reaching maximum values during autumn (Fig. 3g, h).
A total of 130 phytoplankton taxa were identified (118 at least at genus level), including 63 diatoms (Bacillariophyceae), 32 dinoflagellates (Dinophyceae), 25 Chlorophyceae and other groups with less than 10 taxa each (Table 1, 3S). Considering the entire annual cycle, the number of taxa decreased progressively from the bay (St. 1) to the reservoir (St. 10) while the Shannon index reached minimum values in the estuary (St. 5), which also showed the highest abundance (Table 1). There was an even distribution of abundance among taxa in the bay, moderately even in the reservoir but highly uneven in the estuary, as indicated by the values of equitability. The differences in Shannon index were significant for all pairs of stations (Hutcheson t test, t St. 1–5 = 470.73, t St. 5–10 = −1810.8, t St. 1–10 = 386.93, P < 0.001 in all cases). Unique taxa (i.e., those found only at one of the sampling stations) accounted for more than half of all taxa recorded in the bay and the reservoir, but only 35% of those found in the estuary (Table 1).
Most of the variations in abundance were due to cyanobacteria (Cyanophyceae), almost permanent in the reservoir but also present in the estuary and even reaching the bay (Fig. 4a). Cyanophyceae, mainly Chroococcus spp. (Table 3S in the Supplement), reached maximum abundance in February and decreased during spring and summer. The second group in abundance was composed by small (2–8 µm) flagellate monads which increased in abundance from February to December in all zones (Fig. 2b). This group was not employed in further analysis because it was not possible to separate the autotrophic and heterotrophic organisms with the counting technique employed. Apart from these groups, the phytoplankton communities were dominated by Bacillariophyceae (Fig. 4c), Dinophyceae (Fig. 4d) and Cryptophyceae (Fig. 4e) in all zones and Chlorophyceae in the stations under the influence of freshwater (Fig. 4f). The dominant taxa were the diatoms Chaetoceros socialis, C. affinis and Pseudo-nitzschia pungens at St. 1 (spring), and the cyanobacteria Chroococcus spp. (spring) and the Euglenoidea Eutreptia sp. (October) at St. 5. In the reservoir, there were also dominance peaks of Chroococcus spp. in spring that were later replaced by other cyanobacteria, Anabaena spiralis (August), the freshwater diatom Fragilaria crotonensis (June and July) and the Synurophyceae Synura uvella (September) at St. 10 (Table 2S in the Supplement).
The number of taxa and Shannon index values increased in general from spring to late summer at all stations, with the highest values almost always in the bay and the lowest in the reservoir (Fig. 5a, b).There were taxa present in several zones (Fig. 5c); only a few taxa were shared between the estuary and the reservoir at any single sampling time, while the bay and the estuary showed an increase from spring to autumn in the number of taxa present in both zones. Four of the taxa shared were high-level taxa, not resolved at the species level, as Cryptophyceae or unidentified dinoflagellates and diatoms, but in all cases, there were characteristic species that were found in several stations (Table 3S). For instance, F. crotonensis was identified not only in the reservoir and estuary but also in the bay. The highest number of shared species occurred when comparing the bay and the estuary stations. Conversely, β-diversity showed high values for the assemblages of St. 5 and St. 10 through the year and also for those of St. 1 and St. 5 in spring (Fig. 5d) when the number of shared species was relatively low (Fig. 5c). In the later, the decrease of β-diversity in summer and autumn was accompanied by a sharp increase in the number of shared taxa. However, this correspondence was lower in the case of the assemblages of end-member stations (St. 1 and St. 10) showing relatively low β-diversity values and low numbers of shared taxa. Considering the entire sampling period, 8 taxa occurred in all zones and the estuary shared 24 taxa with the bay and 19 with the reservoir, while the corresponding values of β-diversity were 0.61 (all zones combined), 0.64 (St. 1 vs. St. 5) and 0.62 (St. 5 and St. 10).
