Phytoplankton dynamics and bloom formation in the oligotrophic Eastern Mediterranean: Field studies in the Aegean, Levantine and Ionian seas

Journal Pre-proof Phytoplankton dynamics and bloom formation in the oligotrophic Eastern Mediterranean: Field studies in the Aegean, Levantine and Ionian seas I. Varkitzi, S. Psarra, G. Assimakopoulou, A. Pavlidou, E. Krasakopoulou, D. Velaoras, E. Papathanassiou, K. Pagou PII:

S0967-0645(18)30310-2

DOI:

https://doi.org/10.1016/j.dsr2.2019.104662

Reference:

DSRII 104662

To appear in:

Deep-Sea Research Part II

Received Date: 6 January 2019 Revised Date:

3 October 2019

Accepted Date: 6 October 2019

Please cite this article as: Varkitzi, I., Psarra, S., Assimakopoulou, G., Pavlidou, A., Krasakopoulou, E., Velaoras, D., Papathanassiou, E., Pagou, K., Phytoplankton dynamics and bloom formation in the oligotrophic Eastern Mediterranean: Field studies in the Aegean, Levantine and Ionian seas, Deep-Sea Research Part II (2019), doi: https://doi.org/10.1016/j.dsr2.2019.104662. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Phytoplankton dynamics and bloom formation in the oligotrophic Eastern Mediterranean: field studies in the Aegean, Levantine and Ionian seas.

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Varkitzi I. *1, Psarra S. 2, Assimakopoulou G. 1, Pavlidou A. 1, Krasakopoulou E. 1, 3, Velaoras D. 1, Papathanassiou E. 1 and Pagou K. 1

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* Contact author: Dr. Ioanna Varkitzi, [email protected]

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1: Hellenic Centre for Marine Research (HCMR), Institute of Oceanography, 19013 Anavyssos, Greece. 2: Hellenic Centre for Marine Research (HCMR), Institute of Oceanography, 71003 Crete, Greece. 3: present address University of the Aegean, School of the Environment, Department of Marine Sciences, 81132 Mytilene, Greece.

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KEYWORDS: chlorophyll-a, primary productivity; picoplankton; microplankton; diatoms; species abundance; biodiversity.

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ABSTRACT

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This study attempts to elucidate the dynamics of phytoplankton biomass (as Chl-a), productivity, community structure and bloom formation in different regions of the oligotrophic Eastern Mediterranean (EMed). With this scope, several chemical and phytoplankton parameters were studied along a transect of 1300 km across different pelagic environments from North (N) Aegean to South (S) Aegean, Levantine and Ionian seas during spring and summer 2008. An exceptional spring phytoplankton bloom was detected in the S Aegean Sea, dominated by large sized diatoms and spreading almost throughout the euphotic zone. This unprecedented bloom was triggered by the nutrient enrichment of surface water masses due to cyclonic formations and a strong convection event that caused deep mixing. There was a clear decreasing trend of phytoplankton biomass and productivity from the Aegean towards the Levantine and Ionian seas. The same trend was recorded for N:P ratios and increasing P limitation from the Aegean to the Levantine and Ionian seas. Si:N ratio indicated an overall deficiency of N in relation to the available Si. Picoplankton was the dominant and most productive size fraction, except for the S Aegean in spring due to the diatom bloom. Chl-a maxima in the N Aegean were constrained within the surface layer occupied by modified Black Sea water, deepening progressively towards the Levantine and Ionian seas, especially during the stratified period. Nanoflagellates and small-

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sized dinoflagellates were important phytoplankton components in the N Aegean interseasonally, whereas in the S Aegean the large diatoms associated with the spring blooming shifted to nanoflagellates and small dinoflagellates in the stratified period. Coccolithophores and small diatoms were significant components in the Levantine and Ionian seas, whereas the overall microplankton community structure did not demonstrate prominent seasonal patterns. These results confirm that oligotrophy is the main driving force for the distribution of phytoplankters, but also offer evidence of how the low nutrient-low chlorophyll EMed system can occasionally support high phytoplankton biomass and generate prominent blooms. This work will help elucidate the structure and functioning of EMed pelagic food webs and contribute to the analysis of signals related to structural and functional ecosystem changes.

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INTRODUCTION

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The complex morphology and topography of the Mediterranean are mainly responsible for the prevailing complex hydrography. It has narrow continental shelves with an average depth of ~ 1600 m and several areas exceeding 4000 m depth (Bergamasco and Malanotte-Rizzoli, 2010; Coll et al., 2010). It is also characterized by high water transparency, short ventilation rates and relatively warm deep-water masses with long residence times (>100 years). The Mediterranean pelagic realm is thus a highly variable four-dimensional structure, reflected in the structure and dynamics of plankton communities, with considerable diversity and spatial variability in the open epipelagic waters (Ignatiades et al., 2009; Siokou-Frangou et al., 2010; Würtz, 2010; Mazzocchi et al., 2014).

63 64 65 66 67 68 69 70 71

The Mediterranean is also one of the most oligotrophic seas of the world ocean, mainly due to very low concentrations of phosphates (Krom et al., 1991; Thingstad et al., 2005a). It is therefore defined as a Low Nutrient - Low-Chlorophyll system (LNLC) (Durrieu de Madron et al., 2011). This oligotrophy presents a well documented increasing gradient from west to east (Turley et al., 2000; D’Ortenzio and Ribera d’Alcala, 2009). A decrease of the integrated heterotrophic bacterial production, primary production and particulate carbon export has been reported as a function of nutrient availability towards the eastern basin (Van Wambeke et al., 2002; Psarra et al., 2000; 2005; Santinelli et al., 2012, Gogou et al., 2014).

72 73 74 75 76 77 78

The decreasing gradient of subsurface dissolved inorganic N and P concentrations from west to east influences greatly the photosynthetic biomass, chlorophyll-a (Chl-a) concentrations and primary productivity (reviewed in Krom et al., 2010). Phytoplankton dynamics in the eastern basin resemble that in subtropical waters, with low Chl-a variations throughout the year (Lavigne et al., 2015; Barbieux et al., 2018). The Aegean, Levantine and Ionian basins have been classified as “non-blooming” regions with a bimodal dynamic, showing low biomass during late

79 80 81 82 83 84 85 86 87 88 89 90

spring-summer and higher biomass up to the maxima in late fall-winter (D’Ortenzio and Ribera d’Alcala, 2009). The concentration span between minima and maxima increases by a factor of less than or around 2. Within these regions of the EMed, some “intermittently bloom” zones have been identified, which combine oligotrophic conditions with periods of intense biomass accumulation, strongly coupled with physical and chemical forcing, i.e. the north-eastern Aegean frontal area, the Rhodes Gyre and the North Ionian Gyre. The lowest Chl-a and primary production levels and the highest transparency are found in the Levantine basin of the EMed especially during the thermally stratified summer period (Gotsis-Skretas et al., 1999; Bosc et al., 2004; Ediger et al., 2005; Ignatiades et al., 2009). Despite this oligotrophy, phytoplankton blooms are known to occur frequently in open waters of the Western Mediterranean (Barale et al., 2008), whereas our knowledge on bloom incidents of the ultra-oligotrophic EMed waters is limited.

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This study aims to examine the spatiotemporal, vertical and seasonal variations of phytoplankton biomass (as Chl-a), productivity, community structure and bloom formation in the Aegean, Levantine and Ionian seas of the EMed region, along a transect of 1300 km. Within this scope, the distribution of total and size-fractionated Chl-a, as an index of phytoplankton biomass, total and size-fractionated in-situ primary production rates, as an index of phytoplankton productivity, the taxonomic composition of phytoplankton communities, with species identification and abundances (as cells counts), and bloom dynamics were studied in combination with physical and chemical forcing. This work will contribute to fill gaps regarding biochemical data in the Aegean, Levantine and Ionian seas, which to our knowledge are limited in these EMed basins over the last decades.

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MATERIALS AND METHODS

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Study area

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The field work was performed during two multidisciplinary cruises on board R/V Aegaeo during March–April and August–September 2008, representing spring and summer conditions respectively. A subset of 25 stations was sampled for the study of phytoplankton dynamics in the Aegean (A1, A2, A3, A5, A6, A8, A9, A10, A11, A12), Levantine (L1, L2, L3, L4) and Ionian seas (I1, I2, I3, I4, I5, I6, I7, I8, I9, I10, I11) covering a transect of 1300 km (Fig. 1). All seawater samples for the analyses of phytoplankton parameters were collected from discrete depths of 2, 10, 20, 50, 75, 100, 120, 150 m, according to the methodology of Ignatiades et al. (2009), with routine hydrocasts, using 10 L Niskin oceanographic bottles assembled on a CTD rosette sampler, with a Sea-Bird Electronics 11plus CTD deck unit, interfaced with a Sea-Bird Electronics

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9 plus underwater unit and a General Oceanics rosette with twenty four 10 L Niskin bottles, as described in detail by Karageorgis et al. (2012).

