Layout 1 ARTICLE Introduction Mediterranean micro-tidal environments are transitional sys- tems characterized by a tidal range <2m, where salt-wedge intru- sion along the final stretch of rivers plays an important role in shaping biological communities (Boothroyd, 1978; Davies, 1964; Kiriaki et al., 2010; van der Tuin, 1991). The extent of this process depends on several geomorphological and physical factors, such as, for example, the river mouth configuration or man-made bar- rier-like dams (Haralambidou et al., 2010; Lichter et al., 2011). From a geomorphological point of view, a fennel-shaped river mouth can be defined as an estuary, in which the mixing between seawater and freshwater generates strong salinity gradients and the presence of a typical brackish water layer (Perillo, 1995; Pou- los et al., 1993; Valle-Levinson, 2010). These systems are partic- ularly vulnerable to climate change, since strong seasonality and variability in precipitation produce large inter-annual fluctuations in river flow and water mass exchange with the sea (Gasith and Resh, 1999; Kennish, 2002, 2021; Newman et al., 2005; Ustin et al., 2014; Verri et al., 2018;). In the last decade, in fact, a reduction of net rainfall of ~33% of the annual mean was reported for southern Italian regions, mostly due to a reduced con- tribution of winter rainfall (Caloiero et al., 2021; Polemio and Casarano 2004, 2008). The effects of the drought period and human interventions like land reclamation, construction of river embankments, and water extraction activities significantly con- tributed to altering the ecological profiles of these areas (Dugdale et al., 2012; Hearn and Robson, 2001; Kennish, 2021; Newman et al., 2005; Richard et al., 2018;). The combined effect of salt- water intrusion and river discharge produces significant changes in nutrient loads, since the extent to which nutrients are retained in rivers and then made available during the biomass development Phytoplankton community as monitoring tool in the terminal stretch of a micro-tidal estuary facing the Tyrrhenian Sea Francesco Bolinesi1, Emanuela Serino1, Angelo Carotenuto1, Silvia Fanina1 and Olga Mangoni1,2 1Department of Biology, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, Naples; 2NBFC, National Biodiversity Future Center, Palermo, Italy ABSTRACT Mediterranean coastal systems, particularly those characterized by river mouths, have played an important role in the history of human civilization, thanks to unique species assemblages, a high biodiversity, and complex ecological interactions. The overlapping of multiple cross-scale effects, triggered by the ongoing climate change, makes these systems particularly important sites for ecological studies. Here, we have investigated the phytoplankton community structure in the terminal stretch of the Sele River (Tyrrhenian Sea) under two different hydrological regimes: i) with river mouth occluded by sediments preventing water exchange with the sea; ii) with river mouth completely opened, which allowed the maximum water mass exchanges between the river and the sea. The strong variations in physical-chemical properties of the water column, the point-distribution of total phy- toplankton biomass, and the dominance of cryptophytes and cyanophytes under occluded and opened river mouth, respec- tively, indicate the presence of strong allogenic pressures acting on the system. Our data suggest that the monitoring of phyto- plankton communities in the terminal stretch of micro-tidal estu- aries, could be a useful tool for studying the extent of climate change underway in Mediterranean coastal marine areas. Corresponding author: Olga Mangoni, Department of Biology, University of Naples Federico II, Complesso Universitario di Monte Sant’Angelo, via Cinthia 21, 80126, Naples, Italy. E-mail: olga.mangoni@unina.it Key words: anthropogenic pressure, chemotaxonomy, drought, Sele River, Tyrrhenian Sea. Authors’ contributions: all the authors made a substantive intellec- tual contribution. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work. Conflict of interest: the authors declare no potential conflict of in- terest. Funding: none. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Acknowledgments: the authors wish to thank the kind collabora- tion of the Natural Reserve Authority Sele-Tanagro, Fernando Guerra for providing and skippering the boat, “La Foce dei Tra- monti Camping” for logistic facilities, Matilde Di Somma and Erica Errico for their support in sampling activities. Received: 24 December 2022. Accepted: 3 May 2023. Publisher’s note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affil- iated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Advances in Oceanography and Limnology, 2023; 14:11070 DOI: 10.4081/aiol.2023.11070 This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0). Non -co mmerc ial us e o nly F. Bolinesi et al.44 seasons depends on the water retention time (Lancelot and Muy- laert, 2011; Lichter et al., 2011; Newman et al., 2005; Pinckney et al., 1999;). Many authors state that changes in river water regime from lotic to lentic are accompanied by an increase in al- logenic phytoplankton biomass (Lampert and Sommer, 1997; Reynolds, 1984, 1999, 2003; Reynolds and Descy, 1996; Waylett et al., 2013) generating blooms under optimal environmental con- ditions dominated by species able to grow at intermediate salinity as well as tolerant to large salinity fluctuations in brackish waters of terminal stretches (Jackson et al., 1987; Muylaert et al., 2009; Roubeix and Lancelot, 2008). Being passively transported by cur- rents, phytoplankton at the source of the river would require a very rapid replication time so that they can generate blooms. This occurs when net specific growth rates of species exceed the resi- dence time of the water (Lucas et al., 2009; Reynolds, 1984). Hence, phytoplankton represents a significant component of pri- mary production only in slow-moving rivers in lowland regions. The responses of the phytoplankton community to variations in water flow rate, nutrient loads, light availability, and temperature are species-specific and highly dependent on the whole environ- mental context, differing from time to time within each segment of an estuary (Artigas et al., 2014). Several studies report that di- atoms are the dominant phytoplankton group in rivers, with changes in size spectra composition and species associated with changes in nutrient concentration, turbidity, and flushing rate (Genkal 1997; Reylonds et al., 1994b). For example, Schuchardt and Schirmer, (1991) and Muylaert et al., (2000) report the dom- inance of diatoms in turbid estuaries throughout the year under high nutrient availability. Marshall et al., (2006) report the dom- inance of diatoms in spring in estuaries where turbidity is low, when light levels are relatively low and stratification does not occur. Among other groups, dinoflagellates and cyanobacteria are typically more abundant in summer during periods of low water discharge, long water residence time, minimal flushing rates, and low turbidity levels (Lancelot and Muylaert, 2011). Tavernini et al., (2011) report the dominance of diatoms, chlorophytes, and cyanophytes in the terminal stretch of the Po River, with changes in seasonal species assemblages showing an increase of crypto- phytes in late July and of cyanophytes at high water discharge rates, from the end of April to mid-June. Within the problematic framework described above, we have analyzed the phytoplankton community structure through the detection of diagnostic pigments (Ansotegui et al., 2001; Mangoni et al., 2017) in the final stretch of the Sele River (Tyrrhenian Sea) under two distinct hydrological regimes: during a severe drought period in July 2017, in which, for the first time in living memory, the mouth of the river was completely occluded by sediments, and in May 2021, in presence of a high water flow rate and a completely open river mouth. Our aim was to understand to what extent the strong environmental variations influenced the structure of the phytoplankton commu- nity in the terminal stretch of one of the most important river Southern Italy. Materials and Methods Study area The Sele River is the second largest river in southern Italy in terms of average water volume after the Volturno. It is lo- cated within a wide alluvial coastal plain, with a drainage basin of 3235 km2 and a solid flow of 500,000 m3 y-1 (Cocco et al., 1989). 64 km long and tributary of the Tyrrhenian Sea (Di Paola et al., 2014), it has the highest mean annual flow discharge among the rivers of Southern Italy (i.e., ~69.4 m3 s-1) (Magliulo et al., 2021). It has been an important site for ancient popula- tions, with the first archaeological settlements dating back to the second millennium B.C. (Ferrara et al., 2010). The climate of its basis is characterized by prolonged warm and dry sum- mers and wet and mild winters, with mean annual precipitation between 700 to 2000 mm, (average of 1180 mm) showing a marked spatial variation (Diodato et al., 2011). Over the past 150 years, the Sele estuary coastline has been affected by ero- sion. From 1870 to 1984, the coastline gradually receded, with the highest erosion rates occurring in the