Layout 1 ARTICLE INTRODUCTION Picocyanobacteria are small prokaryotes (size range 0.2-2 µm) of the order Chroococcales ubiquitous in fresh- water and marine ecosystems (Zwirglmaier et al., 2008; Callieri, 2008). Picocyanobacteria are mainly constituted by the polyphyletic genus Synechococcus, which is one of the most abundant autotrophic prokaryotes living in oceans and lakes (Flombaum et al., 2013; Callieri et al., 2012). In freshwaters, the genus Synechococcus comprises many uni- cellular strains and isolates that fall into cluster 5 (Casten- holz et al., 2001; Cabello-Yeves et al., 2018). Moreover, their relative abundance on the total autotrophic biomass in freshwater and marine ecosystems increases with a de- crease in the trophic status (Bell and Kalff, 2001; Callieri et al., 2007), indicating global ecological importance in most aquatic systems. In Lake Maggiore, picocyanobacte- ria are likewise mainly composed of the genus Synechococ- cus according to morphological classification (Callieri and Pinolini, 1995), and the isolated strains fall in the subalpine cluster I (Callieri et al., 2013), now subcluster 5.2 (Cyanobium, sensu Dorè et al., 2020). These tiny Gram-negative cyanobacteria have a partic- ular cell membrane composition, with the outer peptidogly- can layer considerably thicker than that of most gram-negative bacteria (Hoiczyk and Hansel, 2000). More- over, Synechococcus spp. can form an external S-layer of glycoproteins on the cell surface (Ernst et al., 1996), impor- tant for cell adhesion and protection (Šmarda et al., 2002), that confers to the cell a suitable site for microgel particle formation starting from certain precursors. These sticky mi- crogel particles are predominantly composed of acidic poly- saccharides stainable with Alcian blue (Alldredge et al., 1993; Passow et al., 2001), and are known to be the main constituent of Transparent Exopolymer Particles (TEP). Such particles are present in oceans and inland waters and have an important role in the food webs, increasing the size of single cells by aggregating autotrophic and heterotrophic cells (Burd and Jackson, 2009; Deng et al., 2015). Algae of different sizes and groups (pico and nano phytoplankton) can release TEP-precursors, generally in periods of senes- cence or decline of the population, such as after a bloom (Passow, 2002a; Grossart et al., 2006; Berman-Frank et al., 2007). Nevertheless, the production of TEP in Synechococ- cus cultures has been recently demonstrated not only under chemical and physical stress conditions but also during cell growth (Callieri et al., 2019a). The process of TEP forma- tion involves abiotic processes like coagulation or gelation from polysaccharide fibrils present on the cell surface (Meng et al., 2013). TEP contain polysaccharides, proteins, lipids, and amino acids (Passow, 2002b), giving it a com- position similar to that of Extracellular Polymeric Sub- stances (EPS). However, unlike EPS, TEP are present in the aquatic environment as discrete particles and not as cell-sur- face attached or dissolved molecules (Meng et al., 2013). TEP may constitute an important fraction of the Total Organic Carbon (TOC) present in aquatic environments, but its seasonal and spatial dynamics are still poorly un- derstood (Callieri et al., 2017). This is the case in Lake Maggiore, where TEP make up to 54% of the TOC (Cal- lieri et al., 2021). In a recent study on the Borromeo Basin (Lake Maggiore, Italy), TEP showed a clear vertical het- erogeneity from surface to bottom, which is related to the distribution of the autotrophic microorganisms that rep- resent the main source of these particles (Callieri et al., 2021). In that annual study, TEP was correlated with di- atom chlorophyll in spring, and with the number of pico- cyanobacteria in autumn. TOC is an important indicator of the trophic state of aquatic environments because it summarizes in a single variable the result of the activities of microbial commu- nities. The study of TOC, moreover, is important due to Spatial-temporal study of cluster 5 picocyanobacteria and exopolymeric microgels in Lake Maggiore Cristiana Callieri*, Raffaella Sabatino, Andrea Di Cesare, Roberto Bertoni CNR–IRSA Water Research Institute, Molecular Ecology Group, Verbania, Italy ABSTRACT In the oligotrophic Lake Maggiore, the majority of picocyanobacteria are phycoerythrin-bearing Synechococcus-type be- longing to cluster 5 (Pcy-5). Their distribution was followed in a seven-year study along a depth gradient from the surface