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Inorganic nutrients

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The seawater samples for the determination of nutrients were filtered through 0.45 μm membrane Millipore filters and collected in polyethylene bottles pretreated with 10% HCl. From each depth, seawater samples were collected in triplicate and kept deep-frozen (-20 °C) until analysis of nitrate, nitrite and silicate in the laboratory according to standard methods (Mullin and Rilley, 1955, for silicate and Strickland and Parsons, 1977, for nitrite+nitrate). Phosphate analysis was performed on board using the method of Murphy and Riley (1962) for phosphate concentrations higher than 200 nmol L-1, whereas the nanomolar method of Rimmelin and Mutin (2005) was used for phosphate concentrations bellow 200 nmol L-1. The limit of quantification (LOQ) for the methods used was 0.126 μmol L-1 for nitrate+nitrite; 0.117 μmol L-1 for silicate; 0.010 μmol L-1 for the classic phosphate analysis and 1.0 nmol L-1 for the nanomolar phosphate analysis.

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Particulate organic matter

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For particulate organic carbon (POC) and particulate nitrogen (PN) determination, seawater samples (4–5 L for each parameter depending on the suspended matter concentration) were filtered through Whatman GF/F filters (nominal pore size 0.7 μm; diam. 25 mm) precombusted at 450 oC. Filters were stored in Petri dishes and kept continuously frozen at -20 oC in the dark until further laboratory analysis. POC and PN were measured using a Thermo Scientific Flash 2000 Elemental Analyzer according to the methodology proposed by Verardo et al. (1990) and Cutter and Radford-Knoery (1991). POC and PN values were corrected on the basis of blank filter measurements; filter blanks were precombusted filters taken on the cruise, but no filtered seawater was passed through them.

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Total and size-fractionated Chl-a

147 148 149 150 151 152 153 154 155

Twenty five stations were sampled for total Chl-a concentrations, as mentioned above, whereas eighteen of them (in the Aegean A1, A2, A3, A5, A9, A10, A12, Levantine L2, L3 and Ionian seas I1, I3, I5, I6, I7, I8, I9, I10, I11) were sampled for size fractionated Chl-a (>0.2 μm, 0.2-2.0 μm and 2.0-5.0 μm fractions) . The Chl-a size fraction range 0.2–2.0 μm is referred to as picoplankton cells, the fraction 2.0–5.0 μm as ultra-phytoplankton cells, the fraction >5.0 μm as nano + microplankton and >0.2 μm as total Chl-a. Seawater samples of 2L were collected from standard sampling depths. Seawater was size-fractionated with separate filtration through polycarbonate membrane filters with 0.2 μm, 2.0 μm and 5.0 μm porosities. The filters were kept deep frozen

156 157

in the dark till the analysis at the laboratory with a TURNER 00-AU-10 or a TURNER TD-700 fluorometer (Holm-Hansen et al. 1965).

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Total and size-fractionated primary production

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Four stations were sampled (A1 in N Aegean, A10 in S Aegean, L2 in Levantine and Ι5 in Ionian sea) for total and size fractionated primary productivity with in-situ experiments (same fractions as for Chl-a). Primary production rates were measured with the in-situ 14C fixation rates method of Steeman-Nielsen (1952). Seawater samples were dispensed in 250 ml transparent polycarbonate bottles (three light and one dark as control for each depth) and each one was inoculated with 5 μCi of NaH14CO3 (Perkin Elmer, 1 mCi mL-1). Immediately after 14C inoculation, the samples were incubated for 2 hours in-situ during midday at the same standard depths as for Chl-a. Fractionation was carried out by filtration, as describe above for Chl-a. Filters were placed in scintillation vials, acidified with 0.5 N HCl and stored in a cool dry place until analyses in the laboratory. The samples were analyzed with a liquid scintillation counter (BECKMAN LS6500).

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Quantitative and qualitative analysis of phytoplankton community structure

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A total of 120 seawater samples were collected from 12 stations in the Aegean (A1, A2, A3, A5, A9, A10), Levantine (L2, L3) and Ionian seas (I1, I5, I8, I11). Seawater samples (150 mL) were collected from the same standard depths as for Chl-a and primary production rates for the determination of phytoplankton species and cell abundance. The samples were fixed with Lugol iodine solution immediately after sampling. The identification of phytoplankton species and the enumeration of phytoplankton cells (> 5 μm) were performed in 25 mL sedimentation chambers with an inverted light microscope (OLYMPUS IX70), according to the standard sedimentation method of Utermöhl (1958). The magnification used was 200-400X, providing good resolution for this cell size range.

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Processing of data and images from Copernicus repository

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In order to examine the distribution of nitrates and phosphates in combination with the dynamic topography and Chl-a over an extended period of time, i.e. before, during and after our sampling cruise, we processed data and images from the E.U. Copernicus Marine Service Information repository (http://marine.copernicus.eu/services-portfolio/access-to-products/). In this view, we processed nitrate and phosphate distributions from model derived values, with monthly means at 10, 50, 75 and 100 m depths for February, March and April 2008 (see Fig. 2a-d and supplementary material). Dynamic topography daily data were processed, with mean values over five-day periods. Ocean color satellite derived data were processed for surface Chl-a

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distributions, with monthly means for Feb, Mar and Apr 2008, and mean values over eight-day periods.

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Integrations and statistics

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For comparison among stations and seasons, the integration of the parameters was calculated with depth discrete values through the 0-150 m water column, by using the trapezoid rule (Hornbeck 1975). The integrated values (per surface area, m−2) were then divided by the integration depth, providing depth-averaged values (per volumetric unit, L−1) (Karageorgis et al., 2005; Lagaria et al., 2017; Varkitzi et al., 2018). The Statgraphics Plus software was used for ANOVA and regression analyses. The PRIMER software was used for the computation of Shannon Η’ diversity index and Bray-Curtis similarity index, using the group average method and log+1 transformation of data. Non-parametrical analyses, such as hierarchical agglomerative clustering (Cluster analysis), multidimensional scaling analysis (MDS) and SIMPER analysis were performed on the basis of Bray-Curtis similarity matrix, according to Clarke and Warwick (1994) and Clarke and Gorley (2006).

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RESULTS

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1. Overview of physical and chemical forcing

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During the spring and summer cruises, the temperature and salinity were within average ranges (Karageorgis et al. 2012). The overall concentration of inorganic nutrients (as nitrates+nitrites, phosphates and silicates) was low within the euphotic zone (Table 1). The thickness of the nutrient depleted layer was about 100-150 m deep in the South (S) Aegean and >150 m in Levantine and Ionian seas, whereas in North (N) Aegean it was about 75 m. Below this depleted nutrient layer, concentrations rose through the nutriclines. The top of the phosphacline was located approximately at the same depth as the nitracline or deeper. Overall, the nutrient concentrations were decreasing from the N Aegean to the S Aegean. In spring, the N Aegean had the highest phosphate and silicate values, while the S Aegean revealed the lowest nitrates and silicates. The Levantine showed the highest nitrates but the lowest phosphate concentrations. In summer, phosphates were similar or lower in all areas except for the Ionian. On the contrary, nitrates and silicates increased or remained the same in all areas, except for the Levantine, where nitrates decreased.

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N:P ratios decreased significantly at the top of the nutricline and reached higher values than the theoretical Redfield ratio of 16:1, indicating the well known P limiting conditions that Eastern Mediterranean (EMed) experiences (Table 1). In spring, the lowest N:P and Si:N values were

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observed in S Aegean, possibly related to nitrate consumption by phytoplankton, whereas N:P values ranged at similar levels in the N and S Aegean in summer. Overall, P limitation increased gradually from the N Aegean to the Levantine and Ionian seas. Therefore, the Levantine and Ionian seas were found to be the most P limited basins. The N:P ratio in Ionian was far higher than 16:1 in summer, which was related to the ultra-low phosphate concentrations (at the detection limit of the method), indicating high variation in N:P ratios and the overall lowest levels of N and P in summer. The wide ranges and the high values of N:P ratios were also related to the different nitracline and phosphacline depths (phosphacline was deeper than nitracline). The Si:N ratio indicated a deficiency of N in relation to the available Si, which was more prominent in summer.

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In order to get an insight into whether there were higher levels of inorganic N and P available prior to the spring oceanographic cruise, which could have triggered and supported the high phytoplankton abundances that were recorded in spring, we examined the nitrate and phosphate distributions from the Copernicus repository (see Materials and Methods for details) over an extended period of time before and after the spring sampling (see Fig. 2b, c. See also supplementary material for more detailed images). There was a clear signal of elevated nitrates in the water column down to 100 m depth in February and March, especially in the S Aegean and partially in the Levantine, whereas these nutrient levels appeared to decrease in April within the surface 75 m (Fig. 2 and 3 in suppl. material). Regarding the phosphate levels, they were higher in February and lower in March throughout the Aegean, the Levantine and part of the Ionian. In April, phosphates were available mainly in deeper layers (100 m).

