Ecological dynamics of anoxygenic phototrophs in stably redox-stratified waters: Intra and inter-seasonal variability of Lake Cadagno

Ecological dynamics of anoxygenic phototrophs in stably redox-stratified waters: Intra and inter-seasonal variability of Lake Cadagno | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ecological dynamics of anoxygenic phototrophs in stably redox-stratified waters: Intra and inter-seasonal variability of Lake Cadagno Nicola Storelli, Oscar Sepúlveda Steiner, Francesco Di Nezio, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3744815/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Lake Cadagno differs from typical alpine lakes as it is stratified into two water layers that never vertically mix. This stratification creates a niche for the development of primordial anoxygenic phototrophs, which thrive in the chemocline of the lake, forming a characteristic bacterial layer (BL). Yet, the relationship between the temporal variation of meteorological factors that regulate stratification and the development of the BL remains unclear. Here, we explored the intra- and inter-seasonal stability of the water column stratification and ecological dynamics of the anoxygenic phototroph community of the BL over three years. Our continuous monitoring showed that the meromixis of the lake is highly stable, with density stratification seemingly unaffected by external meteorological factors. Further reanalysis of the lake’s recent history substantiated this remarkable stability. In contrast, the community of anoxygenic phototrophs showed significant intra- and inter-seasonal variability, modulated by weather events that primarily impacted light penetration. In fact, an exceptional intra-seasonal light increases in September 2020 led to an overgrowth of purple sulfur bacteria compared to commonly dominant, green ones. At the inter-seasonal level, there is a difference in BL development in July 2021, which was characterized by much precipitation and less light, compared with that in 2019/2020. Biological sciences/Ecology/Microbial ecology Earth and environmental sciences/Limnology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The structure of the water column in lakes is primarily influenced by the distribution of temperature and concentration of dissolved ions, which control the water density and, thereby, stratification 1 . The type of stratification is either temporary (holomictic) or permanent (meromictic). Most lakes are holomictic and are divided into three subcategories, according to the water overturning tendency: monomictic lakes, dimictic lakes and polymictic lakes, all related to atmospheric temperature seasonality. In contrast, the stratification of meromictic lakes is mainly controlled by ion content in the water column. There are less than 200 documented meromictic lakes worldwide, although there are probably more, but they are not monitored 2 . Permanently stratified lakes are usually divided into three parts: 1) the lower layer, called monimolimnion, is anoxic and rich in dissolved ions with poor hydrodynamic circulation; 2) the upper layer, known as mixolimnion, behaves essentially as a holomictic lake directly influenced by the meteorological forcings (mainly air temperature); and 3) the region in between referred as chemocline, which hosts a steep chemical gradient with inversion of the redox potential. Due to the lack of mixing, the permanent stratification leads to a stable anoxic environment in the monimolimnion. This is an ideal spot to study biogeochemical processes mediated by microorganisms, such as the sulfur and nitrogen cycles that usually occur in anaerobiosis 3 . In the few cases of meromictic lakes where light can penetrate the anoxic depths of the monimolimnion, dense bacterial layers (BLs) are formed and primarily composed of anoxygenic phototrophic sulfur bacteria 4 – 8 . These anoxygenic phototrophs are also ecologically vital, as they provide an additional food source for the lake's trophic chain 9 , 10 . Cadagno is a crenogenic meromictic lake whose stratification is controlled by deep, ion-rich, subaquatic inflows 9 , 10 . The lake owes this characteristic to the peculiar geological conformation of the Piora Valley (Figure S1 ). This formation comprises Triassic carbonate rocks, including tectonized dolomitic limestones and gypsum deposits with Karstic hydrology. As a result, the water flowing into the lake is enriched with salts, giving it its unique properties 11 , 12 . The water that seeps in through the karst dolomite and re-emerges from sub-lacustrine springs in the southern region of the lake has a high ionic content (9–10 mM; Ca 2+ , Mg 2+ , SO 4 2− and HCO 3 − ; Figure S1 ) and supplies the anoxic lower part (monimolimnion) of the water column 13 . The deep water of the monimolimnion remains isolated from the rest of the