down to 350 m in relation to Transparent Exopolymer Particles (TEP), Total Organic Carbon (TOC), Chlorophyll-a (Chl-a) and water temperature. Pcy-5 abundances exhibit pronounced inter-annual variability, showing years of high numerical abundances as well as years with low numbers. In the upper 20 m, Pcy-5 peaks at around 10-15 m and then progressively decreases. Here, the Pcy-5 presence has been outlined for the first time in the deep layers of a deep lake, thus opening an interesting discussion on these organisms’ survival mechanisms in the absence of the light needed to perform photosynthesis. The relation of Pcy-5 with extracellular microgels was significant in autumn, when peaks of both variables were observed. In the other seasons, TEP was correlated with temperature and Chl-a, indicating the autochthonous origin of this fraction. Non -co mmerc ial us e o nly 58 C. Callieri et al. the conspicuous role of lakes as an active component in the global carbon cycle (Armstrong, 2010). It follows that the study of TEP distribution, as an important fraction of TOC, can also be central to the study of lake ecosystems. The study of the multi-year spatial-temporal evolution of TOC and its fractions from the 1980s to the present date documents the evolution of the trophic state of Lake Mag- giore, highlighting its progressive, albeit discontinuous, transformation, and its return to its original conditions of oligotrophy (Rogora et al., 2021). Although this trend is evident, in recent years the appearance of anomalous blooms of cyanobacteria and green algae has been con- nected to raises in the concentration of TOC and TEP (Callieri et al., 2021). Here we present the results of a seven-year study of TEP and Pcy-5 dynamics in the subalpine Lake Maggiore, also considering the deep hypolimnetic layer. The main aim of this work was to verify the presence of Pcy-5 also in the deep zone of the lake and to ascertain if Pcy-5 re- lation to TEP, already found in Synechococcus cultures (Callieri et al., 2019a), was also evident in the environ- ment. Therefore, we present distribution maps of TEP, TOC, total Chlorophyll-a (Chl-a), and temperature, as well as an analysis of their correlations with Pcy-5 num- bers, along the entire water column from surface to 350 m from 2015 to 2021. MATERIALS AND METHODS Study site and sampling Lake Maggiore is a large, deep, subalpine lake (212 km2, Zmax 372 m) in Northern Italy, recovered from cul- tural eutrophication and now in oligo-mesotrophic condi- tions, with annual average total phosphorus, measured on the whole water column, around 12 μg L–1 (Rogora et al., 2018). Lake Maggiore is one of the most studied lakes in Europe and its long-term chemical, physical and biologi- cal data are published in a variety of journals (Bertoni et al., 2010; Salmaso and Mosello, 2010; Bertoni et al., 2016; Salmaso et al., 2020). Sampling was performed in Lake Maggiore at the station of maximum depth (372 m), using a 5 L Van Dorn type bottle, at 10 depths along the water column (0, 5, 10, 15, 20, 50, 100, 200, 300, 350 m). In the seven years of the study (2015-2021), monthly sampling dates were selected to obtain a detailed picture of the seasonal cycle of TEP, TOC and Chl-a concentrations together with Pcy-5 cell number. In parallel, other limno- logical parameters (for most of the years: water tempera- ture, conductivity, pH, dissolved oxygen) were measured in situ with a multiparametric probe (CTD316, Idronaut, Brugherio, Italy) and presented in the report of the Interna- tional Commission for the Protection of Italian-Swiss Wa- ters (CIPAIS, available at: www. cipais.org). Pcy-5 cell counting and Chlorophyll-a measurements Samples were immediately fixed with filtered formaldehyde (0.2 µm pore size membrane, 1% final con- centration) and stored at 4°C in the dark. A flow cytometer Accuri C6 (Becton Dickinson, Oxford, UK), equipped with a 20 mW 488 nm Solid State Blue Laser and a 14.7 mW 640 nm Diode Red Laser, was used to quantify pic- ocyanobacteria. Pcy-5 were quantified using light scattering signals (forward and side light scatter named FSC-H and SSC-H, respectively), orange fluorescence (FL2-H channel = 585/40 nm) and red fluorescence (FL3-H channel >670 nm and FL4-H channel 675/25). The density plots of FL2- H vs FL3-H allowed for the optimal gating design and the quantification of the phycoerythrin-rich (PE) and phyco- cyanin-rich (PC) Pcy-5 cells (Callieri et al., 2016). All data were acquired at a pre-set flow rate of 35 µL min–1, in order to keep