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The temperature and salinity profiles presented by Karageorgis et al. (2012) showed a well mixed water column in the S Aegean from the surface to ~ 200 m during the spring sampling. By processing the dynamic topography data provided from the Copernicus repository, a persistent cyclonic gyre was traced north-east of the Cyclades plateau in the vicinity of station A5, as well as two cyclonic formations north of Crete in the vicinity of stations A9 and A10 (Fig 2a here and Fig. 1 in suppl. material). The local upwelling caused by the cyclonic gyres may have been an additional factor partially responsible for feeding the S Aegean with nutrient-rich waters prior to the spring sampling, as observed in the Copernicus nutrient images. Moreover, Velaoras et al. (2013), using data from a fixed observatory, reported that during the sampling period of March 2008, a convective mixing in the S Aegean (central Cretan Sea) homogenized the water column down to more than 250 m. The hypothesis of a stronger than ordinary 2008 winter convection was also supported by the fact that intermediate waters of Cretan origin with a density higher than usual had been observed exiting the west Cretan straits during the same cruise (Krokos et al., 2014; Velaoras et al., 2014). Further evidence for this strong mixing in spring were the presence of intermediate waters with a salinity maximum between 400-800 m and an O2 maximum (5.04 ml L-1) that were observed north-west of Crete (station I1). This was apparently

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newly formed intermediate water that was flowing out from the west Cretan Straits. This signal was detected again during the summer cruise (station A12). Indeed, O2 maximum (~5.10 ml L-1) and nutrient minimum values compared to the layers above and below (phosphate 0.10 μmol L1 ; nitrate+nitrite ~0.9 μmol L-1) and low N:P ratio (9.2) were observed in the layer 500-750 m.

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Table 1: Inorganic nutrients, particulate organic matter and phytoplankton parameters in the water column and standard deviations (SD in parentheses) in the Aegean, Levantine and Ionian seas during spring (March-April) and summer (August-September). The depth-averaged values were derived from integrated values divided by the integration depth 0-150 m, except for primary production that was 0120 m and for phytoplankton community structure that was 0-100 m. Spring Summer N. AEGEAN S. AEGEAN LEVANTINE IONIAN SEA N. AEGEAN S. AEGEAN LEVANTINE IONIAN SEA SEA SEA SEA SEA SEA SEA Inorganic nutrients Nitrate+Nitrite 0.316 (0.160) 0.295 (0.147) 0.509 (0.164) 0.335 (0.148) 0.534 (0.288) 0.539 (0.226) 0.285 (0.218) 0.336 (0.253) -1 (μmol L ) Phosphate 13.77 (12.35) 10.73 (6.720) 5.701 (2.929) 5.876 (3.245) 10.82 (7.506) 10.36 (6.199) 3.585 (1.847) 7.374 (10.89) -1 (nmol L ) -1

Silicate (μmol L ) 1.055 (0.443) 0.790 (0.407) 0.927 (0.242 0.945 (0.300) 1.530 (0.091) 1.304 (0.305) 1.195 (0.399) 1.177 (0.354) N:P

67

(55)

Si : N

5.1

(1.8)

37

(12)

122

(31)

101

(64)

73

(32)

69

(28)

179

(127)

139

(109)

3.3

(1.7)

3.7

(1.5)

7.8

(7.6)

14

(5)

11

(6)

7.8

(2.2)

14

(8.8)

-1

Particulate organic matter (μmol L ) Part. org. carbon 3.50

(0.41)

3.55

(1.03)

2.49

(0.03)

2.19

(0.31)

2.98

(0.69)

2.64

(0.25)

2.31

(0.01)

2.83

(0.63)

Part. nitrogen

0.44

(0.07)

0.35

(0.07)

0.29

(0.03)

0.23

(0.02)

0.42

(0.12)

0.31

(0.02)

0.28

(0.03)

0.29

(0.03)

C:N

8.41

(0.80)

10.37

(0.75)

9.25

(0.76)

10.77

(1.85)

7.68

(1.11)

8.97

(0.66)

8.64

(0.95)

9.19

(1.42)

-3

Chl-a (mg m ) Total (>0.2 μm)

0.132 (0.019) 0.156 (0.084) 0.077 (0.017) 0.063 (0.032) 0.069 (0.018) 0.057 (0.023) 0.061 (0.006) 0.054 (0.013)

0.2-2.0 μm

0.082 (0.114) 0.016 (0.012) 0.052 (0.003) 0.045 (0.020) 0.056 (0.015) 0.049 (0.022) 0.048 (0.005) 0.039 (0.014)

2.0-5.0 μm

0.018 (0.009) 0.011 (0.007) 0.012 (0.007) 0.011 (0.010) 0.006 (0.001) 0.005 (0.003) 0.007 (0.004) 0.013 (0.007)

>5.0 μm

0.017 (0.010) 0.088 (0.018) 0.017 (0.009) 0.010 (0.006) 0.007 (0.004) 0.012 (0.006) 0.006 (0.001) 0.007 (0.002)

Chl-a ratios Chl-a : C

0.0024 (0.0009) 0.0018 (0.0004) 0.0019 (0.0006) 0.0025 (0.0017) 0.0015 (0.0002) 0.0017 (0.0004) 0.0022 (0.0002) 0.0026 (0.0010)

Chl-a : N

0.016 (0.005) 0.015 (0.002) 0.014 (0.006) 0.024 (0.019) 0.010 (0.002) 0.012 (0.003) 0.013 (0.001) 0.022 (0.009) -3

-1

Total and size-fractions of primary production rates (mg C m h ) Total (>0.2 μm)

0.261 (0.492) 0.179 (0.492) 0.261 (0.165)

0.237 (0.131) 0.185 (0.190) 0.088 (0.048) 0.075 (0.031) 0.075 (0.042)

0.2-2.0 μm

0.203 (0.263) 0.099 (0.263) 0.155 (0.128)

0.165 (0.089) 0.147 (0.176) 0.052 (0.025) 0.032 (0.020) 0.052 (0.037)

2.0-5.0 μm

0.017 (0.072) 0.006 (0.072) 0.084 (0.060)

0.036 (0.028) 0.027 (0.024) 0.027 (0.015) 0.022 (0.014) 0.017 (0.026)

>5.0 μm

0.072 (0.225) 0.079 (0.225) 0.022 (0.016)

0.035 (0.023) 0.010 (0.014) 0.009 (0.014) 0.021 (0.011) 0.007 (0.007)

-1

Phytoplankton community structure (cells L ) Species number

63

(7.02)

77

(6.56)

65

(10.61)

52

(4.51)

53

(3.51)

48

(6.25)

52

(3.54)

48

(2.75)

Shannon-Wiener Diversity index Diatoms (cells 3 -1 x10 L ) Dinoflagellates 3 -1 (cells x10 L ) Coccolithophores 3 -1 (cells x10 L ) Nanoflagellates 3 (>5μm) (cells x10 -1 L )

283 284

4.078 (0.112) 4.257 (0.095) 4.103 (0.164 3.889 (0.084) 3.903 (0.078) 3.808 (0.139) 3.884 (0.072) 3.811 (0.061) 5.5

(4.1)

123.8

(64.6)

23.3

(20.6)

10.3

(9.1)

5.9

(3.6)

12.4

(10.2)

19.0

(18.4)

9.8

(9.6)

11.9

(3.5)

3.5

(1.2)

9.3

(2.0)

7.5

(2.1)

5.0

(2.5)

6.0

(3.9)

5.8

(0.7)

5.2

(1.5)

2.5

(0.4)

0.7

(0.6)

4.5

(1.5)

4.7

(1.9)

0.6

(0.2)

1.2

(0.7)

0.8

(0.3)

1.5

(0.5)

10.7

(0.4)

1.6

(1.4)

8.1

(4.3)

11.3

(9.6)

14.6

(7.9)

17.3

(15.7)

5.3

(4.6)

6.6

(5.4)

Silicoflagellates

98

(128)

28

(24)

106

(57)

83

(55)

12

(7)

12

(11)

14

(10)

41

(40)

Total abundance 3 -1 (cells x10 L )

30.7

(8.0)

(61.7)

45.4

(26.9)

33.8

(12.6)

26.1

34.7

(8.8)

26.1

(13.4)

22.9

(5.8)

129.6

(13.0)

285

2. Ocean color satellite derived data for Chl-a

286 287 288 289 290 291

In order to compare the aforementioned findings with the Chl-a produced by phytoplankton cells as a response to this nutrient enrichment, ocean color satellite derived images from Copernicus repository were examined. Fig. 2d, shows the well known strong Chl-a signal in N Aegean, as well as some elevated Chl-a concentrations in the S Aegean during March, whereas lower levels appear before and after (see Fig. 4 in suppl. material).

292 293 294 295 296

3. Phytoplankton biomass (expressed as total and size fractionated Chl-a) and particulate organic matter

297

The presentation of results follows the entire transect of 1300 km length from north to south and then from east to west, i.e. moving from N Aegean towards S Aegean, Levantine and Ionian seas (Fig. 3 and 4). During spring (March-April) the Aegean Sea presented elevated total Chl-a and particulate organic matter concentrations, compared to the lower levels of the Ionian and Levantine seas (Fig. 3a). The summary mean values and standard deviations for all stations and depths are presented in Table 1.