main areas around the river mouth. In the last 150 years, the Sele River and its catch- ment area have also been affected by increasing human activi- ties that exerted a significant influence on the evolution of the area. The construction of an artificial drainage channel (Canale di Bonifacimento) (Alberico et al., 2012a, b), with a dense net of artificial drainage channels and the presence of the Dam of Persano strongly enhanced coastal erosion and altered the river environmental conditions (Alberico et al., 2012a, 2012b; Arienzo et al., 2020; Pappone et al., 2011;). The Dam of Per- sano, located 16.2 km from the river mouth, built between 1929 and 1932, created a basin of 1.5 million m3, which affected the downstream sediment deposition causing the consequent retreat of the coast (D’Acunzi et al., 2008; Freeman et al., 2007; Mag- daleno et al., 2018). In most recent years, the significant geo- morphological alterations of the Sele estuary and the presence of pollutants and conspicuous loads of organic matter deriving from anthropogenic activities have been documented, along with a pronounced accumulation of allogenic substances under low flow conditions caused by rainfall deficit (Arienzo et al., 2020; Benassai et al., 2015; De Rosa et al., 2022; Montuori et al., 2022;). Sampling and environmental parameters Water sampling was carried out in the terminal stretch of the Sele River, from the mouth up to 3.5 Km upstream, under two distinct flow-rate conditions (Figure 1a-c). The first sampling was carried out in July 2017 (Arienzo et al., 2020) with the river occluded by sediments, a condition that persisted for 1 week, and the second sampling was carried out in May 2021, with the river’s mouth completely opened. Stations were located taking into consideration the presence of drainage channels flowing into the river (Table 1). At each station, 5 L of water were collected with a Niskin bottle and successively subsampled for the analyses of biological and chemical parameters, as described below. For the determination of total phytoplankton biomass (Chl a), 50-500 mL of water (depending on the presence of particles in the samples) were drawn from the Niskin bottle and filtered onto GF/F Whatman glass-fiber filters (25-mm diameter) im- mediately cryopreserved in liquid nitrogen until the analyses in the laboratory. At each station, water temperature and salinity were deter- mined using Idromar XMAR212 (2017) and Sbe 19 plus (2021) CTD probes interfaced with a GPS (Garmin Map 78S, Garmin, Olathe, USA). Non -co mmerc ial us e o nly Phytoplankton community as monitoring tool in the terminal stretch of a micro-tidal estuary facing the Tyrrhenian Sea 45 Total phytoplankton biomass and chemo-functional groups For the determination of total phytoplankton biomass (Chl a), 50-500 ml of water (depending on the presence of particles in the samples) were drawn from the Niskin and filtered on GF/F What- man filters (25-mm diameter) immediately cryopreserved in liquid nitrogen until the analyses in laboratory (Holm-Hansen et al., 1965; Lorenzen and Jeffrey, 1980; Yentsch and Menzel, 1963). Chlorophyll-a and phaeopigments were determined, after extrac- tion with 90% acetone, with a spectrofluorometer (Mod.RF– 6000; Shimadzu Corporation, Kyoto, Japan), with calibration curves checked daily with fresh Chl a standard solutions (Sigma- Aldrich, St. Louis, USA) (Holm-Hansen et al., 1965). The pheao:chl a ratio was used as a proxy of grazing activity on phy- Table 1. Sampling station with coordinates and bottom depth. Station Latitude N Longitude E Depth (m) 1 40.4814990 14.9448934 3.037 2 40.4812454 14.9488489 6.017 3 40.4810787 14.9506658 3.819 4 40.4812777 14.9538731 3.221 5 40.4829086 14.9582883 3.333 6 40.4850757 14.9623601 3.479 7 40.4877350 14.9655997 3.157 8 40.4902284 14.9688533 3.306 9 40.4920247 14.9718688 3.414 10 40.4962250 14.9724679 2.732 11 40.4997458 14.9718300 3.559 Figure 1. Final stretch of the Sele river with sampled stations (a). Mouth of the river in July 2017 (b) and May 2021 (c). Non -co mmerc ial us e o nly F. Bolinesi et al.46 toplankton cells (Shuman and Lorenzen, 1975). For the determi- nation of the pigmentary spectra, l.5 L of water was filtered on GF/F Whatman filters (47-mm dimeter) and the filters were cry- opreserved as for Chl a. Frozen filters were homogenized and re- suspended in 100% methanol and analyzed by High-Performance Liquid Chromatography (HPLC) (1100 Series, Hewlett Packard, Palo Alto, USA) in a reverse phase (C8 column 3 μm Hyperloop MOS) (Vidussi et al., 1996). The use of HPLC in the study of phytoplankton communities has been largely demonstrated