lake, resulting in the development of a stable anoxic environment that, according to the sediment records, dates back to 10,000 years 14 – 16 . The primary source of the mixolimnion surface water is a small stream flowing from Lake Stabbio, a small lake located 2351 m above sea level (a.s.l.). The crystalline rocks (gneiss) in its catchment area are chemical-resistant, so the water in the mixolimnion has a low ionic strength. The difference in density between the two water layers in Lake Cadagno results in the development of a very stable stratification with a chemocline around 10–12 m depth characterized by a rapid change in the concentrations of chemical components, producing a redox stratification (Fig. 1 ) 17 . The high concentrations of sulfate (> 80 mg l − 1 ) coming from the sublacustrine springs promoted the development of chemoheterotrophic anoxygenic sulfate-reducing bacteria (SRB). The metabolism of SRB leads to the production of hydrogen sulfide (H 2 S) in the deep layers of the lake (e.g., monimolimnion and sediment). When H 2 S reaches the illuminated part of the anoxic layer, it is used by phototrophic sulfur bacteria as an electron donor for anoxygenic photosynthesis 18 . This leads to the development of a highly concentrated bacterial layer (BL; turbidity > 10 FTU) composed of purple (PSB) and green (GSB) sulfur bacteria species (Fig. 1 , red line) 19 – 22 . This light-dependent community is dynamic and concentrated in the photic zone of the anoxic layer. During the year when the lake is not covered by snow and ice, the PSB and GSB communities form a pink-colored BL that can exceed 1 m in thickness. This ecological niche hosts a distinctly heterogeneous community of anoxygenic phototrophic sulfur bacteria utilizing different evolutionary strategies. The BL of Lake Cadagno harbors rich biodiversity, hosting at least 7 species of purple sulfur bacteria (PSB) and 2 species of green sulfur bacteria (GSB) 23 . One important species is the PSB Chromatium okenii , which accounts for most of the BL biovolume due to its large cell size (8–10 µm main rod axis length). Moreover, the microorganism's flagella-driven motility towards light (positive phototaxis) and repulsion to oxygen (negative aerotaxis) results in the accumulation of a high cell concentration zone at the top of the BL. This leads to an increase in the local (physical) density of the water-bacteria mixture that exceeds the ambient water density, causing the heavier fluid to sink and drag the microorganisms down – a phenomenon known as bioconvection 24 , 25 . Among the other PSB species, all sharing a similar spherical cell shape with a diameter of 3–4 µm, Thiodictyon syntrophicum 26 is known for its ability to fix CO 2 27 and to form aggregates with SRB 28 . On the purely numerical level, GSB are the most abundant members of the BL, but because of their small size (< 1 µm), their contribution to the total biovolume is minor 19 . In this study, we analyzed the intra- and inter-seasonal influence of external meteorological factors such as air temperature, rainfall, net radiation and wind on the stability of stratification in the lake water column and the development of the BL anoxygenic phototroph community. The stability of the stratification was analyzed by regular measurements of the main physicochemical parameters of the water column using a multiparametric probe. At the same time, the anoxygenic phototrophs community of the BL was monitored by flow cytometry in combination with turbidity data recorded by the multiparametric probe. This intensive monitoring campaign (2019–2021) first enabled us to assess the stability of the water column and then determine the dynamics of the BL bacterial community. The variety of data collected allowed us to describe the dynamics on a seasonal scale, i.e., the time scale relevant to BL growth and stabilization. Moreover, the three consecutive years of monitoring provided an extended time scale to correlate the influence of abiotic factors, such as weather, on the BL development. Finally, access to more than 30 years of measurement data gave us valuable insights into the effect of climate change on the meromixis and the anoxygenic phototrophs community of the peculiar and intriguing Lake Cadagno. Materials and methods Study site, sampling and meteorological station Lake Cadagno is a crenogenic meromictic lake located in the Piora Valley at 1921 m a.s.l. in the southern Swiss Alps (46°33’N, 8°43’E and depth approximately 21m). The vertical profile of Fig. 1 , showing the redox stratification of Lake Cadagno was determined using a YSI 6000 profiler (Yellow Springs, Inc., USA) especially equipped with dissolved oxygen (mg l − 1 ), oxide reductive potential (ORP, mV) and turbidity (FTU, formazine turbidity unit). All other Physical parameters of the water column were determined using a multiparameter