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Looking closer at the overall spatial distribution of the bulk Chl-a in spring (>0.2 μm fraction), a clear decreasing trend moving from the Aegean to the Levantine and Ionian seas can be seen (R2=0.69, p<0.05) (Fig. 4a). The highest total Chl-a concentration was found in the S Aegean (mean of depth-averaged values 0.156 mg m-3 in S Aegean, max 0.520 mg m-3), while the N Aegean followed with values 1.2 times lower (mean of depth-averaged values 0.132 mg m-3 in the N Aegean, max 0.490 mg m-3) (Table 1 and Fig. 3a). In the blooming waters of the S Aegean, nano+microplankton was the dominant fraction of phytoplankton (as >5μm Chl-a fraction in stations A5, A9, A10), while in all other areas picoplankton dominated (Fig. 3a and 4a).

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The vertical distribution of total Chl-a was mainly driven by picoplankton, except for the S Aegean where nano+microplankton peaked at 75 m (Fig. 5). In the N Aegean there was a prominent surface maximum at 10 m, while in the S Aegean the distribution was more homogenous. In the Levantine and Ionian seas there was a deep Chl-a maximum at 50-75 m. Overall, a gradual shift in the vertical pattern was observed, with high Chl-a values in the N Aegean constrained within the surface layer of the inflowing modified Black Sea Water (BSW), and a deepening of Chl-a maxima towards the south and the southwest.

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During summer, total and size-fractionated Chl-a concentrations were lower than in spring in the Aegean, whereas they were similar in the Ionian and Levantine seas (Table 1, Fig. 3b and 4b). Therefore, when calculating the bulk Chl-a in the water column, the values were rather similar among all sampling areas in summer (Table 1 and Fig. 3b). Picoplankton was the dominant

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fraction in all areas (65-81%) now, giving shape to the vertical total Chl-a profiles (see Fig. 5a in suppl. material). There was a clear pattern for the vertical distributions of total Chl-a with the formation of a Deep Chlorophyll Maximum (DCM) in all the sampling stations, with a progressive deepening from the north (50 m) towards the south (around 75 m) and south-west (around 100 m).

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In spring, particulate organic carbon (POC) and particulate nitrogen (PN) concentrations (as depth-averaged values within the entire euphotic zone) exhibited a gradual decreasing trend from the Aegean towards the Levantine and Ionian seas (Table 1), with the S Aegean exhibiting slightly higher POC values than the N Aegean. Interestingly, maximal POC concentrations were observed within the surface layer (0-20 m) of the N Aegean (7.22-11.46 μmol L-1), decreasing sharply at greater depths (data not shown), whereas in the S Aegean POC was more evenly distributed in the well mixed euphotic zone, following the vertical profile of Chl-a. During summer, there was an overall decrease of particulate organic matter (POM), with the N Aegean showing the highest concentrations. Furthermore, the values of C:N were higher than the classical Redfield ratio value of 6.6 in all cases. All areas showed higher C:N ratios in spring than in summer. The N Aegean demonstrated lower C:N values than elsewhere inter-seasonally, indicating the presence of more nitrogen-enriched organic particles in this area. The Chl:C and Chl:N ratios were low in all areas and seasons. 4. Phytoplankton productivity expressed as total and size fractionated rates of primary production In-situ primary production (PP) rates (mg C m-3 h-1) were measured as a proxy of phytoplankton productivity in the four study areas for both seasonal cruises (see Fig. 6). During spring, elevated total PP rates were demonstrated across all sites with a decreasing trend from north to southsouthwest (R2=0.76, p<0.05) (Table 1 and Fig. 6a). The N Aegean demonstrated approximately two-fold higher total PP rates and picoplankton was the overall most productive fraction. Surface waters (5 m) occupied by the Black Sea originated water mass in the N Aegean were clearly more productive (see vertical profiles in Fig. 7). Picoplankton was driving the vertical distribution of total phytoplankton productivity in all seas throughout the water column, whereas in the S Aegean the nano+microplankton fraction was also closely following the profile shape.

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Phytoplankton productivity declined during summer with the decline of phytoplankton biomass (as Chl-a concentrations) (Table 1 and Fig. 6b). However, the levels of total PP rates in the N Aegean were again approximately two-fold higher than in the other areas. There was a clear decreasing trend towards south-southwest (R2=0.89, p<0.05), where productivity values were similar among them (Fig. 6b). Picoplankton was again the most productive fraction with clearly higher PP rates in surface waters of N Aegean (within 10 m) (Fig. 5b in suppl. material). In the

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other seas, productivity was low throughout the water column. Overall, maximal productivity was observed close to surface (within the first 10 m layer) and did not necessarily coincide with the DCM.

368 369

4. 5. Phytoplankton community structure and bloom identification

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We examined the composition and spatial distribution of phytoplankton communities in the Aegean, Ionian and Levantine seas in spring and summer. A total of 225 phytoplankton species were identified and quantified, comprising of 128 dinoflagellates, 65 diatoms, 28 coccolithophores, 2 silicoflagellates, 1 cryptophyte and 1 raphidophyte (Table 1). Nanoflagellates (> 5 μm) were quantified as one group. The total number of species was similar in all areas for both seasons (ANOVA, p>0.05). However, the diatom and coccolithophore species numbers were higher in spring, while the dinoflagellates were higher in summer. Overall, the diversity was constantly high throughout the study areas. The Shannon-Wiener Diversity index was 3.65-4.36 (mean 3.96 ± 0.17 SD) and did not vary significantly among different stations or seasons (ANOVA, p>0.05).

381 382 383 384 385 386 387 388

In spring, the total abundance of microplankton was high reaching 237.3 x103 cells L-1 in S Aegean (depth-averaged values at 58.4 x103 -167.1 x103 cells L-1, see Table 1 and Fig. 8a). Spring bloom waters in the S Aegean were dominated by diatoms reaching 232.5 x103 cells L-1, (depthaveraged values at 49.3 x103-163.6 x103 cells L-1, see Table 1 and Fig. 8a), while dinoflagellates and nanoflagellates were dominating the N Aegean. In Levantine and Ionian waters there was an overall mixed composition of the microplankton community and coccolithophores were more abundant than in the Aegean waters.

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The total microplankton abundance decreased by almost 4-fold in summer and presented a more homogenous pattern across the study areas (Table 1 and Fig. 8b). There was an overall shift from diatom to dinoflagellate and nanoflagellate dominance (with few exceptions), but their relative contribution increased mainly due to the decrease in diatoms abundance. Coccolithophores were less abundant than in spring. The relative contribution of silicoflagellates remained low inter-seasonally. Overall in the N Aegean, dinoflagellates and nanoflagellates were found to be important phytoplankton functional groups inter-seasonally. In spring, dinoflagellates and nanoflagellates peaked in the surface 2 m of the N Aegean, whereas in the S Aegean the diatoms bloomed homogenously through the water column (Fig. 9). In the Levantine and Ionian waters higher abundances were present in deeper layers (75 m). In general, all phytoplankton groups were represented throughout the water column in all studied areas. In summer, nanoflagellates peaked in surface waters of the N Aegean (2-20 m) and deeper in the S Aegean (50 m) (Fig. 6a in suppl. material). Diatoms dominated throughout

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the water column in the Levantine, whereas in the Ionian all groups were homogenously distributed. Coccolithophores were more abundant in surface waters (2 m), while silicoflagellates decreased across all depths compared to spring. The inter-seasonal vertical pattern (Fig. 6b in suppl. material) demonstrated a clear surface maximum in the N Aegean with more than two-fold lower total microplankton abundances in depths >20 m. In the S Aegean diatoms dominated with high abundances from the surface to 75 m. This dominance was mainly attributed to the elevated spring diatom values. The Levantine and Ionian microplankton groups were more homogenously distributed through the water column with deep maxima at 75 m. It is noteworthy that overall, diatoms displayed the lowest abundances in the N Aegean, while nanoflagellates and dinoflagellates the highest. In Table 2, the most abundant microplankton species are presented. Nanoflagellates, the small diatom Leptocylindrus minimus and small unarmoured dinoflagellates (<15-20 μm) were dominant in the N Aegean inter-seasonally. Mostly large sized diatoms were dominant in the S Aegean in spring (Chaetoceros costatus, C. curvisetus, C. decipiens, C. lorenzianus, Bacteriastrum hyalinum, B. furcatum etc), whereas in summer the small L. minimus and nanoflagellates were dominant. The Levantine and Ionian demonstrated a homogenous pattern with L. minimus and nanoflagellates dominating inter-seasonally. The coccolithophore Emiliania huxleyi was more abundant in spring in all seas except for the S Aegean.

425

Table 2: Range of species abundance in relative contributions (>5%) and cells x103 L-1 in the water column of the most abundant species

426

in the Aegean, Levantine and Ionian seas during spring and summer (nanoflagellates are >5 μm). The depth-averaged values were

427

derived from integrated values divided by the integration depth 0-100 m.