to be a useful tool for the estimation of phytoplankton community com- position through the analyses of photosynthetic pigments (Jeffery and Vesk, 1997; Wright et al., 1996). The method is based on the analysis of accessory pigments, in addition to Chlorophyll-a (Chl- a) or the modified divinyl-Chl-a found in all phytoplankton species, and on the evidence that some of these accessory pig- ments are taxon-specific (Brunet and Mangoni, 2010; Millie et al., 1997; Wright and Jeffrey, 2006). This technique allows to de- tect and identify microscopically overlooked or undetermined ul- traphytoplankton species (Ansotegui et al., 2003; Antajan et al., 2004; Garibotti et al., 2003; Saggiomo et al., 2023), providing re- producible results. For the determination of chlorophylls and carotenoids, a spectrophotometer with a diode array detector was set at 440 nm, making it possible to determine the absorption spec- trum of the 350−750 nm interval for each peak to check the purity of single pigments. The column was calibrated using different pig- ment standards (chlorophyll a, chlorophyll b, chlorophyll c1+c2, chlorophyll c3, MgDVP, alloxanthin, diatoxanthin, prasinoxanthin, 19′-butanoyloxyfucoxanthin, fucoxanthin, 19′-hexanoyloxyfucox- anthin, peridinin, zeaxanthin, neoxanthin, violaxanthin, b- carotene). Standards were provided by the International Agency for 14 C Determination, VKI Water Quality Institute, Copen- hagen, Denmark. Quantification was based on the absorbance at 440 nm and the factor response value (peak area/pigment concen- tration) for each pigment, as described by Mantoura and Repeta (1997). The contribution of main phytoplankton groups to the total Chl a was estimated by CHEMTAX 1.95 software as indicated by Latasa (2007), using an iterative process to find the optimal pigment:Chl a ratios. The chemotaxonomic groups identified in- clude cyanophytes (Cyano), chlorophytes (Chloro), prasinophytes (Prasino), euglenophytes (Eugleno), cryptophytes (Crypto), di- atoms (Diato), pelagophytes (Pelago) haptophytes (Hapto), di- noflagellates (Dino), xantophytes (Xanto). Inorganic nutrient concentrations For the determination of inorganic nutrient concentrations (N-NO3, P-PO4), 10 mL water were filtered on 0.2 μm cellulose acetate filters, and stored in HDP vials at -20C. The analyses in the laboratory were conducted using a discrete sampling ana- lyzer (EasyChem Plus, Systea, Anagni, Italy) equipped with a UV-VIS spectrophotometer following the procedure described by Hansen and Grasshoff (1983) adapted to current instrumen- tation, using a standardized method to detect nitrate (EPA - Ref. National Environmental Methods Index 9171 Nitrate via V(III) reduction), and orthophosphate (ISO 15923 - Ref. International Standard Organization 15923-1 Water quality). Statistical analyses The spatial distribution of physical variables (temperature, salinity) was plotted using ODV 5.6.3 software. To estimate the pairwise dissimilarity between the two con- trasting water flow conditions in a low-dimensional space, a non-metric multidimensional scaling based on a distance matrix computed with the Bray-Curtis dissimilarity has been performed and represented using a bi-plot in which the correlation coeffi- cients between environmental variable and the NMDS scores were presented as vectors from the origin based on algorithm proposed by Taguchi and Oono (2005). The significance of dif- ferences between the two putative conditions of river’s water flow was tested by ANOSIM (Clarke, 1993). All statistical analyses were performed using the PAST 1.95 software. Results Environmental parameters Values of salinity are reported in Figure 2. In 2017 the dis- tribution of salinity showed the presence of a strong water strat- ification, with a net halocline between 1.5-2 m (station 2) and 2-2.5 m (station 9) whose depth increased moving toward the inner part of the river (Figure 2a). In general, values ranged be- tween 39.8 and 0.7. At station 1, near the sandbar occluding the river mouth, salinity reached values up to 39 originating a ‘bub- ble-like’ front probably related to the seawater percolation or episodic waves intrusion from the adjacent sea. The isohalines between 5 and 8, which have been the subject of dispute and de- bate over time (Deaton and Greenberg, 1986; Khlebovich, 1968, 1969), took place between 1 m (station 2) and 1.8 m depth at station 9, with a slightly deepening moving from the mouth to the inner part of the river. Station 1 was the only one with salin- ity >8 in the entire water column. In 2021, with the river mouth completely opened, the entire sampling area was still character- ized