Floriane Delpy
email: floriane.delpy@univ-tln.fr

Bruno Serranito

Jean-Louis Jamet

Gérald Grégori

Christophe Le Poupon

Dominique Jamet
email: d.jamet@univ-tln.fr

Pico-and Nanophytoplankton Dynamics in Two Coupled but Contrasting Coastal Bays in the NW Mediterranean Sea (France)

Keywords: Pico-and nanophytoplankton, Flow cytometry, Coastal environments, Mediterranean Sea, Spatiotemporal distribution

Due to its ecological context, the Toulon bay represents a site of scientific interest to study temporal plankton distribution, particularly pico-and nanophytoplankton dynamics. A monthly monitoring was performed during a two-year cycle (October 2013-December 2015) at two coupled sampling sites, referred to as Little and Large bays, which had different morphometric characteristics and human pressures. Flow cytometry analyses highlighted the fact that pico-and nanophytoplankton were more abundant in the eutrophic Little bay. Furthermore, it evidenced two community structures across the Toulon bays: at times, a co-dominance of picoeukaryotes, nanoeukaryotes, Synechococcus 1-like cells and Prochlorococcus-like cells was found, and at other times, a Synechococcus 1like dominated community existed. The alternation of one structure or the other can be explained by a combined action of temperature regime, nutrient conditions and degree of contamination. This study showed that pico-and nanophytoplankton dynamics were mainly driven by temperature in both sites, as in other temperate Mediterranean regions. Thus, the community was mainly composed of picoeukaryotes and Prochlorococcus-like cells in the winter (< 15 °C), while it was dominated by Synechococcus 1-like cells in the summer (> 20 °C). Additionally, the multiple human stressors in the Little bay seemed to affect the increase in abundance of Synechococcus 1-like cells as they were preferentially observed in the Large bay.

Introduction

Picophytoplankton (size = 0.2-2 μm) and nanophytoplankton (size = 2-20 μm) constitute an important component of aquatic ecosystems. Pico-and nanophytoplankton contribute up to 43-50% and 20-32%, respectively, of total primary production (~0.27 g C m -2 d -1 ) in the Bintermittently blooming watersô f the Mediterranean Sea [START_REF] Uitz | Estimates of phytoplankton class-specific and total primary production in the Mediterranean Sea from satellite ocean color observations[END_REF]. Picophytoplankton are composed of drifting cells, both prokaryotic and eukaryotic ones. Throughout the world's oceans, the most abundant autotrophic cells are often the smallest ones, such as the cyanobacteria Prochlorococcus and Synechococcus (< 1 μm in size), followed by pico-and nanoeukaryotes especially in coastal eutrophic areas [START_REF] Partensky | Differential distribution and ecology of Prochlorococcus and Synechococcus in ocean waters. A review[END_REF][START_REF] Siokou-Frangou | Plankton in the open Mediterranean Sea: A review[END_REF][START_REF] Bec | Distribution of picophytoplankton and nanophytoplankton along an anthropogenic eutrophication gradient in French Mediterranean coastal lagoons[END_REF]. For instance, in the disturbed bay of Marseille (NW Mediterranean Sea), cyanobacteria, pico-and nanoeukaryotes have reached maximum abundances of 7 × 10 4 , 3 × 10 4 and 9 × 10 3 cells mL -1 [START_REF] Grégori | Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry[END_REF]. Pico-and nanophytoplankton constitute an integral compartment at the basis of aquatic food webs, contributing to the microbial loop and, to a lesser extent, to the transfer of energy and matter towards higher trophic levels [START_REF] Stockner | Phototrophic picoplankton: An overview from marine and freshwater ecosystems[END_REF][START_REF] Fogg | Some comments on picoplankton and its importance in the pelagic ecosystem[END_REF]. The increase in biomass of pico-and nanophytoplankton can significantly contribute to the total particulate organic carbon (POC) export fluxes (up to 2-13% for Synechococcus, 1-20% for Prochlorococcus and 6-43% for nanoeukaryotes), especially according to sudden environmental forcing [START_REF] Lomas | Evidence for aggregation and export of cyanobacteria and nano-eukaryotes from the Sargasso Sea euphotic zone[END_REF]. Furthermore, the size spectrum and species composition of phytoplankton have been identified as indicators of the trophic status in aquatic ecosystems [START_REF] Bell | The contribution of picophytoplankton in marine and freshwater systems of different trophic status and depth[END_REF][START_REF] Bosak | Phytoplankton size structure and species composition as an indicator of trophic status in transitional ecosystems: The case study of a Mediterranean fjord-like karstic bay[END_REF].

Pico-and nanophytoplankton populations exhibit an autofluorescence induced by their photosynthetic pigments (e.g. chlorophyll a, phycoerythrin, phycocyanin) that can be detected by analytical flow cytometry [START_REF] Yentsch | Flow cytometry and cell sorting: A technique for analysis and sorting of aquatic particles[END_REF][START_REF] Yentsch | Cytometry in the aquatic sciences[END_REF]. This method performs single-cell analysis at high frequency (up to several thousand cells are analyzed per second) and has been widely used to investigate the spatiotemporal distribution of these organisms [START_REF] Marie | Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I[END_REF][START_REF] Dubelaar | Flow cytometry as a tool for the study of phytoplankton[END_REF][START_REF] Veldhuis | Application of flow cytometry in marine phytoplankton research: Current application and future perspectives[END_REF]. As water samples are analyzed at the single cell level, each cluster of pico-or nanophytoplankton can be differentiated thanks to its optical properties (i.e. types and intensities of fluorescence).

Pico-and nanophytoplanktonic microorganisms are particularly sensitive to external forcing, inducing changes in composition, abundance, cell size and pigment content at an hourly scale [START_REF] Dugenne | Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: An insight into specific population growth rates using a single cell high frequency approach[END_REF][START_REF] Thyssen | Onset of the spring bloom in the northwestern Mediterranean Sea: Influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure[END_REF]. Under specific conditions, they can have faster cell cycles with higher growth rates. Thus, in the presence of warm and nutrient-poor waters, picophytoplankton is the main contributor to the total phytoplankton production in the Mediterranean Sea [START_REF] Agawin | Nutrient and temperature control of the contribution of picoplankton to phytoplankton biomass and production[END_REF]. The composition of pico-and nanophytoplankton communities is influenced by fundamental physico-chemical factors that are characteristic of their environments (e.g. temperature, salinity, nutrients) [START_REF] Moisan | Influences of temperature and nutrients on Synechococcus abundance and biomass in the southern Mid-Atlantic Bight[END_REF][START_REF] Amorim | Controls of picophytoplankton abundance and composition in a highly dynamic marine system, the Northern Alboran Sea (Western Mediterranean)[END_REF]). These factors vary naturally (e.g. depending on the season, rainfall, wind) or can be influenced by human pressures (e.g. massive inputs of freshwater from power plants, byproducts from wastewater treatment plants). Therefore, pico-and nanophytoplanktonic groups may be considered as good biosensors and even as good bioindicators of water quality, especially directly or indirectly in coastal ecosystems affected by various human activities [START_REF] Grégori | Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry[END_REF][START_REF] Caroppo | Impact of heavy metals and PCBs on marine picoplankton[END_REF].