428 Spring

North Aegean Nanoflagellates Leptocylindrus minimus Gymnodinium spp. Emiliania huxleyi Gyrodinium spp.

Summer Nanoflagellates L. minimus Chaetoceros affinis Thalassiosira sp. Gyrodinium spp. Gymnodinium spp. Leptocylindrus mediterraneus 429 430

25-42% 10.1-10.5 0.1-17% 0.04-6.6

South Aegean Chaetoceros spp. L. minimus

0-16% 5-7%

0.04-6.3 1.2-2.1

Bacteriastrum spp. 0-6% Pseudo-nitzschia spp. 0-6%

0-7%

0.01-2.0

29-60% 6-13% 0-12% 0-10% 0-6% 0-6% 0-6%

2.1-23.7 0.1-3.5 0-0.9 0-2.6 0.01-2.2 0.02-1.5 0.0-0.5

L. minimus Nanoflagellates Gyrodinium spp. Emiliania huxleyi

0-26% 0-16%

0.2-30.1 0.06-26.9

Levantine L. minimus Nanoflagellates

1-44% 8-42%

0.3-28.6 5.1-11.2

Ionian L. minimus Nanoflagellates

1-53% 4-52%

0.2-21.8 1.6-23.2

0.15-10.6 0.04-9.5

Emiliania huxleyi Chaetoceros spp.

6-8% 0-8%

2.0-4.0 0.02-5.2

Emiliania huxleyi Chaetoceros spp.

5-12% 0-5%

1.7-3.9 0.2-2.2

Gyrodinium sp. Gymnodinium spp. Katodinium glaucum L. minimus Nanoflagellates Gyrodinium spp. Thalassiosira sp. Emiliania huxleyi Gymnodinium spp.

3-5% 1-5% 1-5%

0.7-1.2 0.02-2.1 0.3-0.9

16-81% 1-49% 0-10% 0-7% 3-6% 0-5%

3.5-23.5 0.2-11.1 0.1-1.1 0.1-1.0 0.6-1.4 0.01-1.2

0-88% 0.3-23.9 3-73% 0.8-32.1 0-6% 0.01-2.7 0.4-5% 0.1-1.5

L. minimus Nanoflagellates Thalassiosira sp. Gymnodinium spp.

10-80% 18-42% 0-7% 0-5%

2-31.1 0.8-8.3 0-1.3 0.02-1.0

431 432 433 434 435 436 437 438 439 440 441 442 443 444

The hierarchical clustering with Bray-Curtis similarity index and group averages were based on the species abundance data. The cluster analysis showed that there are three groups of stations with a similarity higher than 50% on the basis of the seasonal factor (Fig. 10). Group 1 was made up of stations of the S Aegean in spring (similarity at 60.56%). Species analysis with Simper routine showed that these stations were similar mainly due to the presence of large-sized diatoms at high numbers, e.g. Chaetoceros spp. (~24 x103 cells L-1), Bacteriastrum spp. and Cerataulina pelagica. The similarity of Group 2 with the Ionian and Levantine stations in spring (at 60.33%) was attributed to high abundances of small-sized Leptocylindrus minimus (~25 x103 cells L-1) and the presence of small Gymnodinioids and Emiliania huxleyi. Group 3 (at 59.53%) included the rest of the areas on the basis of L. minimus, but at lower densities (~6 x103 cells L-1), and small Gymnodinioids, E. huxleyi and Prorocentrum minimum. Station A1 in the N Aegean was separated from all the others (48.81% similarity with Group 2) mainly due to the high density of the nanoflagellate Leucocryptos marina, the presence of large Gymnodinioids and the absence of large diatoms and some coccolithophores.

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DISCUSSION

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The Aegean Sea is known to have a complex hydrographic and ecological structure due to its geographical position between the Black Sea, the Levantine and Ionian seas (Theocharis et al., 1999; Psarra et al., 2000; Ignatiades et al., 2002; Siokou-Frangou et al., 2002). During the spring cruise, the Aegean Sea demonstrated elevated phytoplankton biomass (as Chl-a) despite the low levels of inorganic N and P recorded. Previous studies have documented that the N Aegean can support high plankton stocks and productivity due to the enrichment from the inflowing Black Sea Water (BSW, Zervoudaki et al., 2011; Lagaria et al., 2013; Frangoulis et al., 2017). In the thermohaline front of the NE Aegean, modified BSW masses with lower salinity (∼30) and temperature outflow through the Dardanelles straits and meet the warmer more saline (∼38.5) Levantine water (Theocharis and Georgopoulos, 1993; Zervakis et al., 2000; Zervakis and Georgopoulos, 2002). The less dense modified BSW remains mostly close to the surface layer and therefore maxima of Chl-a, phytoplankton productivity and abundances are mostly constrained within the surface. Our findings further support this feature of high phytoplankton biomass and productivity in the N Aegean within the surface 10 m layer. A lower Chl-a peak existed at 50 m giving to the vertical profile a “complex shape” according to the classification of Chl-a vertical profiles in the Mediterranean (Med) by Lavigne et al. (2015). The “complex shape” often displays several peaks and a relatively high surface Chl-a. Maxima of Chl-a close to the surface (less than 10 m) have also been documented for the northwestern Mediterranean basin, during the spring bloom period (Marty et al., 2002; Manca et al., 2004).

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Surface Chl-a peaks and the influence of the BSW in spring were decreasing with increasing distance from the N Aegean. Chl-a was homogenously distributed in the S Aegean water column due to the vertical mixing, showing a homogenous vertical profile, typical of mixed conditions (Lavigne et al., 2015). The S Aegean has been recognized as a more oligotrophic environment than the N Aegean (Ignatiades et al., 2002; Lykousis et al., 2002). In the Levantine there was a subsurface peak and a Deep Chlorophyll Maximum (DCM) in 50 m, forming a “modified DCM”, which describes profiles with relatively high values in the mixed layer and with a peak of Chl-a just below the mixed layer depth (Lavigne et al., 2015). This represents an intermediate condition between the DCM and homogeneous conditions. In the Ionian, a clear DCM was evident at 75 m depth. The two latter regions occupy the most oligotrophic and transparent waters in the Mediterranean, with a permanent DCM. The existence of the DCM in 100 m or more has been reported in the Levantine Sea (Yilmaz et al., 1994; Christaki et al., 2001). In summer, surface Chl-a peaks disappeared and clear DCMs were formed in all the study areas in deeper waters than in spring, forming the typical DCM of the stratified period (Lavigne et al., 2015).

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DCM is a well-defined feature of the Mediterranean Sea, which is linked to the high light penetration, the stratification of water masses and the low nutrient concentrations in the euphotic zone (Cullen, 2015). During a trans-Mediterranean cruise in summer 1999, Ignatiades et al. (2009) reported higher values of total cell abundance, Chl-a and phytoplankton taxa below the seasonal thermocline (~50 m). This was linked with hydrographic features and significantly higher concentrations of phosphates and nitrates below the thermocline (Pavlidou et al., 2010; Pavlidou and Kontoyiannis, 2011). DCM formation is more prominent in summer, due to higher light irradiance and the formation of the thermocline and the nutricline (Crise et al., 1999; Dolan et al., 2002; Mignot et al,. 2014). During winter, the DCM disappears in the whole Mediterranean due to winter mixing and the so-called “mixed” shape is often observed, characterized by a constant Chl-a from the surface to the bottom of the mixed layer depth. This seasonal pattern was confirmed by our results. Karageorgis et al. (2012) reported a distinct DCM variation across the Aegean and the Ionian in spring (based on in-situ fluorescence data), whereas in early summer the DCM was clearly formed everywhere due to the decrease of BSW inflow and the thermal stratification in all areas. Besides, the whole Mediterranean is known to be more oligotrophic and uniform in summer, and therefore both phytoplankton and mesozoplankton standing stocks are lower and more equally distributed among regions than in spring (Mazzocchi et al., 2014).