by a net water stratification, with a 5-8 salinity boundary cline placed between 1.5 and 2.5 m depth, deepening moving from the mouth (station 1) to the inner part of the river (station 11) (Figure 2b). In the entire area, salinity ranged between 36.2 and 0.01. All stations presented two distinct layers: a freshwater one, between 0 and 1.5 m, and a marine one, below 2.5 m. The only exception was station 10, where values reached maximum salinity of 10 on the sea bottom, and the freshwater layer reached its maximum thickness. As far as the temperature is concerned, it showed clear dif- ferences between the two sampling periods (Figure 3). In July 2017 water temperature exceeded 28.5 °C in the first meter of the water column, generating a marked thermocline at ~1.3 m depth in the overall sampling area (Figure 3a). The bottom layer was characterized by the presence of the coldest water reaching the minimum of 24.59 °C at station 2. The surface layer showed a slight increasing trend of temperature moving from the mouth to station 11, with values ranging between 28.5°C and 31.34°C. In May 2021, the water column appeared to be rather homeothermic (Figure 3b). In general, values decreased moving from the mouth to the station 11, where temperature reached the minimum of 19.3 °C. A weak hermos-stratification was present in the first 30 cm between stations 1 and 5, where the water re- tention time reached its maximum. Nevertheless, a slightly in- creased temperature in the bottom water layer was observed in vertical profiles between stations 5 and 9, in accordance with the increase of salinity linked to seawater inflow. The concentration of N-NO3 and P-PO4 in July 2017 led to Non -co mmerc ial us e o nly Phytoplankton community as monitoring tool in the terminal stretch of a micro-tidal estuary facing the Tyrrhenian Sea 47 a mean N:P ratio of 12.12 in the entire area, showing a point- distribution depending on the influence of drainage channels (Arienzo et al., 2020). In particular, N-NO3 ranged between 12.85 μmol L-1 (station 5) to 23.80 (station 10) with a mean con- centration of 18.2 μmol L-1 ; P-PO4 ranged between 0.81 μmol L-1 (station 3) and 8.39 μmol L-1 (station 10) with a mean con- centration of 2.84 μmol L-1 (Figure 4a). In May 2021, values of N-NO3 showed a mean concentra- tion of 15.76 μmol L-1 ranging between 4.57 μmol L-1 (station 2) and 23.51 μmol L-1 (station 9), and displaying a point-shaped distribution, as observed in the first sampling (Figure 4b, c). P- PO4 were characterized by very low concentrations, with a mean of 0.2 μmol L-1 and values ranging between 0.08 μmol L-1 (sta- tion 2) and 0.39 μmol L-1 (station 6). These aspects led to a mean N:P ratio of 83, a value far from what was observed in 2017 and from the Redfield one. In particular, the N:P ratio showed values ranging between 148 (station 3) and 30 (station 5), with the high- est values in the surface layer. Phytoplankton biomass and chemo-functional groups The distribution of total phytoplankton biomass (Chl a) in July 2017 showed the presence of eutrophic condition in the Figure 2. Distribution of salinity along the water column of the entire sampled area in July 2017 (a) and May 2021 (b). Y axis: depth (m); X axis: station distance; Z colored axis: salinity. Non -co mmerc ial us e o nly F. Bolinesi et al.48 overall sampling area (Figure 5a). Values ranged between a min- imum of 15.05 µg L-1 at station 9, and a maximum of 95.73 µg L-1 at station 10, highlighting a strong variability in the area, es- pecially near drainage channels. This aspect was also empha- sized by the fluctuation of grazing index values (Phaeo:Chl a), which showed a mean of 0.59 ranging between 0.20 (station 11) and 2.54 (station 9). The overall picture that emerged from Chl a concentration indicated the presence of an increasing trend moving from the mouth to the inner part of the river. In May 2021 (Figure 5b, c), values of Chl a were at least one order of magnitude lower compared to what was reported before, with values usually observed in oligotrophic/mesotrophic systems. The mean value for the entire area was 1.40 µg L-1, with con- centrations ranging between 0.55 µg L-1(station 10) and 3.11 µg L-1(station 11). The grazing index ratios were very high at all stations, with a mean of 1.21 and values ranging between 0.62 (station 2) and 1.87 (station 10). As far as the pigment spectra composition (expressed as pig- ment:Chl a ratios) is concerned, we observed high variability in both sampling periods (Table S1). In July 2017, fucoxanthin was the dominant pigment, with a mean ratio of 0.67 and values ranging between 