Reports of studies in the Northwestern part of the Mediterranean Sea, an area particularly influenced by anthropogenic constraints [START_REF] Karydis | Eutrophication and environmental policy in the Mediterranean Sea: A review[END_REF], are still scarce in the literature due to the lack of ultraplankton monitoring compared to other oceanic regions [START_REF] Denis | Short-term variations of the vertical distribution of cyanobacteria in the open Mediterranean Sea[END_REF][START_REF] Grégori | Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry[END_REF][START_REF] Goffart | Drivers of the winter-spring phytoplankton bloom in a pristine NW Mediterranean site, the bay of Calvi (Corsica): A long-term study (1979-2011)[END_REF]. On the French Mediterranean coast, the Toulon bay represents a site of special scientific interest in the Marine Strategy Framework Directive (MSFD, European Commission) and in the French national network RESOMAR (REseaux des Stations et Observatoires MARins) [START_REF] Serranito | Proposition of indicators of anthropogenic pressure in the Bay of Toulon (Mediterranean Sea) based on zooplankton time-series[END_REF]. Its functioning is determined by typical climatic conditions observed in this temperate Mediterranean region, with large intra-annual variations of temperature. Influenced by inputs from Toulon city (ca. 170,000 inhabitants), this site is composed of two systems known as Little bay and Large bay, which are separated by a breakwater built in 1882. Previous studies highlighted that both sites are affected by various human pressures. The Little bay is a semi-enclosed ecosystem affected by many human activities (i.e. commercial and military harbors, industries, shipyard), as well as by natural inputs from the Las River and the Large bay [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon Bay (France)[END_REF][START_REF] Pougnet | Sources and historical record of tin and butyl-tin species in a Mediterranean bay (Toulon Bay, France)[END_REF]. Conversely, the Large bay waters are influenced by the North current and, to a lesser extent, by an outfall pipe of a wastewater treatment plant and occasional freshwater inputs from the Eygoutier River [START_REF] Jouan | Modélisation de la Rade de Toulon: test méthodologique[END_REF][START_REF] Nicolau | Transfer of nutrients and labile metals from the continent to the sea by a small Mediterranean river[END_REF]. Some planktonic compartments observed in the Toulon bays, such as microphytoplankton and zooplankton, have been the subject of many studies [START_REF] Jamet | The zooplankton community in bays of Toulon area (northwest Mediterranean Sea, France)[END_REF][START_REF] Rossi | Structure and succession of plankton communities in two Mediterranean neighbouring coastal ecosystems (Toulon Bays, France)[END_REF][START_REF] Serranito | Proposition of indicators of anthropogenic pressure in the Bay of Toulon (Mediterranean Sea) based on zooplankton time-series[END_REF]. These communities appeared to be more abundant but less diversified in the Little bay than in the Large bay. Microphytoplankton was investigated and blooms evidenced in the area had a typical pattern of temperate marine regions, reaching their highest densities in the spring and occasionally in the autumn [START_REF] Rossi | Écologie des communautés planctoniques méditerranéennes et étude des métaux lourds (Cuivre, Plomb, Cadmium) dans différents compartiments de deux écosystèmes côtiers[END_REF]. Some phytotoxic species, such as Alexandrium and Dinophysis, were regularly observed [START_REF] Belin | Toxic and harmful phytoplankton species on the French coast from 1984 to[END_REF][START_REF] Joanny | Qualité du milieu marin littoral[END_REF]). There is no information available concerning ultraphytoplankton (cell size < 10 μm; [START_REF] Strickland | Phytoplankton and marine primary production[END_REF]. In this context, the present work complements previous studies, by focusing on the understudied pico-and nanophytoplankton compartments. The main objectives are (a) to evaluate how m u l t i p l e h u m a n s t r e s s o r s i nf l u e nc e d p i c o-a n d nanophytoplankton communities in coastal environments and (b) to identify which main environmental variables drove their phenology. Spatial and temporal variabilities of the picoand nanophytoplankton communities are expected in relation to the different human pressures observed in coastal areas and to large temperature variations typical of temperate regions.

Material and Methods

Study Sites

Located on the French Mediterranean coast (43°05′ N/05°55′ E), Toulon bay consists of two contrasting systems, the Little bay and the Large bay, separated by an artificial breakwater 1.4 km long and 3 m wide (Fig. 1). The Little bay is a semienclosed basin of 9.8 km 2 (mean depth of 15 m) [START_REF] Dang | Sedimentary dynamics of coastal organic matter: An assessment of the porewater size/ reactivity model by spectroscopic techniques[END_REF], whereas the Large bay is more open to the sea (42 km 2 , 5 to 30 m deep). These two sites only communicate by two passes: a small one at the north of the breakwater (30 m wide, 10 m deep) and a larger pass at the south (~1 km wide, maximum depth of 30 m) which constitutes the shipping channel.

They are submitted to the same typical climatic conditions of temperate Mediterranean regions, with two prevailing winds, i.e. the Mistral (W-NW) and a South-East wind. Hydrodynamic models of the Toulon bay showed that water circulation is directly driven by wind events inducing sediment resuspension [START_REF] Jouan | Modélisation de la Rade de Toulon: test méthodologique[END_REF][START_REF] Dufresne | Wind-forced circulation model and water exchanges through the channel in the Bay of Toulon[END_REF]. Without any wind, water circulation is much less active, since then only the constant North current generates a cyclonic circulation of the order of a few centimetres per second [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon Bay (France)[END_REF][START_REF] Dufresne | Wind-forced circulation model and water exchanges through the channel in the Bay of Toulon[END_REF]. The water residence time was from 3 to 6 days in the Little bay and about 1-2 days in the Large bay [START_REF] Jouan | Modélisation de la Rade de Toulon: test méthodologique[END_REF][START_REF] Duffa | An operational model to simulate post-accidental radionuclide transfers in Toulon marine area: Preliminary development[END_REF].

A monthly sampling was carried out from October 2013 to December 2015 at two stations: one in the Little bay (LiB, 43°06′30″ N/05°55′00″ E, 12 m deep) and one in the Large bay (LaB, 43°05′45″ N/05°56′40″ E, 17 m deep) (Fig. 1). All samples were collected between 08:00 and 10:00 h.