probe (CTD115M, Sea&Sun Technology, Germany) equipped with several sensors such as pressure, temperature, conductivity, dissolved oxygen, turbidity, Blue Green Algae Sensors: Phycocyanin (BGA-PC), in combination with a Tygon tube (20 m long, inner diameter 6.5 mm and volume 0.66 L) and a peristaltic pump (KNF Neuberger Inc., USA) for the BL sampling, as described in Di Nezio et al. 19 Turbidity served as a proxy for determining the position of the BL in the water column. Specifically, a consistent peak (> 10 FTU) in the turbidity profile was used as a physical signature of the BL and sampling depths were determined accordingly. Water samples were collected one meter above, at the top, 50 cm within, at the bottom and one meter below the BL. Samples were stored in 1.5 ml Eppendorf tubes for the flow cytometry analysis, in 50 mL Falcon tubes for the chemical analysis and in 12.0 ml glass vials containing zinc solution (4.0% ZnCl 2 ) to avoid the oxidation of the hydrogen sulfide. All samples were kept in the dark and analyzed within a few hours at the CBA facilities directly in Piora. Chemical analyses were done using Merck colorimetric kits following the user's manual and quantified by Spectroquant spectrophotometer. Atmospheric radiation data at 10 min resolution were retrieved from a meteorological station (istSOS; https://hydromet.supsi.ch/ ) close to the lake shore. This station is equipped with temperature and humidity sensors (Rotronic), a rainfall meter (1518 H3, Lambrecht), a pyranometer (CNR-4, Kipp&Zonen), and a weathervane-oriented anemometer (L14512, Lambrecht). Despite some technical issues in the first year of monitoring (2019, pyranometer and anemometer), we collected regular air temperature, solar radiation, and rainfall data for 3 consecutive years (Fig. 1 , left, missing red graph in 2019). The meteorological data used for analysis for the past 30 years were taken from the Copernicus Climate Change Service (C3S) (2017): ERA5- Fifth generation of ECMWF atmospheric reanalyses of the global climate. Copernicus Climate Change Service Climate Data Store (CDS). Flow cytometry A BD Accuri C6 cytometer (Becton Dickinson, San Jose, CA) equipped with two lasers (488 and 680 nm), two scatter detectors and four fluorescence detectors (laser 488 nm: FL1 = 533/30, FL2 = 585/40, FL3 = 670; laser 640 nm: FL4 = 675/25) was used for samples analysis. A threshold of 2000 on FSC-H was applied to exclude most of the unwanted abiotic particles. Furthermore, an FL3-A > 1100 threshold was applied to FL3 (red fluorescence) to discriminate cells emitting autofluorescence due to chlorophyll and bacteriochlorophyll. Phototrophic sulfur bacteria were enumerated by flowcytometry (FCM), measuring chlorophyll-like autofluorescence particle events as described by Danza et al. 22 . Definition of the cellular biovolume Cell volumes were calculated as in Tonolla et al. (2003) 29 . Biovolumes of bacterial cells were analyzed on images captured with a Zeiss Axiocam 305 color camera connected to a Zeiss Axio Scope A1 epifluorescence microscope (Zeiss, Germany) using the ZEN 2.6 (blue edition) imaging software (Zeiss, Germany). For each species, between 30 and 40 cells in the exponential phase were considered to determine the mean size. To give bacterial biovolumes a lake-wide representation, we converted concentrations obtained from the analysis above into dimensional volumes (m 3 ) by considering the BL thickness and the lake's surface area at its position. Physical Calculations The water density ( \({\rho }_{w}\) ; without bacteria) was calculated using the ionic water composition of Lake Cadagno 30 as implemented in Sepúlveda Steiner et al. (2021) 25 , represented as: $${\rho }_{w}\left(T,S\right)={\rho }_{w}^{{\prime }}\left(T\right)+\beta S$$ where \({\rho {\prime }}_{w}=999.84\hspace{0.17em}+\hspace{0.17em}6.55\times {10}^{-2}\hspace{0.17em}\text{T}\hspace{0.17em}-\hspace{0.17em}8.56\hspace{0.17em}\times \hspace{0.17em}{10}^{-3} {\text{T}}^{2}\hspace{0.17em}+ 5.94\hspace{0.17em}\times \hspace{0.17em}{10}^{-5}{\text{T}}^{3}\) is the temperature (T) dependent water density and β = 0.96 × 10 −3 kg g −1 is Lake Cadagno’s water haline contraction coefficient. Salinity (S) is obtained using the expression S = ακ 20 , where α = 0.72 × 10 −3 kg m −3 (µS cm −1 ) −1 is the ion-specific conductivity to salinity factor for Lake Cadagno 30 and κ 20 (µS cm −1 ) is conductivity normalized to 20°C. To quantify the overall water column stability, we calculated the Schmidt stability index (Sc) 31 , 32 . This quantity represents the amount of mechanical work per unit area required to vertically mix the water column of a density-stratified lake. Using the CTD data, Sc was calculated as 33 : $$Sc=\frac{1}{{A}_{o}}{\int }_{0}^{{z}_{bot}}g{\rho }_{w}\left(z-{z}_{v}\right)A\left(z\right) dz$$ where A o = 0.23 km 2 is the surface