the number of total events below 1000 per second. The BD Accuri C6 resident software (v.1.0.264.21) was used for cytogram gating and data processing. Chl-a concentration was measured by Pulse-Ampli- tude-Modulation Phytoplankton Analyzer (PhytoPAM, Heinz Walz, GmbH, Effeltrich, Germany). PhytoPAM was equipped with the Optical Unit ED 101US/MP, the phyto ML (25 measuring LED in the 4 wavelenghs and 12 actinic LED 655nm), and the phyto AL (37 actinic LED 655nm) (Schreiber et al., 1986). The Chl-a concen- tration (µg l–1) was obtained using specific Chl calibration as reference and reference spectra using pure culture (per- formed by Walz H.). Total Organic Carbon (TOC) and Transparent Exopolymer Particles (TEP) TOC concentration was measured using a total organic carbon analyzer (TOC-L High-Sensitivity Model, Shi- madzu, Kyoto, Japan) equipped with ASI-L autosampler. The CO2 derived from high-temperature oxidation is measured with a Non-Dispersive Infrared Detector (NDIR). Two replicates for each sample were analyzed. Running in TOC mode, the Shimadzu analyzer removes the inorganic carbon from the sample by acid addition and bubbling. For each analysis 250 µL of the sample are in- jected in the oxidation furnace and, for each sample, the TOC concentration is computed by averaging five analy- ses. The concentration of TEP was measured spectropho- tometrically (787 nm) following the protocol of Alldredge et al., (1993). In short, the samples were filtered onto polycarbonate membranes (0.4 μm pore size) under low vacuum (<10 mbar, 150mm Hg), stained with 500 µL of 0.22 μm pre-filtered 0.02% Alcian Blue (8GX, Fluka; Sigma-Aldrich, St. Louis, USA) aqueous solution, in 0.06% glacial acetic acid (pH 2.5) for 30 second (All- Non -co mmerc ial us e o nly 59Spatial-temporal study of cluster 5 picocyanobacteria and exopolymeric microgels in Lake Maggiore dredge et al., 1993). Subsequently, the membranes were left immersed for 2 hours in 6 mL of 80% (v/v) sulphuric acid and removed before reading the resulting liquid frac- tion at the spectrophotometer. The concentration of TEP was expressed as Gum Xan- than (GX) equivalent (µg GX eq L–1) following Passow and Alldredge (1995) and converted in µg C L–1 using the conversion factor of 0.63 (Engel, 2004; Berman-Frank et al., 2016). Statistical analyses Statistical analyses were carried out to: 1) assess which biotic (Pcy-5 abundance, Chl-a concentration) and abiotic (TOC concentration, depth, temperature, and season) vari- ables can affect TEP concentration during the sampling years (2015-2021); 2) deepen our understanding of the role of Pcy-5 in the production of TEP in a natural environment. We applied a Linear Mixed Effect Model (LMEM) and extrapolated partial R2 values, which represent the extent of the variance explained by a variable in the model, and p values, which express the significance of the variable. All statistical analyses were conducted in the R environment v3.6.0 (R Core Team, 2019; R Foundation for Statistical Computing, Vienna, Austria), using the package lme4 v1.1-31 (lmer) to perform the LMEM and car v3.1-1 (Anova) and partR2 v0.9.1 (partR2) to obtain the partial R2 and p values, respectively. RESULTS Pcy-5 number, Chlorophyll-a concentration and temperature In Lake Maggiore the study of Pcy-5 distribution began in 2000, but only recently have these microorganisms been counted at different depths of the water column thanks to the acquisition of a flow cytometer, which allows fast and accurate counting. Examples of cytograms are presented in Figure 1, which show the clouds of PE and PC cells in 2016 at different depths, including the 200 and 350 m depths (upper graphs), and the typical presence of eukaryotic cells in May and of microcolonies and filamentous forms in Sep- tember (lower graphs), as recurrently observed in late sum- mer during all the years of study. The abundance map for the seven-year period 2015- 2021 (Figure 2) showed a decrease in their numbers as the depth increases, but it also confirmed the presence of these cyanobacteria even at high depths. In the layer around 75 m, particularly in 2017 and 2019, there is a clear increase in cell number, also visible, even with lower numbers, around 250 m. The highest numbers of Pcy-5 were measured in August 2019 at 15 m (6.75x105 cells mL–1) and in May 2020 at 10 m (5.42x105 cells mL–1). If we compare the map of Pcy-5 with that of Chl-a in the upper 100 m (Figure 3), we notice that in the 20-40 m water layer they are very scarce and other fractions