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The DCM deepens gradually from west to east in the Med, especially in the Ionian and Levantine seas where it develops consistently around 100 m depth (Estrada et al., 1993; Moutin and Prieur, 2012; Lavigne et al., 2015). In this study, the DCM deepened from the N Aegean towards the south and south-west. As discussed above, the BSW enriches the surface layers of the N

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Aegean with mineral nutrients, especially dissolved organic matter (Lykousis et al., 2002; Sempéré et al., 2002), whereas in the S Aegean, Ionian and Levantine seas nutrients enter the euphotic zone mostly from deeper layers through convection events (Siokou et al., 2010; Pedrosa-Pàmies et al., 2016) or via atmospheric deposition (Kouvarakis et al., 2001; Markaki et al., 2010; Christodoulaki et al., 2013; Djaoudi et al., 2018). This is in accordance with our findings in the Levantine and Ionian seas, which experienced the deepest DCM inter-seasonally (max 75100 m) together with the lowest phytoplankton biomass (as Chl-a) and productivity and the most severe P-limitation (N:P ratios ~100-180). Salgado-Hernanz et al. (2019) reported a very low increase in winter Chl-a concentration (~ 0.1 mg m−3) resulting in an overall low variability of Chl-a (< 0.06 mg m−3), dominated by seasonality. Barbieux et al. (2018) also reported that Chl-a is essentially constant throughout the year. Indeed the Levantine basin is one of the most transparent and oligotrophic areas in the world oceans, with very low nutrient and low chlorophyll (LNLC) concentrations and an average annual primary productivity approximately half that of the ultra-oligotrophic Sargasso Sea (Krom et al., 2003). The Ionian presents a gradient from north to south due to the influence of Adriatic waters, oscillating between cyclonic and anticyclonic modes per decade and forming the well known Adriatic–Ionian bimodal oscillating system (BiOS) and the North Ionian Gyre (NIG) (Gacič et al., 2010). This circulation regime affects the vertical dynamics and the entire north Ionian biogeochemistry (Civitarese et al., 2010). Recent findings report that a significant winter–spring bloom is observed when the circulation is cyclonic and the winter mixing is relatively strong (Lavigne et al., 2018). Our sampling stations were located in the south Ionian during an anticyclonic phase of the NIG, characterized by an early winter bloom onset and absence of a Chl-a peak in March.

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Higher Chl-a concentrations in the deeper layers of the Mediterranean might also be connected to other factors: (i) photo-inhibition and photo-degradation processes of Chl-a at 5–10 m depth (Cuny et al., 2002; Bellacicco et al., 2016), (ii) grazing pressure of planktonic ciliates proliferating at 0–50 m (Pérez et al., 2000), and (iii) higher sinking rates of phytoplankton cells in the clear stratified Med waters (Huisman and Sommeijer, 2002). It is noteworthy that DCM did not necessarily coincide with maximal productivity (observed close to surface within the first 10 m layer) in our study, implying that the Chl-a content of the cells was higher rather than an actual increase in biomass of phytoplankton. This could be a result of adaptation by the deep phytoplankton communities to sub-optimal light intensities. This miss-match was recorded also during a summer trans-Mediterranean cruise, with max Chl-a below the thermocline (50 m) and max productivity above the thermocline (Ignatiades et al., 2009). Recent analysis of the DCM in the Ionian sea suggests that the almost constant total phytoplankton biomass throughout the year implies that the summertime DCM biomass increase is not due to DCM photo-acclimation, nor an increase in DCM production, but instead of the "migration" − with photoacclima_on − of surface phytoplankton into the DCM (Palmiéri et al., 2018).

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Picoplankton was the dominant phytoplankton fraction in all seas inter-seasonally (with the exception of S Aegean in spring) accounting for 64-81% of the total Chl-a and 42-80% of the total primary production. Therefore most carbon was fixed by picoplankton throughout the study area and in both seasons. In the Aegean, previous studies have shown that almost 60–70% of autotrophic biomass and primary production was performed by cells < 3 μm (Ignatiades et al., 2002; Sioukou et al., 2002). Small phytoplankton cells have a higher surface area-to-volume ratio, nutrient assimilation efficiency, photosynthetic activity, faster division rates and lower sinking rates (Raven, 1998; Finkel et al., 2009). With such characteristics, the photosynthetic picoplankton can proliferate in nitrogen-limited and clear oceanic waters, comprising >50% of the overall plankton community, primary production and Chl-a biomass in oligotrophic tropical and subtropical open ocean waters (Clark et al., 2013).

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The extreme oligotrophy of the EMed is known to favor small sized phytoplankters which have lower nutrient requirements for growth and can take up inorganic nutrients fast, whenever they might enter the euphotic zone (Moutin and Raimbault, 2002; Psarra et al., 2005; Tanaka et al., 2007). Phytoplankton size structure plays a major role for the carbon fluxes in microbial pelagic communities (Santinelli et al., 2012). Small-sized phytoplankton (<3 µm) form the basis of the microbial food web by recycling the organic matter within the ecosystem, whereas larger phytoplankton (>3 µm) sustain the classical food chain by exporting the organic matter, either to adjacent systems or to upper trophic levels. For example, in the N Aegean a large part of the fixed carbon has been found to be channeled through the microbial food web towards copepods, where picoplankton production was sufficient to cover the carbon demand of nanoand micro-heterotrophs (Siokou et al., 2002; Zervoudaki et al., 2007; 2011). In contrast, there was a multivorous food web with lower energy transfer in the S Aegean (Siokou et al., 2002).

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Surprisingly, higher values of total Chl-a and phytoplankton abundances were found in the south rather than the north of the Aegean in spring. This is not a common pattern for the Aegean waters and actually the Chl-a values in this study are among the highest reported so far (Gotsis-Skretas et al., 1999; Psarra et al., 2000; Ignatiades et al., 2002; 2009, Lagaria et al., 2013; 2017). This pattern was also rather inconsistent with the low availability of inorganic N and P at the time, so some source of nutrients must have been used to trigger phytoplankton growth in this area where no rivers or other significant nutrient point sources exist. Siokou et al. (2010) suggest that in the Med it is the vertical flux of nutrients to the euphotic zone that allows for new primary production, and this is mainly driven by the mixing depth and the subsurface nutrient availability. The presence of cyclonic structures and the winds affecting winter mixing and coastal upwelling are considered to be the main physical driving forces that promote the phytoplankton biomass build-up through the induced increase of nutrient availability.

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A well mixed water column in the S Aegean was identifiable in the temperature and salinity vertical profiles during the spring oceanographic cruise, showing also a dome shaped signature of increased in-situ fluorescence (Karageorgis et al. 2012). In this study, total Chl-a and phytoplankton populations were also spread homogenously throughout the mixed water column. As already mentioned, Velaoras et al. (2013) showed that during the sampling period, a strong convective mixing in the central Cretan Sea (S Aegean) reached depths of more than 250 m, while the outflow of denser than usual intermediate waters of Cretan origin from the Cretan Straits towards the open Med (Krokos et al., 2014; Velaoras et al., 2014) further supports strong convection episodes in winter-early spring 2008. As a consequence, waters containing nutrient concentrations typical of intermediate waters (~100-140 nmol L-1 phosphate and ~2.0 μmol L-1 of nitrate+nitrite, with an N:P ratio close to 16:1 and σθ ~ 29.2 kg m-3) reached the upper layers of the water column (Pavlidou et al., 2011). Recent studies show that N and P atmospheric inputs can also be considerable, especially during periods with intense precipitation events, and thus can play an important role for the nutrient availability in the euphotic zone in the Med (Krom et al., 2014; Rahav et al., 2018). The atmospheric input of dissolved inorganic N and dissolved inorganic P accounts for 61% and 28% of the total budget of N and P respectively in the EMed (Krom et al., 2004). D’Ortenzio and Ribera d’Alcala (2009) suggest that the Levantine is the bioregion most subjected to the influence of atmospheric deposition in the Med.

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To be able to further examine the duration and impact of this vertical mixing over an extended time period, we processed dynamic topography, inorganic nutrient and Chl-a data from Copernicus repository before, during and after the spring sampling. The dynamic topography images that were processed, showed cyclonic structures before our sampling in the S Aegean, namely in the area north of Crete and north-east (NE) of the Cyclades plateau, mixing the water masses vertically. The latter is known as the Chios Gyre (Olson et al., 2007). The Cyclades plateau separates the Aegean Sea in the two sub-basins of the N Aegean and the S Aegean, with significantly different hydrographic characteristics due to the influence of Black Sea waters and Levantine Sea waters respectively (Zervakis et al., 2000). According to the nutrient images, most possibly as a consequence of the convective winter mixing, which was further enhanced in the presence of cyclonic gyres like those appearing in the dynamic topography images (Fig.2 and supplementary material), high nitrate (~ 1 μmol L-1) and phosphate (~ 0.06 μmol L-1) concentrations were introduced to the euphotic zone in February prior to sampling. These high nutrients decreased later in April, most probably due to the consumption by phytoplankton in March. Therefore this vertical transfer of nutrient rich waters from the deep must have supported the dense phytoplankton populations that we found in the euphotic zone of the S Aegean in March.

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This is actually the first time that such high phytoplankton abundances are reported from the EMed open waters, constituting a distinct and unprecedented spring bloom in the S Aegean,

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reaching ~237 x103 cells L-1 (as mean of depth-averaged values for the S Aegean ~130 x103 cells L-1). Ignatiades et al. (2002) reported phytoplankton total abundances of 17 x103 – 51 x103 cells L-1 (depth-averaged values ) in S Aegean in spring 1998, which were higher than in the N Aegean. In the present study, we report values of ~58 x103 - 167 x103 cells L-1 (depth-averaged values ) which are more than 3 times higher than those reported by Ignatiades et al. (2002). D’Ortenzio and Ribera d’Alcala (2009) divided the entire Med basin into trophic regions (based on 10 years satellite data) according to the type of seasonal cycle of the surface phytoplankton biomass, into bloom, intermittent and non blooming areas. The entire Aegean Sea was characterized as a non blooming area with the exception of the NE Aegean, which was characterized as intermittent. Unlike the above mentioned literature, this study is the first record of a prominent phytoplankton bloom in the open waters of the EMed. Furthermore, it was shown recently that large regional variations exist in the Med, suggesting that the response of Chl-a plenology to environmental and climate forcing may be complex and regionally driven (Lavigne et al., 2018; Salgado-Hernanz et al., 2019).