Environmental Parameters

Temperature, salinity (conductivity) and dissolved oxygen concentration were measured in the Little and Large bays at 3 and 10 m depths with a calibrated multiparametric probe (Hydrolab-Quanta). Concentrations of nitrite/nitrate (N-NO x -= NO 2 -+ NO 3 -) and reactive phosphorus (P-PO 4 3-) were also measured in 2015. Water samples were collected at 3 and 10 m depths in both sites, with a 10-L Niskin bottle, then transferred into 50-mL polypropylene centrifuge tubes and immediately frozen at -20 °C until further analysis [START_REF] Clementson | The effect of frozen storage of open-ocean seawater samples on the concentration of dissolved phosphate and nitrate[END_REF][START_REF] Dore | Freezing as a method of sample preservation for the analysis of dissolved inorganic nutrients in seawater[END_REF]. All reagents were at least of analytical grade. Reagent solutions were prepared in distilled-deionized water (Millipore, Milli-Q system) and used as diluent for all reagents and to establish the baseline. Nitrite/nitrate analyses (N-NO x -thereafter) were performed using a continuous flow Technicon Autoanalyzer II system, following the method by [START_REF] Tréguer | Manuel d'analyse des sels nutritifs dans l'eau de mer (utilisation de l'AutoAnalyzer II Technicon®)[END_REF]. These autoanalyses have a detection limit of 0.03 μM and an approximate analytical precision of ± 0.3%. Reactive phosphorus in seawater determination is based on the method by [START_REF] Murphy | A modified single solution method for the determination of phosphate in natural waters[END_REF], with modifications of [START_REF] Strickland | A practical handbook of seawater analysis-Determination of reactive phosphorus[END_REF]. The resulting complex gives a blue-colored solution, and its absorbance can be measured with a spectrophotometer (UV-1800, Shimadzu) at a wavelength of 885 nm in a 10-cm cell, against a blank of Milli-Q water. These analyses have a detection limit of 0.02 μM and an approximate analytical precision of ± 0.5%.

Pico-and Nanophytoplankton

Sampling

To determine the abundance of pico-and nanophytoplankton, water samples were collected with a 10-L Niskin bottle at 3 and 10 m depths in both sites (LiB and LaB). Samples were first filtered through a 90-μm mesh to eliminate grazers and macroparticles (i.e. debris). Sub-samples of 4.5 mL were then immediately fixed with a glutaraldehyde solution (final concentration at 0.25%), incubated for 15 min in the dark and flash-frozen in liquid nitrogen. These sub-samples were thawed at 37 °C just prior to analysis by flow cytometry.

Flow Cytometry Analyses

Flow cytometry analyses were performed using an Accuri C6 flow cytometer (BD Biosciences) equipped with 488 and 640 nm laser beams. Ultrapure water filtered through a 0.2-μm filter was used as the sheath fluid. For each particle analyzed, two light scattering intensities were collected: the forward scattered light (FSC) is related to the size of the particles, while the side scattered light (SSC) is related to their shape and inner structure. Three natural fluorescence intensities induced by the 488 nm laser beam were recorded. The flow cytometer measured red fluorescence (FL3) mainly emitted by chlorophyll (> 670 nm), orange fluorescence (FL2) mainly resulting from phycoerythrin emission (585 ± 20 nm) and green fluorescence (FL1) (530 ± 15 nm). The FL4 far red fluorescence (675/25 nm) is induced by the 640-nm red laser beam. It is worth noting that the abundance of the smallest cells, especially cyanobacteria, can be underestimated with a BD Accuri C6 because of their low content of photosynthetic pigments and only partially solved from background noise [START_REF] Ribeiro | Estimating microbial populations by flow cytometry: Comparison between instruments[END_REF]. Beads of 2 μm diameter (Fluoresbrite YG, Polysciences) were periodically added to the samples as an internal standard. All data were collected on a log scale using CFlowPlus software (BD Biosciences). A total of 500 μL sub-samples were analyzed at a flow rate of 60 μL min -1 . Data acquisition was triggered using the red fluorescence signal to focus on autofluorescent particles containing chlorophyll a (i.e. pico-and nanophytoplankton). The optical resolution of the various clusters was based on their ranges of light scattering and fluorescence intensities using two-dimensional data displays called cytograms [START_REF] Olson | Phytoplankton analysis using flow cytometry[END_REF][START_REF] Marie | Enumeration of phytoplankton, bacteria, and viruses in marine samples[END_REF][START_REF] Marie | An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples[END_REF]. Median values were used to describe the optical characteristics of each group.

Statistical Analyses

As data were not normally distributed (Shapiro-Wilk test), non-parametric Mann-Whitney U tests (MWU thereafter; Mann and Whitney 1947) were used to estimate the effect of sampling sites (depth or station) on environmental parameters (temperature, salinity, dissolved oxygen, N-NO x -and P-PO 4 3-concentrations) and on communities studied (pico-and nanophytoplankton abundance, fluorescence intensities). Sampling sites were considered independent of each other. Therefore, as no significant difference was recorded between 3 and 10 m d e p t h s ( p > 0 . 0 5 ) , a b i o t i c d a t a , p i c o -a n d nanophytoplankton abundance and fluorescence intensities were averaged. These statistical analyses were performed using Statistica 7.1 (StatSoft, Inc.). The relationships between the temporal variations observed in the Little bay and those observed in the Large bay were examined for total pico-and nanophytoplankton abundance and for each cluster using Pearson correlation analyses (R software).

A PERmutational Multivariate ANalysis Of VAriance (PERMANOVA) was conducted on Bray-Curtis distances to evaluate the impact of the site (LiB and LaB), the season and the year on the structure of pico-and nanophytoplankton communities, using the BVegan^package of R software [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. For each site, the linear relationship between pico-and nanophytoplankton groups and environmental conditions was evaluated on data from 2015, using constrained analysis (Redundancy Analysis) on an exploratory matrix (standardized abiotic data) and response data (logtransformed abundance). The evaluation of global analysis and factor significance was performed according to stepwise model selection using a 1000 permutation test (BOrdistep^function from the BVegan^package of R software). Results were summarized in triplot ordination and represented in scaling 2 so that the angles of the different vectors corresponded to an approximation of the correlations observed between the variables. Finally, two-dimensional surface plots were drawn to show the effects of temperature and salinity on the abundance of the pico-and nanophytoplankton groups using pooled datasets of the Little and Large bays. They were then summarized by a global analysis on the total pico-and nanophytoplankton assemblage. These kriging analyses were performed with a linear variogram model using Surfer 11 (Golden Software).

Results

Hydrological Parameters

Seasonal variations of the hydrological variables, i.e. temperature, salinity, dissolved oxygen, N-NO x -and P-PO 4 3concentrations, are displayed in Fig. 2. Similar ranges of temperature were observed in the Little bay (LiB) and Large bay (LaB): 13.1 to 23.1 °C and 12.9 to 23.0 °C, respectively (Fig. 2a). Maximal values were recorded in the summer or early autumn and minimal ones in the winter, corresponding to typical seasonal variations observed in the temperate Mediterranean areas. Nevertheless, a delay in the warm season was evidenced, with the highest temperatures (> 22 °C) observed from September to October 2014 and from June to August 2015. The coldest period (< 13 °C) only lasted one month in 2014 (February) and three months in 2015 (January to March).