area, g = 9.81 m s − 2 is the gravitational acceleration, z bot is the lake bottom depth (21 m), A(z) is the lake’s hypsometric curve as a function of depth (z), and z v = 5.3 m is the depth of the lake’s center of volume. Results Weather during the 3 years of monitoring Weather readings were collected at a meteorological station near Lake Cadagno (< 100 m) at the Alpine Biology Center (CBA, coordinates 46°54’N, 8°71’E) foundation, equipped with different sensors such as air temperature and humidity, rainfall, wind, and irradiance (Fig. 2 ). Due to its geographical position in the Alps at 1921 m a.s.l., Lake Cadagno is covered with ice for almost half the year. In an average year, the lake freezes in early December and remains covered until mid-May (Fig. 2 , left, blue shadow) for about 5 months. Consequently, the sampling timeframe was constrained to the period between June and October. Access to the Piora Valley is only possible in snow-free conditions, precluding an extension of the sampling period into November or December. The meteorological conditions varied notably across the years 2019, 2020, and 2021, especially in spring 2020, which was extraordinarily warm, and in summer 2021, which recorded much more precipitation than usual in July (Table 1 , left panel). The 2019 spring was icy and rainy, resulting in a late lake defrost (June 2, 2019). On the contrary, 2020 saw an early ice melt (April 27, 2020) due to warm and sunny weather. Lastly, 2021 featured a fairly cold springtime with a late defrost (May 27, 2021), followed by an unusually cold and wet summer, particularly in July. In any case, the air temperature measured during the sampling period (Fig. 2 , left, blue graphs within the dashed lines) shows little difference between the three years considered. In this region of the Southern Alps, precipitation in July is usually low, under 100 mm per month, as shown by 2019 and 2020 data (Table 1 , left panel). Therefore, the values recorded in 2021 should be considered relatively uncommon, with almost 3 times more mm of water falling (Table 1 , left panel). Instead, August saw 3 times less precipitation than 2019 and 2020, and more generally, the situation found in August in the Piora Valley (Table 1 , left panel). One consequence of the high rainfall in July 2021 is the lower net radiation measured during that period, which was almost 40% inferior to that in 2020 (Table 1 , central panel). Interestingly, there is no increase in the total energy reaching the lake in August 2021 despite low precipitation, which remains similar to what was measured in 2021 (Table 1 , central panel). Note that measurements in 2019 from May to September are missing due to a pyranometer breakdown. The total sum of wind speed measured for each month does not appear to be correlated with either total rainfall or net radiation (Table 1 , right panel). Interestingly, however, the strong winds measured in August and September of 2019 were not found in the various months of 2020 and 2021. The wind sensor was installed in July 2019, so measurements for June and July are missing. In summary, we were confronted with three relatively different weather conditions during our monitoring. This allowed us to evaluate the effect of various abiotic factors on the physicochemical and microbiological stability of Lake Cadagno. Stratification stability: Water column monitoring When the lake was accessible for water column measurements (June to October), physicochemical parameters relevant to meromixis were monitored on a regular basis (Fig. 3 ). Temperature (Fig. 3 , first row, Temp. °C) and conductivity (Fig. 3 , second row, C 20 µS cm − 1 ) profiles provide information on the density structure defined by two distinct layers. The chemocline of the lake is located at 10–12 meters depth, with a transition marked by an increase in the concentration of dissolved ions, a water temperature nearly constant around 4°C, and oxygen dropping to anoxic conditions (Fig. 3 , third row, DO ml l − 1 ). These three physical-chemical parameters define the permanent stratification of Lake Cadagno. Even when the temperature of the upper layer becomes similar to that of the lower layer, the lake remains stratified, as shown in Fig. 3 . Even when the temperature profile shows homogeneous temperatures of about 4 degrees along the entire depth (0–18 m), meromixis is persistent. In fact, despite the same temperature, the density gradient is maintained by the difference in ion concentration between mixolimnion and monimolimnion (Fig. 3 , second row, C 20 µS cm − 1 ). Development of anoxygenic phototrophs forming the characteristic bacterial layer (BL; turbidity > 10 FTU) can be observed at about 12.0 m depth, where oxygen levels drop to zero (Fig. 3 , third row, DO ml l − 1 ). The turbidity data (Fig. 3 , fourth row, FTU) can serve as a proxy of the intra