0.43 (station 5) and 0.97 (station 1). Alloxan- Figure 3. Distribution of temperature along the water column of the entire sampled area in July 2017 (a) and May 2021 (b). Y axis: depth (m); X axis: station distance; Z colored axis: temperature (°C). Non -co mmerc ial us e o nly Phytoplankton community as monitoring tool in the terminal stretch of a micro-tidal estuary facing the Tyrrhenian Sea 49 thin showed the highest ratio at stations 5 (0.64) and 10 (0.64) and, together with Chl c2 and Diadinoxanthin was among the most represented ones. Chl c3, peridinin, phaeophytin, and 19’HF were the only pigments completely absent in the sam- pling area. In May 2021, fucoxanthin and MgDVP were the most abundant pigments, with mean ratios of 0.49 and 0.41, re- spectively. aFucoxanthin ranged between 0.36 (station 2, bottom layer) and 0.56 (station 5, bottom layer); MgDVP between 0.29 (station 4) and 0.59 (station 8, bottom layer). The contribution of fucoxanthin and MgDVP to the total pigmentary spectra was higher at the bottom layer than 0 m. Neoxanthin was the only pigment to be absent at all stations. Peridinin and neoxanthin were completely absent only at surface layer, while other pig- ments showed weak and nonlinear changes as highlighted by colored bars. The pigmentary spectra composition descripted so far, determined a different chemotaxonomically composition of phytoplankton community in the two sampling periods (Figures 6a-c). In July 2017, cryptophytes strongly dominated the com- munity in the entire area, with a mean of 58% and values ranging between 29% (station 2) and 79% (station 11). Diatoms and chlorophytes were the second most representative groups, with a mean of 16% and 11% respectively, and higher percentages at station 7. Dinoflagellates and haptophytes were completely ab- sent, while xanthophytes were only reported with a weak per- centage (2%) at station 11. In May 2021 (Figure 7b-c), the community was dominated by cyanophytes and diatoms, with mean percentages of 28.5% and 27.3% respectively. Cyanophytes showed a strong gradient at station 4, ranging between 9% (surface layer) and 43% (bot- tom layer); diatoms ranged between 6% (station 3, bottom layer) and 47% (station 11). Chlorophytes and euglenophytes showed similar mean percentage (7%), with the first group that was more represented at the surface layer, and the second one more repre- sented at deeper layer where reaches percentages up to 16% (sta- tion 11). Haptophytes was the only groups to be absent at surface layer and to be present exclusively at bottom of the station 4. Figure 4. Box plots with nutrient concentrations of P-PO4 and N-NO3 in July 2017 (a); surface layer in May 2021 (b); bottom layer in 2021 (c). Figure 5. Chart bar with chlorophyll a concentration (Chl a μg/L) and grazing index (Phaeo:Chl a) at each station (X axis) in July 2017 (a); surface layer May 2021(b); bottom layer in May 2021(c). Non -co mmerc ial us e o nly F. Bolinesi et al.50 Discussion Mediterranean microtidal estuaries have played an important role in human civilization (Haidvogl 2018). The present study aims at providing the first insights into changes in phytoplankton community structure in the terminal stretch of the Sele River, a microtidal estuary subjected to multiple anthropogenic pressures, as the result of contrasting conditions of the river’s water flow. The reclamation works carried out in the region during the last century have profoundly altered its morphology and degree of in- teractions between the river, the surrounding lands, and the sea. The extent of these changes is reported today in several geomorpholog- ical studies, though with a considerable lack of information on the ecology of the area, especially regarding microalgal communities in the terminal stretch, where phytoplankton plays a pivotal role as primary producers in lentic regime waters. Our data represent the first attempt to describe the phytoplankton community structure in one of the most important hydrographic basins of southern Italy, under two distinct hydrological phases: with the mouth of the river completely occluded by sediments - for the first time in living mem- ory–preventing the water exchange with the sea, and under a high hydrological regime with mouth completely opened. Under two different hydrological conditions, the terminal stretch of the Sele River was characterized by the coexistence of two distinct water layers. A shallow one, showing typically fresh- water salinity whose thickness tends to be higher in the inner part Figure 7. Non-metric multidimensional scaling based on Bray-Curtis index. Environmental variables