Salinity was similar to that observed in the coastal areas of the Northwestern Mediterranean basin, with mean values of 37.7 ± 1.2 in LiB and 37.8 ± 1.1 in LaB (Fig. 2b). For both sites, exceptional highest values (~39.7) were recorded in October 2014, whereas the lowest ones (~35.6) were observed in February-March 2014. No data was recorded from May to July 2015 because of a dysfunction of the sensor.

Dissolved oxygen concentrations varied from 5.3 to 9.1 mg L -1 with minimal values seen between September 2014 and January 2015 (Fig. 2c). Oxygen concentrations were significantly different between the two sites,with highervaluesin LaB(7.6 ± 0.9 mgL -1 ) than in LiB (7.3 ± 0.8 mg L -1 ) (MWU, p < 0.05).

Nutrients were not analyzed in the first year of this study. However, N-NO x -and P-PO 4 3-concentrations were measured in 2015. N-NO x -concentrations had major variations (0.4 to 3.2 μM) with maximal values (> 1 μM in LiB and > 2 μM in LaB) displayed in March, September and November 2015 (Fig. 2d). As for dissolved oxygen, N-NO x -concentrations were significantly higher in LaB (1.2 ± 0.9 μM) than in LiB (0.7 ± 0.5 μM) (MWU, p < 0.05).

Considering P-PO 4 3-concentration, no significant difference in the average was highlighted between the two sites (MWU, p > 0.05) (Fig. 2e). Nevertheless, LiB exhibited two lower values (< 0.05 μM) and five upper values (> 0.08 μM), whereas LaB displayed five lower values (< 0.02 μM) and three upper values (twice about 0.08 μM and once > 0.1 μM). The median values illustrate more clearly the differences between the sites with 0.07 μM for LiB and 0.05 μM for LaB, showing that LiB is generally more concentrated in P-PO 4 3-during the period studied.

Time Series of Pico-and Nanophytoplankton Groups as Defined by Flow Cytometry

Five different clusters of pico-and nanophytoplankton were discriminated by flow cytometry thanks to their optical properties: Synechococcus-like (SYN), Prochlorococcus-like (PROCHL), picoeukaryotes (PICOEuk), nanoeukaryotes (NANOEuk) and Cryptophyceae (CRYPTO) (Fig. 3). As we did not include any molecular biology approach to identify the species present in the area, we used the term Blike^to characterize cyanobacteria. Typical flow cytometric signatures based on both light scattering and fluorescence intensities were used to define each cluster [START_REF] Olson | Phytoplankton analysis using flow cytometry[END_REF][START_REF] Marie | Enumeration of phytoplankton, bacteria, and viruses in marine samples[END_REF]). The analysis of these signatures showed that Synechococcuslike was composed of two sub-clusters defined by different optical characteristics: Synechococcus 1-like (SYN1) and Synechococcus 2-like (SYN2). Cyanobacteria (PROCHL and SYN) were the smallest cells (FSC < 11,000), followed by PICOEuk (~43,000), NANOEuk (~413,000) and CRYPTO (~913,000). This order was confirmed with the Pico-and nanophytoplankton abundance in the Toulon bays is displayed in Fig. 4. From October 2013 to December 2015, total abundance showed significant positive correlation between the two sites as shown byPearson correlation tests (r = 0.42, p = 0.02) (Fig. 4a). Comparable timing of maximal values was observed in November 2013 (51,996 cells mL -1 in LiB vs. 43,056 cells mL -1 in LaB), in February 2014 (34,493 cells mL -1 in LiB vs. 36,631 cells mL -1 in LaB), in June 2014 (42,689 cells mL -1 in LiB vs. 39,196 cells mL -1 in LaB) and in February 2015 (50,629 cells mL -1 in LiB vs. 37,615 cells mL -1 in LaB). Nevertheless, in autumn 2014 and spring-summer 2015, three peaks occurred slightly later in LiB (October-November 2014, May and August 2015) than in LaB (September-October 2014, April and July 2015). Furthermore, the range of total abundance was shifted towards higher values in LiB which displayed one minimum value (< 20,000 cells mL -1 ) and six maximum ones (> 40,000 cells mL -1 ), than in LaB that exhibited eight minimum values and only four maximum ones.

Strong positive correlations were also highlighted between the temporal variations observed in LiB and LaB for PROCHL, SYN2, PICOEuk and NANOEuk (Pearson correlation tests, r = 0.68, r = 0.74, r = 0.62, r = 0.51, respectively, with p < 0.01). At both sites, the lowest abundance of PROCHL occurred in the summer (1829 ± 768 cells mL -1 in LiB vs. 529 ± 354 cells mL -1 in LaB) and the highest ones in late autumn-winter (5160 ± 2901 cells mL -1 in LiB vs. 3348 ± 1749 cells mL -1 in LaB) (Fig. 4c). The abundance of SYN2 did not exhibit a clear seasonal pattern with maximal values recorded in September 2014 (659 cells mL -1 in LiB vs. 435 cells mL -1 in LaB) (Fig. 4d). Concerning PICOEuk, the highest abundance was observed in February 2014 (13,630 cells mL -1 in LiB vs. 14,711 cells mL -1 in LaB) and in February 2015 (29,602 cells mL -1 in LiB vs. 19,645 cells mL -1 in LaB) (Fig. 4e). It is worth noting that two autumnal peaks also occurred in LiB in November 2013 (26,978 ± 1448 cells mL -1 ) and in November 2014 (16,966 ± 6771 cells mL -1 ). NANOEuk were particularly abundant in June and October (Fig. 4f).

Conversely, an absence of correlation was identified between the two sites for the two remaining groups: SYN1 and CRYPTO (MWU, p > 0.05). The highest abundance of SYN1 (defined as > 30,000 cells mL -1 ) was recorded in September-October 2014 (39,679 ± 4953 cells mL -1 ) and in April 2015 (42,917 ± 1079 cells mL -1 ) in LaB, while a single peak occurred in July-August 2015 (32,396 ± 2021 cells mL -1 ) in LiB (Fig. 4b). Maximal values of CRYPTO were found in summer-early autumn in LiB, whereas no clear seasonal pattern was detected in LaB (Fig. 4g).

The differences in the abundance of pico-and nanophytoplankton between the two sites were confirmed as shown in Fig. 5 . Total abundance of pico-and nanophytoplankton were significantly different between the two sites studied, with a higher median value in LiB (31,280 cells mL -1 ) than in LaB (25,122 cells mL -1 ) (MWU, p < 0.05) (Fig. 5a). A similar spatial distribution was observed for almost all groups: PROCHL (2892 cells mL -1 in LiB vs. 1668 cells mL -1 in LaB), PICOEuk (10,239 cells mL -1 in LiB vs. 2930 cells mL -1 in LaB), SYN2 (181 cells mL -1 in LiB vs. 142 cells mL -1 in LaB), NANOEuk (4424 cells mL -1 in LiB vs. 2078 cells mL -1 in LaB) and CRYPTO (552 cells mL -1 in LiB vs. 152 cells mL -1 in LaB) (p < 0.05) (Fig. 5c-g). Only SYN1 were significantly more abundant in LaB (16,732 cells mL -1 ) than in LiB (11,467 cells mL -1 ) (MWU, p < 0.05) (Fig. 5a).