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Phytoplankton blooms are ephemeral events of exceptionally high phytoplankton biomass that are known to regulate the carbon flux across marine food webs (Field et al., 1998; Behrenfeld et al., 2006; Boyce et al., 2010). In the review of Siokou-Frangou et al. (2010), phytoplankton distribution in the open Med has confirmed the existence of regular large bloom formations in late winter–early spring in the NW Med basin exclusively and not the Eastern basin. Regarding the possible linkage of this EMed bloom to higher trophic levels, Mazzocchi et al (2014) found high mesozooplankton standing stocks in the S Aegean during spring. This provides evidence of coupling of high phytoplankton biomass with mesozooplankton grazers.

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While picoplankton dominated all the other basins inter-seasonally, microplankton was the dominant fraction during the bloom event in the S Aegean, comprising mostly of large-sized diatoms (Chaetoceros costatus, C. curvisetus, C. decipiens, C. lorenzianus, Bacteriastrum hyalinum, B. furcatum etc). This peculiarity was reflected in the multidimensional analysis of phytoplankton community structure and distribution. S Aegean stations in spring formed a distinct similarity cluster mainly due to high numbers of the large-sized diatoms. Communities dominated by large diatoms are often found in regions of upwelling and variable environmental conditions (Malviya et al., 2016). Diatoms prefer nutrient rich environments with high turbulence in order to keep theirheavier cells in suspension. These environments are characterized by a well mixed water column similar to the one found in the S Aegean in spring in terms of T, S and Chl-a vertical profiles (see also Karageorgis et al., 2012). Diatoms are known to have a number of ecophysiological advantages to outcompete other phytoplankton groups under such conditions . For example, diatoms can take advantage of Si rapidly when it becomes available, grow fast and dominate the phytoplankton assemblage, forming a bloom (Bates and

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Trainer, 2006). This ecological success may lie in their use of Si to form siliceous cell walls, which require less energy to synthesize compared to organic cell walls (Raven, 1983).

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Another ecological advantage of diatoms is their high affinity for nitrates thanks to the complete urea cycle that they inherited from the heterotrophic host of the secondary endosymbiosis, giving them the ability to outcompete all other species as soon as a nitrate pulse occurs (Allen et al., 2011). The formation of nutrient storage vacuoles can also provide a growth advantage of large diatoms over smaller cells under pulsed nutrient supply regimes (Raven, 1997). A ferritin from the pennate diatom Pseudo-nitzschia multiseries (PmFTN) is also known to facilitate blooming after iron inputs in iron-limited ocean regions, linking iron uptake to many other metabolic reactions, such as photosynthesis, nitrate assimilation, the urea cycle and carbohydrate synthesis (Marchetti et al., 2009; 2012). Predation, parasitism, viruses and other trophic interactions could also regulate the size structure of phytoplankton communities in favour of large diatoms (Smetacek, 2001; Raven and Waite, 2004; Thingstad et al., 2005b). Phytoplankton grazers seem to also follow a similar pattern in the Mediterranean, where large mesozooplankters are known to thrive in spring rather than autumn (Siokou et al., 2002; Zervoudaki et al., 2007; Mazzocchi et al., 2014).

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The high diatom abundances that we found coincided with low in-situ nitrate and phosphate concentrations. This coupling indicates that the bloom had been fuelled by upwelling masses, while our sampling was performed in the aftermath of the conditions that triggered the cell growth. This has also been observed previously by Varkitzi et al. (2018) for spring diatom blooms which coincided with low nutrient levels in coastal waters of the EMed. Bargu et al. (2016) have also reported the same pattern with high abundances of the diatom Pseudo-nitzschia associated with low nutrient conditions in the Mississippi delta. In spring the S Aegean had the highest cell abundances but the lowest levels of nitrates and silicates and the lowest N:P and Si:N ratios than any other area. It is likely that the inorganic nutrients had been consumed for the build-up of the diatom bloom in the S Aegean. Smayda (1990) showed that the Redfield ratio of Si:N=1:1 is a sensitive pivot point below which diatoms, their predators, and zooplankton fecal pellet production declines, whereas the flagellate food web is competitively enabled, with a potential consequent disruption of the diatom-zooplankton-fish food web.

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This pronounced bloom in the S Aegean had x3 times higher cell abundances than in the N Aegean, but was not immediately followed by increased Chl-a values (x1,2 times higher than the N Aegean) nor by primary production (x1.5 times lower than the N Aegean), potentially revealing a less active community with less Chl-a per cell content. This feature can be attributed to the growth phase and/or the physiological condition of the phytoplankton cells. Many of the large blooming diatoms in our study had shrunk cytoplasm or some others had resting spores within their cell walls, when observed under the microscope. The senescent cells present during

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the bloom demise phase, demonstrate low intracellular Chl-a content (Bidle and Falkowski, 2004; Reynolds, 2006). Light-harvesting components are rich in nitrogen, therefore making Chl-a an expensive energy investment when nitrogen and other nutrients are scarce (Hasley et al., 2015; Liefer et al., 2018). Varkitzi et al. (2018) have also observed high numbers of large sized Pseudo-nitschia spp. but comparatively low Chl-a concentrations during spring blooms in the EMed coastal waters. The possible virus infection of blooming phytoplankton cells can be a mortality agent causing rapid bloom termination (Suttle, 2007; Lehahn et al., 2014; Sharoni et al., 2015). At least 18 diatom viruses have been isolated from marine waters, most frequently infecting bloom-forming Chaetoceros and Rhizosolenia species (Nagasaki, 2008; Tomaru et al., 2015; Yamada et al., 2018).

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Larger cells, like the large diatoms in this study, tend to have lower cellular pigment concentrations than smaller cells under similar environmental conditions, in order to counteract the so-called “package effect” (Geider, 1987; Finke,l 2001; Finkel et al., 2004; Alvarez et al., 2017). In this study, the levels of the ratios between Chl-a and chemical elements of the particulate matter (e.g. carbon C and nitrogen N) denoted this decoupling between the bulk POM and Chl. These ratios of Chl-a:C, Chl-a:N and C:N are indicative properties of the nutritional and physiological state of the phytoplankton cells (Goldman, 1980; Halsey and Jones, 2015). The cellular process of photo-acclimation is known to have a great impact on phytoplankton cells by changing the intracellular Chl-a, C and N concentrations as the combined effect of light and nutrient availability (Geider et al., 1997; Moore et al., 2006; Behrenfeld et al., 2015). Photoacclimation is known to play a dominant role over the phytoplankton seasonal cycle in the Med (Siegel et al., 2013; Mignot et al., 2014). In this context, Bellaccico et al. (2016) report a dramatic decrease of Chl:C ratio from March to April in the Med because Chl-a values start to drop in March due to high solar irradiance, whereas C highs still persist due to the phytoplankton buildup in late winter-early spring, when nutrients are available. This is consistent with the low Chl:C ratios found in this study (0.0015-0.0026 μg g-1) during the same period (late Mar-early Apr). The same authors also reported that as irradiance continues to increase, the water column stratifies and nutrients become depleted, further decreasing Chl:C to minimum values in summer (~0.0013 μg g-1).

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In this study, the values of Chla:C ratio were approximately an order of magnitude lower than the conventional levels that represent a balanced physiological state of the phytoplankton cells (Banse, 1977; Geider et al., 1997). Such low Chla:C values indicate that the cells were deprived of N, which is known to result in the decline of Chl-a synthesis, as explained above. This N deficiency of the phytoplankton cells in our study was further supported by C:N ratios higher than the Redfield ratio for the particulate matter, suggesting rather the synthesis of carbonenriched compounds (e.g. lipids, carbohydrates) than nitrogenous ones due to N limitation (Falkowski, 2000). The S Aegean in spring demonstrated the lowest Chla:C values that were

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coupled with the highest particulate C:N values and the lowest inorganic N, Si, N:P and Si:N values in the surrounding water masses, indicating a severe N deficiency of the phytoplankton cells there. All these findings support our suggestion that the initially high N consumption by the blooming cells (mostly large diatoms), was followed by a deficit of N which finally caused the cells to decrease their metabolism (e.g. Chl-a synthesis) and to enter the bloom decay that was encountered during this sampling.