The taxonomic composition defined the characteristics of each sampling zone throughout the year, as shown both by the MDS (Fig. 6) and cluster analysis (Fig. 1S in the Supplement). As observed for environmental variables, the composition of phytoplankton communities varied mainly with the spatial component (i.e., station) while the temporal variability was comparatively smaller (Table 2S in the Supplement). The samples from the bay (St. 1) were at all times clearly separated from the other stations (Fig. 6) and their similarity was mainly due to medium-sized Cryptophyceae, the diatom Nitzschia longissima and small dinoflagellates (Table 2). Samples from the estuary (St. 5) and the reservoir (St. 10) were also separated but in this case, there were more similarities in the composition of the communities between stations in some periods of the year. For instance, half of the samples from the reservoir clustered with either summer and autumn samples or winter and spring samples of the estuary at the 20% similarity level (Fig. 6). The main contributors to the similarity of St. 5 were Cryptophyceae, small diatoms, N. longissima, and F. crotonensis, which was also the main contributor to the similarity of St. 10 along with the Chlorophyceae Ankistrodesmus falcatus, Cryptophyceae and small dinoflagellates (Table 2). It must be noted that the contribution of most taxa to similarity within stations was small (<1%), while the main contributors were none of those identified above as bloom producers.
Environmental effects on the phytoplankton communities
Salinity, nitrate, and dissolved organic carbon concentration were the main variables correlated with the composition of phytoplankton communities when all zones were considered (Table 3). In the bay, where variations in salinity were relatively low, the main variables contributing to the correlation between environmental and taxonomic data were the concentration of phosphate and dissolved organic matter. In the estuary, salinity, phosphate, and concentrations of humic acids and tryptophan-like substances contributed to a relatively high correlation, compared to that found in other stations. In turn, nitrate, phosphate and organic matter components were the main environmental variables correlated with phytoplankton composition in the reservoir.
In general, the meteorological variables showed a low correlation with phytoplankton taxa, but there was asymmetric covariation of zonal diversity indices with the meteorology (Fig. 7; Table 4S; Fig. 2S in the Supplement). Rainfall and river flow were related with both the number of shared taxa and β-diversity by a saturation-type function, but the pattern was different for the combination of the bay and estuarine stations or the estuarine and the reservoir stations. In the former case, there was a rapid decrease of the number of shared taxa (and conversely an increase in β-diversity) with the increase in river flow (Fig. 7a, b). Apparently, the effects on zonal diversity depend on a critical value of the flow (ca. 20 hm3 per 15 days). River flow values larger than this critical value had little effect on zonal diversity while there were large changes at lower flow values. For instance, when the flow was lower than the critical value there were more taxa shared between the bay and the estuary than during periods of high flow (Mann–Whitney test, p < 0.05, n = 10). Conversely, there was a slight increase in the number of shared taxa (and a decrease in β-diversity) between the estuary and the reservoir when the river flow exceeded the critical value. Similar patterns could be applied to the accumulated rainfall, but with lower confidence than for the river flow (Fig. 7c, d; Table 4S). In contrast, upwelling did not show the described saturating response and its covariation with zonal diversity indices was less clear (Fig. 7e, f; Table 4S). The only significant effect of upwelling was a linear and negative effect on the number of taxa shared between the estuary and the reservoir (Fig. 7e).
The combination of river flow and upwelling conditions thus affected the number of phytoplankton taxa found in nearby zones (PERMANOVA+ test, Table 5S in the Supplement). As summarized in Fig. 8, the number of taxa shared between the bay and the estuary peaked in periods of upwelling and low river flow (<20 hm3 in 15 days) when also the number of estuarine taxa reached a maximum. In turn, the maximum number of taxa shared between the estuary and the reservoir was found in downwelling conditions and during periods of high river flow (≥20 hm3 in 15 days). For all combinations of upwelling and river flow, the number of taxa found only in the estuary was higher than the number of taxa shared with the other zones.