Besides the abundance of cells, the fluorescence intensities of both photosynthetic pigments characterizing the populations of Toulon bay (chlorophyll a and phycoerythrin) were then investigated. Inter-annual variations of these features can reflect photoacclimation, blooms of small cells or a shift in species [START_REF] Durand | Phytoplankton population dynamics at the Bermuda Atlantic-Time-series station in the Sargasso Sea[END_REF][START_REF] Six | Prochlorococcus and Synechococcus have evolved different adaptive mechanisms to cope with light and UV stress[END_REF]. The intensity of chlorophyll a-related red fluorescence displayed clear seasonal variations for four groups: SYN1, PICOEuk, NANOEuk and CRYPTO (Fig. 6). The highest values were observed in the winter, whereas the lowest ones corresponded to summer periods. The same seasonal pattern was observed for the intensity of phycoerythrin-related orange fluorescence, which was characteristic of SYN1 and CRYPTO. It is worth noting that NANOEuk gave significantly higher red fluorescence values in LiB (237,462 ± 94,194) than in LaB (173,473 ± 71,721) (MWU, p < 0.05).

Driving Factors of the Pico-and Nanophytoplankton Structure

A PERMANOVA analysis was performed to determine the influence of the sites (LiB or LaB), the seasons and the years on the structure of the pico-and nanophytoplankton community (Table 1). The factor BSite^explained most of the variance (R 2 = 17.9%), followed by the factor BSeason( R 2 = 15.2%). The factor BYear^was slightly significant, at 3.3% of the variance. Two interactions involving the seasons were also significant: BSeason^and BYear^(8.1%) and BSite^and BSeason^(3.9%).

The relative contribution of pico-and nanophytoplankton groups highlighted similar seasonal patterns of abundance at both sites but with different intensities, as shown in Fig. 7. In LiB, SYN1 were predominant in the summer-autumn for both years (48.0 ± 17.4%), whereas the community was dominated by PICOEuk (42.8 ± 12.5%) and SYN1 (22.5 ± 12.2%) in the winter. In LaB, SYN1 dominated the community with 74.1 ±

LiB LaB

Total pico/nanophytoplankton Fig. 4 Temporal variations of abundance (cells mL -1 ) for total pico-and nanophytoplankton (a) and each identified cluster (b to g) in LiB and LaB. Data were averages of that obtained at depths of 3 and 10 m 14.8% in the summer-autumn, whereas their relative contribution dropped to 43.0 ± 21.9% in the winter. This drop correlated with an increase of PICOEuk contribution: 32.4 ± 16.9% in the winter vs. 11.4 ± 8.6% in the summer-autumn. At both sites, the relative contribution of CRYPTO was weak (< 2.5%).

Variability was observed between 2014 and 2015 at both sites. From the spring to the autumn, a strong growth of SYN1 population occurred in 2015 (48.9 ± 21.7% in 2014 vs. 67.4 ± 17.5% in 2015, values for pooled sites) to the detriment of PICOEuk (24.5 ± 12.2% in 2014 vs. 16.7 ± 11.4% in 2015), PROCHL (9.1 ± 5.7% in 2014 vs. 4.7 ± 4.5% in 2015) and NANOEuk (14.9 ± 7.3% in 2014 vs. 9.4 ± 6.8% in 2015). Conversely, in the winter, the relative contribution of SYN1 was not as large, either in LiB (28.3% in 2014LiB (28.3% in vs. 16.6% in 2015) ) or in LaB (45.7% in 2014LaB (45.7% in vs. 40.3% in 2015)).

Redundancy analyses (RDA) were then performed for both sites to identify the environmental factors driving the abundance of the various pico-and nanophytoplankton groups (Fig. 8). The axis significance test (ANOVA-like) identified axis 1 as the only significant one (50.5% of the total variance in LiB and 57.0% in LaB). Among the five environmental parameters considered in these analyses (i.e. temperature, salinity, dissolved oxygen, N-NO x -and P-PO 4 3-concentrations), three displayed a significant influence to explain the model tested for LiB, and these were represented in Fig. 8a. A negative correlation between temperature and N-NO x -concentration was observed. While SYN1 and CRYPTO were correlated positively with temperature and salinity, PROCHL and PICOEuk were significantly linked with N-NO x -concentration and inversely correlated with temperature. For LaB, only temperature and salinity had a significant influence (Fig. 8b). A seasonal signal was observed, with a negative correlation between temperature and most pico-or nanophytoplankton populations (i.e. PROCHL, CRYPTO and PICOEuk). Only SYN1 were correlated positively with temperature, but to a lesser extent. Temperature and salinity were identified as being significantly influential for both sites. These two fundamental factors serve as proxies for monitoring environmental changes as they and large phytoplankton) have already been investigated along long-term time series [START_REF] Jamet | The zooplankton community in bays of Toulon area (northwest Mediterranean Sea, France)[END_REF][START_REF] Rossi | Structure and succession of plankton communities in two Mediterranean neighbouring coastal ecosystems (Toulon Bays, France)[END_REF][START_REF] Serranito | Proposition of indicators of anthropogenic pressure in the Bay of Toulon (Mediterranean Sea) based on zooplankton time-series[END_REF], there has been no study devoted to the pico-and nanophytoplankton communities. Considered as good bio-indicators of water quality [START_REF] Grégori | Phytoplankton group dynamics in the Bay of Marseilles during a 2-year survey based on analytical flow cytometry[END_REF][START_REF] Caroppo | Impact of heavy metals and PCBs on marine picoplankton[END_REF], pico-and nanophytoplankton exhibited an important dichotomy in terms of abundance and composition depending on the sampling sites. Higher pico-and nanophytoplankton abundance was observed in the Little bay than in the Large bay, following the trends observed for other higher planktonic compartments [START_REF] Jamet | The zooplankton community in bays of Toulon area (northwest Mediterranean Sea, France)[END_REF][START_REF] Rossi | Structure and succession of plankton communities in two Mediterranean neighbouring coastal ecosystems (Toulon Bays, France)[END_REF][START_REF] Serranito | Proposition of indicators of anthropogenic pressure in the Bay of Toulon (Mediterranean Sea) based on zooplankton time-series[END_REF]. Concerning the composition, microphytoplankton and zooplankton communities were generally less diversified in the Little bay than in the Large bay [START_REF] Jamet | The zooplankton community in bays of Toulon area (northwest Mediterranean Sea, France)[END_REF][START_REF] Rossi | In situ heavy metals (copper, lead and cadmium) in different plankton compartments and suspended particulate matter in two coupled Mediterranean coastal ecosystems (Toulon Bay, France)[END_REF]. Pico-and nanophytoplankton exhibited in the Little bay a community represented by several co-dominant groups (i.e. Synechococcus 1-like, picoeukaryotes, nanoeukaryotes and Prochlorococcus-like), while in the Large bay, the community was largely dominated by Synechococcus 1-like.