of phytoplankton contribute more significantly to Chl-a con- centration: likely the eukaryotic fraction. During the years 2017 and 2021 we observed particularly low numbers of Pcy-5, even though at high depths they were always pres- ent. In the 100-350 m layer, generally considered not suit- able for the growth of Pcy-5, we calculated an average number of 1333 cells mL–1 in the seven years of the study, with a significant trend toward a decrease from 2015 to 2021 (Tau=-0.3539, p<0.001) (Figure 1S). Chl-a concentrations peaked in April during 2015 with 12.6 µg L–1, mainly due to diatoms (pers. comm. G. Morabito). The mean value in the 0-20 m zone in the seven years was 2.25 µg L–1, while at 100 m was 0.35 µg Figure 1. Examples of cytograms (FL2 phycoerythrin vs FL3 chlorophyll) obtained by flow cytometer analysis and showing the fingerprints of autofluorescent picocyanobacteria in Lake Maggiore at 5, 10, 200 and 350 m in July 2016 (upper 4 graphs) and in the euphotic zone in May and September (lower 2 graphs). In violet the more numerous fraction of PE Pcy-5, in green of PC Pcy-5, in red the microcolonies, and in blue the pi- coeukaryotes. Non -co mmerc ial us e o nly 60 C. Callieri et al. L–1. It is interesting to note a significant trend toward a decrease in concentrations in the deep layers from 2015 to 2017 followed by an increase in 2018 and a drop to values around 0.40 µg L–1 that remained constant till 2021 (Tau=-0.0678, p=0.4280) (Figure 1S). Temperature showed the usual pattern of a deep sub- alpine lake with a maximum value of 25.6°C at the sur- face on July 2019 and a minimum of 6.5°C at 350 m in January 2015 (Figure 4). Complete overturn has not been observed since the seventies due to the increase in winter air temperatures and the decrease in the wind (Dresti, 2022). Over the seven years, a significant trend towards an increase in values was observed in the layer 100-350 m (Tau=0.9260, p<0.001) (Figure 1S). Total Organic Carbon (TOC) and Transparent Exopolymer Particles (TEP) The TOC dynamics in Lake Maggiore in the seven- year period studied are well evident in the concentration map (Figure 5). The first observation is that no recurring and repeatable annual trend could be found; instead, we found a clear interannual variability, that was not so marked in previous decades (Bertoni et al. 2016). The maximum concentration measured during the period was 1953 µg C L–1 and the minimum 600 µg C L–1. The second observation is a pronounced decrease in concentration with depth: the mean value in the 0-20 m layer was 1161 µg C L–1, while in the 100-350 m layer it was of 853 µg C L–1. In 2021 the concentrations were particularly low in the deep layers. The TEP concentration map shows a more recurrent pattern in contrast with TOC, with two years of very low concentration (2017 and 2021) and the other years (2018-19-20) with the highest concentrations (up to 3000 µg C L–1) reached in September (Figure 6). In par- ticular, in 2019 high concentrations were also measured in the deep layers, where usually TEP concentrations are very low. One characteristic of TEP is that during winter they are very low and only begin to appear from June/July; however, in some years, these particles reach very high concentrations in the first 10/15 m in Septem- ber, in coincidence with the appearance of pico- cyanobacteria bloom and foam events (Callieri et al., 2022). The scatterplots on a log:log scale between TEP:Chl-a and TEP:Pcy-5 showed the relations among these parameters and how the highest values are preva- lent in summer and autumn (Figure 7). Statistical analyses Before analyzing the data, we performed a correla- tion test in order to reduce the number of variables to use subsequently, retaining only one variable of each pair in case of correlation coefficients ≥0.65. Tempera- ture and TOC showed a correlation (Figure 2S), there- fore we decided to discard the latter from the following analysis. Then, we applied a LMEM to explain TEP vari- ations, using Pcy-5, Chl-a, depth, temperature, and sea- son as fixed effect variables, and the year as a random effect variable to minimize annual fluctuations. The re- sults showed that temperature is the variable that ex- plains most of the variance of TEP (p=0.0010). Also Chl-a was significantly correlated to TEP (p=0.0209) (Table 1). Moreover, we further investigated the effect of Pcy-5 on TEP variations, introducing in the model, as explanatory factors, the interaction between these mi- croorganisms and each of the other variables (Table 1). Figure 2. Total Pcy-5 cell number at Ghiffa station in Lake Maggiore. Non -co mmerc ial us e o nly 61Spatial-temporal study of cluster 5 picocyanobacteria and exopolymeric microgels in Lake Maggiore There was a significant correlation between Pcy-5 and TEP only if season and temperature were introduced as explanatory factors (Table 1). The results showed that TEP and Pcy-5 were correlated on a seasonal basis (p=0.00002): in autumn, when Pcy-5 abundances reached their peak, also high TEP concentrations were found (Figure 7B). DISCUSSION This is the first detailed study of Pcy-5 spatial and temporal distribution with maps of their abundance from the surface to the bottom (350 m) over the medium-term (seven years). Figure 3. Chlorophyll-a concentration map (upper panel) compared with the Pcy-5 number (up to 100 m, lower panel) at Ghiffa station in Lake Maggiore. Table 1. Statistical results of the Linear Mixed Effect Model (LMEM) assessing the influence of the biotic and abiotic vari- ables on TEP concentration over the years (2015-2021). For Pcy- 5 a significant p-value on TEP was obtained by isolating the temperature and the season effect. p-value R2 MODEL 0.3216 Pcy-5 0.1467 0.0043 Chl-a 0.0209* 0.0099 Depth 0.6267 0.0021 Season 0.0753 0.0123 Temperature 0.0010** 0.0255 Pcy-5 : Chl-a 0.8619 Pcy-5 : Depth 0.3412 Pcy-5 : Season 0.00002*** 0.0421 Pcy-5 : Temperature 0.00003*** 0.0351 Non -co mmerc ial us e o nly 62 C. Callieri et al. The first observation we can make is that Pcy-5 abun- dances exhibit pronounced inter-annual variability, showing years of high cell numbers (2015, 2019) as well as years of very low numbers (2017, 2021) (Figure 2). In the first 20 m, Pcy-5 peak around 10-15 m and then progressively de- crease. However, in the deeper, lightless water mass, below 50 m, we observe the presence of Pcy-5 in well-defined niches between 50 and 100 m and around 250 m. The pres- ence of Pcy-5 in deep waters without the light necessary to perform photosynthesis has already been observed, but mainly in marine systems (Sohorin et al., 2011; Miller et al., 2017; Callieri et al., 2019b). Pcy-5 have been found in the subtropical NW Pacific and in the Gulf of Aqaba in meso- and bathypelagic waters, transported from the epipelagic zone down the water column by convective mix- ing (Sohorin et al., 2011; Miller et al., 2017), as well as in the deep anoxic water of the Black Sea (Callieri et al., 2019b; Di Cesare et al., 2020). However, to our knowledge, the presence of Pcy-5 in deep lakes has not yet been re- ported. This is most likely due to the fact that the study of autotrophic communities is normally restricted to the eu- photic zone, leaving out the deep aphotic zone. The cy- tograms of samples from different depths (Figure 1) clearly show that the autotrophic communities at 350 m are pre- Figure 4. Temperature (°C) map at Ghiffa station in Lake Maggiore. Figure 5. Total Organic Carbon (TOC) map at Ghiffa station in Lake Maggiore. Non -co mmerc ial us e o nly 63Spatial-temporal study of cluster 5 picocyanobacteria and exopolymeric microgels in Lake Maggiore dominantly composed of picocyanobacteria, while pi- coeukaryotes or larger cells are generally absent. In marine systems, the presence of Prochlorococcus and Synechococ- cus in the deep layer was explained by assuming that they could adapt and tolerate repeated periods of light energy deprivation due to periodic deep mixing that transports cells from the euphotic layer to deeper waters (Coe et al., 2021). Small subpopulations of dark-tolerant cells could be trans- ported again to the surface and contribute to the dispersal of new genotypes dark-tolerant (Coe et al., 2021). In the case of the meromictic Black Sea the genome of the Synechococ- cus strains (BS55D and BS56D) isolated from the 750 m, contained genes encoding various compounds that enable them to exploit heterotrophic or fermentation pathways (Callieri et al., 2019b). In general, we can hypothesize that adaptation mechanisms similar to those found in marine en- Figure 6. Transparent Exopolymer Particles (TEP) map at Ghiffa station in Lake Maggiore. Figure 7. Scatterplots on a log:log scale between A) TEP:Chl-a and B) TEP:Pcy-5 data of the whole water column of Lake Maggiore, during the period 2015-2021. The different symbols refer to the seasons. Non -co mmerc ial us e o nly 64 C. Callieri et al. vironments can also be conceivable for Pcy-5 in a deep lake. The comparison of Pcy-5 abundances with TEP concen- tration maps indicates a substantially