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In the N Aegean the particulate organic matter (POM) was found less N deprived than elsewhere probably due to the influence of freshwater input from local rivers as well as the low salinity waters of Black Sea origin (BSW). Τhe BSW, being enriched in particulate and dissolved organic matter, results in enhanced planktonic biomass and primary production in the N Aegean (Meador et al., 2010; Lagaria et al., 2013; Petihakis et al., 2014; Souvermezoglou et al., 2014) and an efficient microbial food web (Pitta and Giannakourou, 2000; Giannakourou et al., 2014). Furthermore, the BSW inputs along with the riverine discharges are responsible for the high probability of occurrence of a secondary phytoplankton growing period in autumn (SalgadoHernanz et al., 2019). The C:N ratio in the N Aegean being higher than in the other regions during both seasons implies an effective N assimilation in photosynthetically produced particulate matter and/or reflects the presence of relatively fresher POM possibly linked to the fertilizing effect of BSW. In summer, there was an overall decrease of C:N, Chl-a:C and Chl-a:N ratios, which in combination with the lower Chl-a and primary productivity, imply that phytoplankton accounted for a relatively small fraction of the bulk POM. This is consistent with the general pattern that nano- and micro-heterotrophs are known to play a very important role in the whole Aegean Sea, with a gradual decreasing contribution of autotrophs and a parallel increasing contribution of heterotrophs from the N to the S Aegean (Siokou et al., 2002). On a temporal scale, the role of heterotrophs (mostly micro-heterotrophs) is more important during the stratification period (September) than the mixing period (March). At this point it is worth to mention that the interpretation of Chl:C and Chl:N ratios is not straightforward since there will always be some contribution to POC from material other than phytoplankton (e.g. heterotrophic bacteria, small zooplankton, detritus), and therefore some underestimation of Chl-a per carbon unit is expected (Sathyendranath et al., 2009; Graff et al., 2012).

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It is noteworthy that overall nanoflagellates and small-sized dinoflagellates displayed the highest abundances in the N Aegean, while diatoms the lowest. For example, the high nanoflagellate abundance (max ~ 23 x103 cells L-1) and the presence of unarmoured dinoflagellates mostly in surface waters distinguished the N Aegean from the other regions. Previous research has shown that the modified BSW in the N Aegean carries mostly dissolved organic nutrients (Sempéré et al. 2002) and these properties favor heterotrophic nanoflagellates and dinoflagellates grazing on bacteria. There are also indications that nanoflagellates can vary in space and time and contribute significantly to blooms in the WMed (Marty et al., 2002). In contrast, both in Ionian

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and Levantine seas microplankton composition was similar and dominated by the small diatom Leptocylindrus minimus, which preferred depths around 75 m. This is a common species for the ultra-oligotrophic EMed waters (Ignatiades et al., 2009). Furthermore, the contribution of the coccolithophore Emilinia huxleyi was more pronounced there than elsewhere, probably due to the properties of these areas resembling open ocean waters.

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Differences in the community structure of microplankton were most prominent in spring, whereas in summer an overall shift from diatom to dinoflagellate and nanoflagellate dominance was observed across all seas. Phytoplankton communities were more homogenous with low densities of small-sized Leucocryptos marina, Gymnodinioids, L. minimus and E. huxleyi. The vertical stability of the water column in the warm season is determining the prevalence of dinoflagellates over diatoms because of their competitive advantage of motility, allowing them to accomplish vertical migrations to acquire nutrients and/or optimum photosynthetic conditions (Smayda and Reynolds, 2001). During the stratified period in the EMed, the inorganic nitrate and phosphate levels are further decreased in the euphotic zone and this is known to promote the growth of heterotrophic nanoflagellates and mixotrophic/heterotrophic dinoflagellates feeding on bacteria (Christaki et al., 2001). Together with these patterns of phytoplankton dynamics that was recorded in the different basins of the EMed, the biodiversity was constantly high and similar over space and time. The EMed is profoundly known to host high biodiversity despite its extreme oligotrophy (Bianchi and Morri, 2000).

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Conclusions

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This study verified the heterogeneous patterns and functioning of phytoplankton biomass, productivity and community structure along a gradient of oligotrophy in the EMed. In summary, the impact of the productive Black Sea waters in the N Aegean seems to fade out towards S Aegean, following the deepening of the DCM distribution and the decreasing trend of phytoplankton biomass and productivity. This trend is even more apparent in the Levantine and Ionian seas. Furthermore, an episodic nutrient enrichment of the S Aegean oligotrophic surface waters due to deep mixing appears to trigger the formation of a prominent spring bloom. This nutrient enrichment was highlighted as the driving mechanism for this exceptional bloom with the help of satellite and model derived data from the E.U. Copernicus repository by examining the sequence of events before, during and after the bloom we found in S Aegean. With our results, we further confirm that oligotrophy is the main driving force for the distribution of phytoplankters, but we also offer evidence of how the low-nutrient-low-chlorophyll euphotic zone of the EMed can occasionally support high phytoplankton biomass and generate prominent blooms.

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Acknowledgements

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The authors wish to thank the captain and crew on R/V Aegaeo of HCMR for their valuable help. We are grateful to Mrs. A. Konstantinopoulou and Mr. T. Zoulias for their assistance on board and during laboratory analyses. We express our sincere thanks to the anonymous reviewers and the handling editor for their helpful comments and suggestions. This work was supported by the European Union through the integrated project Southern European seas: Assessing and Modeling Ecosystem changes (SESAME; contract no. 036949). This study has been conducted using E.U. Copernicus Marine Service Information.

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Supplementary material

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Supplementary material associated with this article can be found in the online version at http://dx.doi.org/...................

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FIGURE CAPTIONS

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Fig. 1. Study area with the sampling stations during spring (March-April) and summer (AugustSeptember) 2008 in the Eastern Mediterranean: Aegean, Levantine and Ionian seas (a) sampling stations for the phytoplankton parameters, and (b) the major patterns of surface circulation in Eastern Mediterranean (from Karageorgis et al. 2008).

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Fig. 2. Dynamic topography prior to in-situ sampling processed from Copernicus repository, the scale is in cm (a); Nitrates (b) and phosphates (c) monthly means in ~10m depth from model derived data and images processed for February from Copernicus repository, the scale is in μmol L-1. Surface Chl-a (d) monthly means for March processed from Copernicus satellite ocean color imagery, the scale is in mg m3 (more images can be found in the Supplementary material).

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Fig. 3. Total (>0.2 μm) and fractionated Chl-a (mg m-3) in the Ionian, Levantine, South and North Aegean seas in (a) spring (March-April) and (b) summer (August-September). The means of depth-averaged values were derived from integrated values divided by the integration depth 0-150 m per sampling station.

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Fig. 4. Spatial distribution per sampling station of total Chl-a (>0.2 μm as solid line in mg m-3, left Y axis) and fractionated Chl-a (as stacked bars in % of total Chl-a, right Y axis) in the North Aegean (A1-A3), South Aegean (A5-A12), Levantine (L2-L4) and Ionian (I1-I11) seas in (a) spring (March-April) and (b) summer (August-September). The means of depth-averaged values were derived from integrated values divided by the integration depth 0-150 m per sampling station.

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Fig. 5. Vertical distribution of total (>0.2 μm) and fractionated Chl-a (mg m-3) in the N and S Aegean, Levantine and Ionian seas in spring (March-April). Go to Suppl. material for summer (AugustSeptember).

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Fig. 6. Spatial distribution of total (>0.2 μm) primary production (PP) rates (as solid line in mg C m-3 h-1, left Y axis) and fractionated PP rates (as stacked bars in % of total PP rates, right Y axis) in N and S Aegean, Levantine and Ionian seas during (a) spring (March-April) and (b) summer (August-September). The depth-averaged values were derived from integrated values divided by the integration depth 0-120 m per sampling station.

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Fig. 7. Vertical distribution of total (>0.2 μm) and fractionated primary production rates (mg C m-3 h-1) in N and S Aegean, Levantine and Ionian seas during spring (March-April). Go to Suppl. material for summer (August-September).

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Fig. 8. Abundance of phytoplankton cells per station (as solid line in cells L-1, left Y axis) and phytoplankton groups (as stacked bars in % of total phytoplankton abundance, right Y axis) in N and S Aegean, Levantine and Ionian seas during (a) spring (March-April) and (b) summer (August-September). The means of depth-averaged values were derived from integrated values divided by the integration

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depth 0-100 m per sampling station. Total abundance refers to the sum of all the phytoplankton groups. Stacked bars refer to the right Y axis.

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Fig. 9. Vertical distribution of the phytoplankton groups’ abundance (cells L-1) in N and S Aegean, Levantine and Ionian seas during spring (March-April). Go to Suppl. material for summer (AugustSeptember).

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Fig. 10. Similarity dendrogram (a) and MDS plot (b) deriving from hierarchical clustering with Bray-Curtis similarity index and group average on the basis of species abundance data with the factor of season.

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Athens, 01 October 2019

From: Dr. Ioanna Varkitzi, Hellenic Center for Marine Research (HCMR) Institute of Oceanography PO BOX 712 Anavyssos 19013 Athens, Greece tel: +30 22910 76387 fax: +30 22910 76347 e mail: [email protected]

Dear Professor Aristegui, All authors of the Manuscript DSR2_2018_172 with the title ‘’Phytoplankton dynamics and bloom formation in the oligotrophic Eastern Mediterranean: field studies in the Aegean, Levantine and Ionian seas’’ declare that there is no conflict of interest. With kind regards, Ioanna Varkitzi