Previous studies described the Little bay as being more eutrophic than the Large bay because of significantly higher nutrient and chlorophyll a concentrations [START_REF] Rossi | Écologie des communautés planctoniques méditerranéennes et étude des métaux lourds (Cuivre, Plomb, Cadmium) dans différents compartiments de deux écosystèmes côtiers[END_REF][START_REF] Rossi | Structure and succession of plankton communities in two Mediterranean neighbouring coastal ecosystems (Toulon Bays, France)[END_REF][START_REF] Bogé | Role of DOP on the alkaline phosphatase activity of size fractionated plankton in coastal waters in the NW Mediterranean Sea (Toulon Bay, France)[END_REF]. P-PO 4 3-concentration measured in this study confirmed this trend, even if values were close to the detection limit. As the Little bay is a shallow basin (mean depth of 15 m) with a high water residence time (3-6 days), phosphates can be released from sediments during resuspension phenomena induced by an intense maritime traffic (i.e. commercial and military harbors) and by occasional strong wind events. In the Large bay, waters are regularly mixed with offshore seawaters and exhibited lower P-PO 4 3concentrations. Furthermore, the predominance of Synechococcus 1-like in this site, which can contribute up to 35% of the total Pi uptake, could participate in these low P-PO 4 3-values [START_REF] Talarmin | Vertical partitioning of phosphate uptake among picoplankton groups in the low Pi Mediterranean Sea[END_REF]. The opposite situation was observed for nitrite/nitrate with higher concentrations in the Large bay than in the Little bay. In the Large bay, nitrate intakes come mainly from the Eygoutier River with a baseflow ranging from 1 to 10 mg L -1 depending on season [START_REF] Nicolau | Transfer of nutrients and labile metals from the continent to the sea by a small Mediterranean river[END_REF]). In the Little bay, N-NO x -concentration was identified as an explicative variable by redundancy analysis. This site knows regular phytoplankton blooms whose growth depends on a sufficient supply of nutrients [START_REF] Rossi | Écologie des communautés planctoniques méditerranéennes et étude des métaux lourds (Cuivre, Plomb, Cadmium) dans différents compartiments de deux écosystèmes côtiers[END_REF]. Pico-and nanoeukaryotes constituted the most successful groups in coastal eutrophic waters and were particularly abundant in this site [START_REF] Pan | Picophytoplankton, nanophytoplankton, heterotrophic bacteria and viruses in the Changjiang Estuary and adjacent coastal waters[END_REF][START_REF] Jyothibabu | Ecology and trophic preference of picoplankton and nanoplankton in the Gulf of Mannar and the Palk Bay, southeast coast of India[END_REF]. With respect to cyanobacteria, N-NO x -concentration had a positive correlation with Prochlorococcus-like cells. Šantić et al. (2011) highlighted that the abundance of Prochlorococcus can be influenced by nutrient availability, but as a consequence of the movement of water mass. Other environmental conditions, such as temperature, salinity, water turbidity and irradiance, could thus explain these spatiotemporal variations, leading to the presence of high abundances during periods of heavy rains and floods. Furthermore, numerous studies evidenced the intense contamination of the Little bay by organometallic (e.g. TBT, DBT and MBT), organic (e.g. HAP and PCB) and metallic (e.g. Hg, Cu, Pb, Zn and Cd) pollutants [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon Bay (France)[END_REF][START_REF] Tessier | Diagnostic de la contamination sédimentaire par les métaux/métalloïdes dans la rade de Toulon et mécanismes contrôlant leur mobilité[END_REF][START_REF] Pougnet | Sources and historical record of tin and butyl-tin species in a Mediterranean bay (Toulon Bay, France)[END_REF]. In surface water, [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF] highlighted clear decreasing gradients of dissolved copper and lead from the Little bay (7.9 to 121.0 nM Cu and 0.4 to 12.7 nM Pb) to the Large bay (2.4 to 16.0 nM Cu and 0.08 to 0.73 nM Pb). Phytoplankton resistance to metallic contaminants was s t r o n gl y r e l a t e d t o c e l l s i z e , h i gh l i g h t i n g t ha t picocyanobacteria were more sensitive than other functional groups [START_REF] Echeveste | Toxic thresholds of cadmium and lead to oceanic phytoplankton: Cell size and ocean bassin-dependant effect[END_REF]. In Toulon bays, pico-and nanophytoplankton community exhibited either a cod om i na n ce of p i co e uk ar y ot e s , n an oe u ka r y o t es , Synechococcus-like and Prochlorococcus-like cells in the most contaminated site (LiB), or a Synechococcus 1-like dominated community in the site near the open sea (LaB). Synechococcus can be actually impacted by copper with a major inhibition of its growth rate [START_REF] Debelius | Toxicity of copper in natural marine picoplankton populations[END_REF], 2 0 11 ) . C o c l et e t a l . ( 20 1 7 ) e x p o s ed p i c o -a n d nanophytoplankton collected in the less impacted area of Large bay to a trace metal cocktail (Cu, Pb and Zn). These laboratory experi ments reveal ed a shi ft from a Synechococcus-like dominated community towards a community such as that observed in Little bay, confirming the sensibility of this strain to metallic contaminants. These results were compared to the pico-and nanophytoplankton composition observed at the neighboring SOLEMIO-Marseille station (43°14′30″ N/05°17′30″ E), used as external reference. This station belongs to the SOMLIT (Service d'Observation en Milieu LITtoral, INSU-CNRS) French National Network. Its aims are to coordinate the observation activities (e.g. in situ sampling, measurements and analyses) in marine laboratories along the French coastlines and to assure the distribution of acquired data for the different parameters monitored by SOMLIT (T, S, O 2 , NH 4 , NO 3 , NO 2 , PO 4 , SiOH 4 , COP, NOP, MES, CHLa, δ 15 N, δ 13 C, pico-and nanoplankton) via its website (http://somlit.epoc.u-bordeaux1.fr/fr/). Located in the Northwestern Mediterranean basin (Marseille, France), t h e S O L E M I O s t a t i o n c o n s t i t u t e s a n e x t e r n a l Mediterranean coastal reference site located in the v i c i n i t y o f To u l o n . O v e r t h e w h o l e y e a r, t h e predominance of Synechococcus 1-like was intensified in the Marseille bay (mean value of ~23,737 cells mL -1 ) in comparison to the Large bay (~18,106 cells mL -1 ), whereas the other groups were significantly less abundant than in the Little bay (~3612 and 10,867 cells mL -1 for picoeukaryotes, ~1362 and 4501 cells mL -1 for n a n o e u k a r y o t e s , ~9 7 a n d 6 0 2 c e l l s m L -1 f o r Cryptophyceae, respectively, for the Marseille bay and t h e L i t t l e ba y ) ( d at a p r o v i d ed b y th e BS e r v i c e d ' Observation en Milieu Littoral, INSU-CNRS, Marseille^). These observations outline a major impact from the local context on the pico-and nanophytoplankton s t r u c t u r e a n d c o n f i r m t h e p r e p o n d e r a n c e o f Synechoccocus-like cells in the farther offshore environments. These cells are likely close to more open sea strains since genomic sequencing analyses have revealed the existence of coastal Synechococcus strains that are tolerant to metals, especially copper and iron [START_REF] Palenik | Genome sequence of Synechococcus CC9311: Insights into adaptation to a coastal environment[END_REF].