similar distribution. During 2017 and 2021, TEP and Pcy-5 were very low, whereas both peaked in 2019 and 2020. This picture is con- firmed by the model used in the statistical analyses, from which Pcy-5 resulted to be significantly correlated to TEP if the season effect is removed (Table 1) and it is particularly evident in autumn. This correlation, however, is not present when all seasons are considered. On the other hand, Pcy-5’s ability to produce extracellular substances has been demon- strated in laboratory experiments: under both stress and nor- mal growth conditions, marine and freshwater Pcy-5 were able to form TEP and EPS (Thornton and Chen, 2017; Deng et al., 2016; Callieri et al., 2019a). In Lake Maggiore, on a smaller dataset, TEP variability was explained mainly by Chl-a, a proxy for phytoplankton biomass, and to a lesser extent by picocyanobacteria (Callieri et al., 2017). With our large seven-year dataset, we showed that in nature a corre- lation between TEP and Pcy-5 is visible in autumn, when this component of phytoplankton is dominant over other groups. In a littoral to pelagic gradient in Lake Maggiore similar results were obtained (Callieri et al., 2021). Septem- ber was shown to be the month in which numerous colonial forms like Microcystis aeruginosa, Aphanothece spp. and Aphanocapsa spp. and even single cells of Synechococcus spp. appear in the lake (Callieri et al., 2021). We can there- fore assume an essential role in TEP production of the colo- nial forms of Pcy-5 that are often immersed in mucilaginous organic matrices. Although TEP is they are a fraction of TOC, the concen- tration of mucilaginous substances seems to exceed that of the TOC during lake foam production (Callieri et al., 2022), probably because the analytical protocol for measuring TEP implies concentration trough filtration, whilst the total TOC analysis via high-temperature oxidation utilizes microliters amount of sample. This could lead to an over-accumulation of TEP above the filters in one case and a loss of mucilagi- nous TEP particles in the other. TOC is the variable that best illustrates, alongside nutri- ents, the trophic evolution of Lake Maggiore. In fact, the current TOC concentration in the lake is the result of the input of organic matter of autochthonous and allochthonous origin, net of TOC decomposed by bacteria and sequestered at the bottom by sedimentation. It is currently difficult to determine the different weight of meteoclimatic and anthro- pogenic forces in determining the interannual variability ob- served, because of the absence of macroscopic pollution phenomena that would suggest a causal relationship with the increase in TOC. However, it is clear that the trophic state of Lake Maggiore is in a precarious balance between oligotrophy and mesotrophy, and therefore the utmost atten- tion must be paid to the management of this ecosystem to avoid a shift towards worse conditions. CONCLUSIONS The results of our multi-year study on the spatial and temporal dynamics of Pcy-5 in Lake Maggiore and its rela- tionships with TEP, TOC, Chl-a, and temperature suggest the existence of more robust causal relationships between these variables than what can be assumed on the basis of an- nual observations. This prompts further efforts to move from a speculative to an empirical view of the relationship be- tween TEP and picoplanktonic populations. Corresponding author: Cristiana Callieri, CNR–IRSA Water Re- search Institute, Microbial Ecology Group, Verbania, Italy. E-mail: cristiana.callieri@irsa.cnr.it Authors’ contributions: All the authors made a substantive intel- lectual contribution, performed part of the experiments. All the au- thors have read and approved the final version of the manuscript and agreed to be accountable for all aspects of the work. Conflict of interest: The authors declare no potential conflict of in- terest. Funding: CIPAIS - International Commission for the Protection of Italian-Swiss Waters. Availability of data and materials: All data generated or analyzed during this study are included in this published article. Key words: Picocyanobacteria cluster 5, Synechococcus spp., TEP, TOC, Lake Maggiore, deep picocyanobacterial. Acknowledgements: We thank Filippo Bertoni for assistance with editing the text, and Gabriele Tartari for providing temperature data. Received: 29 November 2022. Accepted: 15 December 2022. Publisher’s note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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