are shown in green; the correlation coefficients between each environmental variable and the NMDS scores are presented as vectors from the origin. Stations of July 2017 are reported as black circles; stations of May 2021 are reported as filled (bottom layer) and empty (surface) orange squares. Figure 6. Chemotaxonomically composition of phytoplankton community in July 2017 (a); surface layer in May 2021 (b), bot- tom layer in May 2022 (c). Non -co mmerc ial us e o nly Phytoplankton community as monitoring tool in the terminal stretch of a micro-tidal estuary facing the Tyrrhenian Sea 51 of the river, and a deeper one with salinity values that typically exceeded that of the adjacent seawater, especially in deepest ponds and during summer. These thickness changes were accompanied by changes of 5-8 isohaline depth, which was absent near the mouth occluded by sediments and highest at station 10 in the inner part of the river. Salinity variation represents a key factor for the biotic component of ecosystems and the presence of brackish or- ganisms is a typical property of estuarine environments. Changes in salinity over river mouths depends on the tides, coastal flood- ing, and season of the year (Reynolds, 1984; 2006), so the forma- tion of sandy bar occluding the mouth and preventing the water exchange with the sea, can have significant effects on the func- tioning and ecology of such a complex system, with multi-scale effects still unknown. Since dramatic rainfall-decreasing trends were reported for the southern Italian Tyrrhenian coast, causing droughts during summer (Polemio and Casarano, 2004, 2008; Romano et al., 2022), the results presented here could be the first sign of a new condition towards which this system will likely tend more fre- quently in the near future. Contrarily to the salinity, the thermal profile of the water col- umn showed marked differences between the two sampling peri- ods in accordance with the season. In July 2017, a net thermocline was present at ~ 1.3 m depth, with water temperature exceeding 28.5 °C in the first meter of the water column reaching colder val- ues on the bottom (minim 24.59 °C, at station 2). In spring 2021 the water column was rather homeothermic, with lower values in the inner part of the river and relatively warmer water on the bot- tom linked to the seawater intrusion. A weak stratification was ob- served in the first 30 cm between stations 1 and 5, and despite the fact current meters data are not available, we hypothesize that this warmer layer reflects the hydrological dynamics of the area, where the increase in water retention time favors surface waters warming. It must be noted that stations were sampled ~10 minutes apart from each other, moving from the mouth to station 11, so sampling activities should not have influenced these data. Many authors indicate that any potential alteration to river flows would increase the temporal and spatial variability of estu- arine fronts, water stratification, and mixing, with potentially neg- ative impacts associated with eutrophication (Reynolds, 1984; 2006). The impact of nutrient enrichment on rivers is often season specific and complicated by their dynamic nature (Newman et al., 2005). Differences in nutrient concentration observed in our study, with extremely large spatial and temporal variations in N:P ratios, suggest how complex and articulated are the dynamics of nutrient supply in this area, where the presence of drainage channels (in addition to the combined effects of water retention times) can strongly influence the chemical properties of the water column (Albanese et al., 2007; Arienzo et al., 2020; Montuori et al., 2022). Nitrates did not show drastic changes between the two sampling periods, as instead observed with the phosphate load, which led to higher N:P ratios in 2021, with values up to 148 at station 3, compared to those in 2017. The first biological effect of nutrient enrichment in pelagic environments is the growth of phy- toplankton species, whose blooms extent and community structure strongly depend on local conditions, for example, the extent to which the nutrient load is retained by the receiving water (New- man et al., 2005; Reynolds, 2006). This is the reason why rivers are considered highly selective environments, where flow rate and turbidity, along with temperature, are the most critical factors lim- iting the development and structuring the phytoplankton commu- nity composition (Rojo et al., 1994; Reynolds, 1994, Tavernini et al., 2011). The higher the flow rate and turbidity, the lower the amount of phytoplankton can be expected: where the water