As highlighted by the PERMANOVA analysis, the differences in terms of composition between the two sites are explained by the two sites themselves. Thus, the different human pressures discriminating the sites mostly perturb the seasonal dynamics of Synechococcus 1-like cells and Cryptophyceae and confirm their greater sensitivity [START_REF] Debelius | Toxicity of copper in natural marine picoplankton populations[END_REF][START_REF] Debelius | Toxicity of copper, nickel and zinc to Synechococcus populations from the Strait of Gibraltar[END_REF][START_REF] Kirkham | Basin-scale distribution patterns of photosynthetic picoeukaryotes along an Atlantic Meridional Transect[END_REF]). However, strong positive correlations were evidenced between the connected Little and Large bays for the other groups (i.e. Prochlorococcus-like, picoeukaryotes, nanoeukaryotes and Synechococcus 2-like). Temperature and salinity, which were comparable in the two sites, were identified by our RDA analyses as environmental factors influencing the pico-and nanophytoplankton distribution. In the Toulon bays, pico-and nanophytoplankton community had two structures either with Synechococcus 1-like cells correlated to high temperature and high salinity or with picoeukaryotes/Prochlorococcus-like cells correlated to low temperature and low salinity. Synechococcus-like populations had two optimal temperatures corresponding to conditions observed in early spring and late autumn (~15 °C) for Synechococcus 1-like, and in the summer (> 21 °C) for Synechococcus 1-like and Synechococcus 2-like. The genetic diversity of marine Synechococcus is complex with several clades widely distributed in the Mediterranean Sea [START_REF] Mazard | Is the distribution of Prochlorococcus and Synechococcus ecotypes in the Mediterranean Sea affected by global warming?[END_REF]. [START_REF] Amorim | Controls of picophytoplankton abundance and composition in a highly dynamic marine system, the Northern Alboran Sea (Western Mediterranean)[END_REF] suggested that, in the Northern Alboran Sea (Spain, W Mediterranean), there were different ecotypes of Synechococcus which were adapted either to summer conditions or to winter ones. However, Synechococcus-like populations are known to further develop in the summer demonstrating a growth rate which was maximized by warm and stratified waters, as well as a limitation of nutrients [START_REF] Agawin | Growth and abundance of Synechococcus sp. in a Mediterranean Bay: Seasonality and relationship with temperature[END_REF][START_REF] Moisan | Influences of temperature and nutrients on Synechococcus abundance and biomass in the southern Mid-Atlantic Bight[END_REF]. During this time, Synechococcus-like cells would be too abundant for the grazing capacity of their predators, and so represent a major source of organic carbon and nutrients for coastal Mediterranean food webs [START_REF] Agawin | Growth and abundance of Synechococcus sp. in a Mediterranean Bay: Seasonality and relationship with temperature[END_REF]. Conversely, picoeukaryotes and Prochlorococcus-like cells were particularly abundant in the winter (i.e. < 15 °C). Marine picoeukaryotes could have a higher photosynthetic activity inducing a better use of light and allowing them to be more competitive at low temperature and low light intensity [START_REF] Somogyi | Winter bloom of picoeukaryotes in Hungary shallow turbid soda pans and the role of light and temperature[END_REF]. As for the Prochlorococcus-like cluster, their proliferation at temperature < 15 °C is unusual. At a global scale, their higher abundances were observed in warm waters as demonstrated by [START_REF] Flombaum | Present and future global distributions of the marine cyanobacteria Prochlorococcus and Synechococcus[END_REF]. Nevertheless, several studies reported abundances of 10 3 -10 4 cells mL -1 in the winter (temperature < 15 °C) in the Mediterranean Sea [START_REF] Vaulot | Winter presence of prochlorophytes in surface waters of the Northwestern Mediterranean Sea[END_REF][START_REF] Babić | Distribution and diversity of marine picocyanobacteria community: Targeting of Prochlorococcus ecotypes in winter conditions (southern Adriatic Sea)[END_REF][START_REF] Salhi | Seasonal variability of picoplankton under contrasting environments in northern Tunisian coasts, southwestern Mediterranean Sea[END_REF]). Prochlorococcus has even been detected within temperatures ranging from 6.33 to 26.93 °C in the Adriatic Sea (Šantić et al. 2011). Our datasets also showed that the largest cells (i.e. nanoeukaryotes and Cryptophyceae) were more abundant in warmer waters (> 19 °C).

Moreover, seasonal variations were observed on red and orange fluorescence intensities of some pico-and nanophytoplankton groups, namely: Synechococcus 1like, picoeukaryotes, nanoeukaryotes and Cryptophyceae. Red and orange fluorescence intensities are related to the amounts of two photosynthetic pigments: chlorophyll a and phycoerythrin, respectively. Maximum values were first observed in the autumn-winter, when irradiance is low, as phytoplanktonic cells need to produce more antennas for a better efficiency of the photon collection that is mandatory for photosynthesis. Then, fluorescence intensities reached minimum values in late spring-early summer when cells are developing and more irradiance is available, thus less pigments are needed. These seasonal changes of fluorescence intensities appeared to be inversely related to those of cell abundances, reflecting probable blooms of small cells in late spring-early summer [START_REF] Durand | Phytoplankton population dynamics at the Bermuda Atlantic-Time-series station in the Sargasso Sea[END_REF]. This decrease also illustrates a photoacclimation strategy to prevent any damage of the photosynthetic apparatus, particularly of the Photosystem II complex [START_REF] Six | Prochlorococcus and Synechococcus have evolved different adaptive mechanisms to cope with light and UV stress[END_REF][START_REF] Murphy | Photoinactivation of photosystem II in Prochlorococcus and Synechococcus[END_REF]. It is worth noting that nanoeukaryotes were the only group with a significant difference of fluorescence intensities between the two sites. The higher values observed in the Little bay can be explained by either a higher turbidity limiting the amount of light perceived by cells which synthetized in response more pigments, or by the presence of different species depending on the environmental conditions of each site.

Combining all these observations, we have demonstrated the specific situation of the Toulon bays. On the one hand, pico-and nanophytoplankton composition and dynamics were driven by temperature. On the other hand, the various contaminations reported in the literature and the nutrient conditions influenced by human pressures might deeply modulate the predominance of one or more clusters. Indeed, the redundancy analyses evidenced a negative correlation between Synechococcus 1-like and the other picoand nanophytoplankton groups in the Little bay. When environmental conditions were not suitable for the growth of a m aj o r S y n e c h o c o c c u s 1 -l i k e p o p u l a t i o n , Prochlorococcus-like cells reach their highest abundance. The opposition of these two cyanobacteria clusters reflects differences in life strategy, as well as ecological niches [START_REF] Partensky | Differential distribution and ecology of Prochlorococcus and Synechococcus in ocean waters. A review[END_REF][START_REF] Six | Prochlorococcus and Synechococcus have evolved different adaptive mechanisms to cope with light and UV stress[END_REF]. Likewise, pico-and nanoeukaryotes proliferate in the Little bay where Synechococcus 1-like was less abundant as they develop successfully in coastal eutrophic waters [START_REF] Pan | Picophytoplankton, nanophytoplankton, heterotrophic bacteria and viruses in the Changjiang Estuary and adjacent coastal waters[END_REF][START_REF] Jyothibabu | Ecology and trophic preference of picoplankton and nanoplankton in the Gulf of Mannar and the Palk Bay, southeast coast of India[END_REF].

Conclusion

The monitoring of Toulon bay contributes to an effort to survey marine environments through two French national networks (i.e. RESOMAR-REseaux des Stations et Observatoires MARins, SOMLIT-Service d'Observation en Milieu LITtoral-INSU CNRS) and a European one (i.e. MSFD-Marine Strategy Framework Directive-European Commission). This effort takes place within a harmonisation process of protocols for sampling, measurem e n t a n d a n a l y s i s t o o b t a i n e x p l o i t a b l e a n d intercomparable datasets. Multiplication of such lowfrequency monitoring are essential for understanding the natural dynamics of plankton communities and for assessing the evolution of marine ecosystems in the context of the global change and human pressures. Thus, in Toulon bay, we emphasized the typical presence of Synechococcus 1-like in summer and the unusual one of Prochlorococcus-like in winter. At spatial scale, the low abundance of Synechococcus 1-like in the site the most impacted by human activities is probably related to chronic exposures of pico-and nanophytoplankton to contaminants, such as trace metals. This Synechococcus strain could be used as a bioindicator of sudden environmental perturbations in this area, as demonstrated in laboratory experiments by [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. To this end, such monitoring could be completed by in situ automated flow cytometers capable of performing automated high frequency sampling and analysis (several times per hour) to track any significant sudden change in their abundance and/or their optical properties [START_REF] Dugenne | Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: An insight into specific population growth rates using a single cell high frequency approach[END_REF][START_REF] Thyssen | Onset of the spring bloom in the northwestern Mediterranean Sea: Influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure[END_REF]. The seasonal patterns pointed out by our study contribute to a better understanding of the pico-and nanophytoplankton role in the environments under multiple human stressors. Their monthly monitoring should then be continued in Toulon bay to supply the existing long-term dataset to t h e o t h e r p l a n k t o n c o m p a r t m e n t s ( i . e . microphytoplankton and zooplankton), allowing to refine knowledge and predictions of coastal ecosystem functioning in the context of the global warming in the Mediterranean Sea.

Fig. 1

 1 Fig. 1 Map of Toulon bay, and location of the two sampled stations: LiB (Little bay) and LaB (Large bay)

Fig. 2

 2 Fig. 2 Temporal variations of temperature (°C) (a), salinity (b), dissolved oxygen (mg L -1 ) (c), N-NO x -(μM) (d) and P-PO 4 3-(μM) (e) concentrations for LiB and LaB. Data were averages of that obtained at depths of 3 and 10 m

Fig. 3

 3 Fig. 3 Cytograms of orange (FL2) vs. red (FL3) fluorescence intensities in parts a and c. Cytograms of size (FSC) vs. red fluorescence intensity (FL3) in parts b and d. Data corresponded to two natural water samples collected in December 2014 in LiB (a and b) and LaB (c and d) at a depth

Fig. 5 Fig. 6

 56 Fig. 5 Abundance (cells mL -1 ) of total pico-and nanophytoplankton (a) and of each identified cluster (b to g) in LiB and LaB. The central boxes show the data between the 25th and the 75th percentiles with the median

  population (Fig.9a-f). SYN1 had two ranges of optimal values (~15.5 °C and > 21 °C), whereas a reverse pattern (< 15 °C and 16 to 20 °C) was observed for PROCHL and PICOEuk. SYN2 and CRYPTO reached their highest abundance during warm periods (> 19 °C). Likewise, NANOEuk were particularly observed at 18.5, 20 and > 22 °C. SYN1, PROCHL, PICOEuk and NANOEuk were present in high abundance whatever the salinity, while SYN2 and CRYPTO seemed to prefer a salinity of about 38. The pooling of previous two-dimensional surface plots highlighted a clear succession on the structure of the pico-and nanophytoplankton assemblage, depending on temperature (Fig.9g). Below 14 °C, PICOEuk and PROCHL were particularly abundant. An inverse trend was observed at 15 °C with a community dominated by SYN1. Between 16 and 19 °C, PICOEuk and PROCHL reappeared, this time accompanied by some NANOEuk. Above 20 °C, the pico-and nanophytoplankton community was mainly composed of SYN1, joined SYN2, NANOEuk and CRYPTO above 21 °C.

Fig. 8 Fig. 7 Fig. 9

 879 Fig. 8 Triplots of redundancy analyses in scaling 2 for LiB (a) and LaB (b). Explanatory variables (environmental parameters) were represented by solid vectors and response variables (pico-and nanophytoplankton

Table 1

 1

	Results of PERMANOVA analysis carried		Df	Sum of	Mean sum	F.Model	R 2	p value
	out on Bray-Curtis distances for			squares	of square
	the structure of the pico-and
	nanophytoplankton communities	Site	1	1.621	1.621	32.965	0.179	< 0.001	***
	in relation to the site, the season	Season	3	1.376	0.459	9.329	0.152	< 0.001	***
	and the year. Df, degrees of freedom; F.Model, F value by permutation	Year Site:Season	1 3	0.299 0.351	0.299 0.117	6.071 2.376	0.033 0.039	0.003 0.018	** *
		Season:Year	3	0.738	0.246	5.002	0.081	< 0.001	***
		Site:Year	1	0.071	0.071	1.449	0.008	0.228
		Residuals	94	4.622	0.049	0.509
		Total	106	9.078			1.000

* 0.01 < p < 0.05 ** 0.001 < p < 0.01 *** p < 0.001

Acknowledgements This research was financed by in-house funding from the EBMA (Marine Ecology and Biology) research team (PROTEE laboratory EA 3819, University of Toulon, France). We are grateful to the Regional Flow Cytometry Platform for Microbiology (PRECYM) of the Mediterranean Institute of Oceanography (MIO UM 110, Aix-Marseille University, France) for their support concerning the arrangement of our flow cytometry protocols and sample preparation. The authors thank Jingwei YANG (PhD) for help in implementing flow cytometry in the laboratory, as well as the two anonymous reviewers that provided helpful comments on the earlier draft of this manuscript.

Funding This research was financed by in-house funding from the EBMA (Marine Ecology and Biology) research team (PROTEE laboratory EA 3819, University of Toulon, France).