reten- tion time is longer than the generation time of the phytoplankton, large blooms can occur (Ibeling et al.,1998; Lampert and Sommer, 1997; Newman et al., 2005). Nevertheless, the extent to which nutrient loads trigger phytoplankton blooms in micro-and macro- tidal estuaries is different, with small nutrient enrichment produc- ing larger effects in the microtidal ones (Warwick et al., 2018). In general, allogenic factors (i.e. inoculum of phytoplankton species transported downstream, temperature, light regime, discharge rate, turbidity) are considered the main drivers of phytoplankton suc- cession in lowland rivers (Reynolds, 1994, 2006), with autogenic ones becoming more relevant with increasing eutrophication lev- els (del Giorgio et al., 1991). The distribution of total phytoplankton biomass (Chl a) in July 2017 points to a speckled distribution with an increasing trend of concentration moving toward the inner part of the river, where values are typical of eutrophic waters reaching the maximum of 97.73 μg L-1. The station with lowest concentrations of Chl a (15.0 μg L-1) showed the highest phaeo:Chl a ratio (2.7), indicating the presence of large amounts of degraded biomass, most likely be- cause of the adjacent drainage channel transport. On the contrary, in May 2021, values of Chl a were at least one order of magnitude lower, with a mean of 1.40 μg L-1, appearing more homogeneously distributed in the entire sampling area and being characterized by very high phaeo:Chl a ratios, the latter supporting the hypothesis that much of the phytoplanktonic biomass was photosynthetically inactive. The overall picture emerging from nMDS analyses clearly shows the differences between the two samplings periods, with data from July 2017 (black points) clustering in the left side of the plot, and those from May 2021 (orange squares) in the right part. Temperature, P-PO4, and the N:P ratios are among the main environmental drivers shaping the phytoplankton community, with high Chl a concentration and cryptophytes characterizing the first sampling period, and high N:P ratios characterizing the sec- ond one. In May 2021 (orange squares), euglenophytes, cyanophytes, and xantophytes were the dominant groups, espe- cially at the surface layer (filled orange squares). The correlation between salinity and depth highlights the role of seawater inflow in shaping the structure of the phytoplankton communities, with dinoflagellates, haptophytes, and pelagophytes dominating in the deepest and saltiest layers, and high loads of N-NO3 that were in- stead associated to the shallow layer, because of inputs from drainage channels. A high and positive R value (0.9777) in the ANOSIM analysis (p=0.0001) indicates the strong and significant dissimilarity between the phytoplankton communities’ composi- tion in the two sampling periods (Table 2). Altogether our data Table 2. Results of the ANOSIM (ANalyses Of SIMilarities) test carried out to ascertain differences between the two sam- pling periods, representing contrasting water flow conditions. ANOSIM Test – Bray-Curtis Permutation N 9999 Mean rank within 132 Mean rank between 334.6 R 0.9777 p (same) 0.0001 Non -co mmerc ial us e o nly F. Bolinesi et al.52 agree with the results reported by Tavernini et al., (2011), who, in the Po River, observed the highest concentration of crypto- phytes biovolume in late July, concomitantly with an higher con- tribution of cyanophytes between April and June: these patterns provide a further confirmation that the terminal stretch of the Sele River is exposed to strong allogenic pressures. Conclusions In addition to what discussed so far, we pinpoint here that the phytoplankton community represents a promising and effec- tive monitoring tool for the study of estuarine environments, in which cross-scale interactions produce large fluctuations in physical, chemical, and biological properties. Nevertheless, the large variability of the phytoplankton community structure both in space and time, and the extremely ample variations in N:P ra- tios underline how much allogenic pressures can influence com- munities’ functional traits in the terminal stretch of the Sele River and, supposedly, in other estuarine areas, with similar combinations of natural and anthropogenic pressure. The pres- ence in both study periods of a saltier layer near the bottom high- lights the role played by the seawater inflow in the ecology of the area, characterized by a strong summer thermoaline stratifi- cation, which exacerbates the difference between superficial and bottom water layers. 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