and inter-seasonal variability of the BL, both in terms of spatial distribution and intensity. We note the presence of a blue-green algae (BGA) community containing phycocyanin (PC), a photosynthetic pigment typical of these microorganisms (Fig. 3 , fifth row, BGA-PC ppb) about one meter above the BL, yet still in the chemocline zone with low concentration of oxygen (Fig. 3 , third row, DO ml l − 1 ). This population of aerobic photosynthetic microorganisms is constantly present throughout the summer season, exhibiting varying concentrations (Fig. 3 , fifth row, BGA-PC ppb). Fine-scale monitoring of the anoxygenic phototrophs community in the BL. One of the unique characteristics of the meromictic Lake Cadagno is the development of a BL with a high concentration of cells (up to 10 7 cells ml − 1 ) mainly composed of a heterogeneous community of anoxygenic phototrophic sulfur bacteria. The BL is best localized by the turbidity profile and is defined when a value greater than 10 FTU is observed (Fig. 3 , fourth row, FTU). The increase in turbidity above 10 FTU is generated by the wide variety of anoxygenic phototrophs (7 PSB and 2 GSB) concentrated in the upper part of the anoxic zone where light penetrates, referred to as the anoxygenic photosynthetic zone (APZ) 34 . During the sampling season (June to October), the BL can be as thick as 1.5 meters (Fig. 4 A, pink shadow). During the three years of monitoring, the main characteristics of the BL, such as depth, thickness and composition of the main populations of anoxygenic phototrophs, both in biovolume and cell number, were collected (Fig. 4 ). In addition, we measured the concentration of hydrogen sulfide (H 2 S), which is not only necessary for anoxygenic photosynthesis but also defines the redox gradient of the chemocline, which, however, does not seem to show specific patterns (Fig. 4 A, grey square graph). The biovolume of the whole BL is dominated by PSB C. okenii (Figs. 4 A, blue bars), mainly due to their large cell size (about 8.0–10.0 µm). The biovolume values contrast with the purely numerical counts (cells ml − 1 ), where the two GSB species ( Chlorobium phaeobacteroides and chlatratiforme ) dominate instead (Fig. 4 B-C-D, orange graph), although with a much smaller cell size (about 0.8-1.0 µm). Small cells PSB populations have lower biovolumes (approx. 2.0–4.0 µm) and numerical counts. Interestingly, both intra- and inter-seasonal population dynamics in the BL show different profiles. In 2019, C. okenii was the dominant population in the BL until the end of August, after which it appears that the GSB population increased in importance (Figs. 4 A and 4 B). Notably, with the decline in the number of PSB C. okenii and the increase in GSB, a substantial 50% reduction in the total biovolume was observed by the end of September compared to July. While less conspicuous, the small-cell PSB also exhibited an uptick in numbers during September (Fig. 4 B, red graph). This shift of populations reported in 2019 and in previous years 22 is no longer observed in 2020 and 2021. Up until the end of August 2020, the situation was similar to what we had observed in the past and 2019. However, we then witnessed a second growing phase of the PSB C. okenii population, which continued to dominate the BL at the expense of GSB (Figs. 4 A and 4 C). As a consequence, we did not observe a decrease in biovolume in September; instead, biovolume remains as high as in July. For their part, small cells PSB behaved similarly to 2019, with slight growth in September. However, the exact cause of this deviation from the typical ecological dynamics observed in September remains unknown. The lack of light associated with the exceptional rainfall in July 2021 (Table 1 ) affected the phototrophic communities in the BL, with a marked reduction in both biovolume and total cell number (Fig. 4 A and D). However, a significant increase in biovolume is observed (Fig. 4 A), mainly related to PSB C. okenii , which, in contrast, is not observable when considering cell numbers (Fig. 4 D). The calculation of biovolume is based on the amplitude of the BL measured in the lake (see material and methods), which is then multiplied by the number of cells counted by the flow cytometry and then normalized to the different cell sizes of the 3 distinct populations. Sampling on July 14, 2021, was carried out under adverse weather conditions, i.e., with rain and wind (a situation also encountered in the days before), which destabilized the BL by showing a turbidity value greater than 10 FTU for more than 2 meters (from 11.25 to 13.33 m). As a result, an unlikely biovolume exceeding 3.0 m 3 was generated, more than twice the value typically observed. Intense rainstorm events can alter the ecological dynamics of the BL. As shown before, weather conditions can influence the development of the BL anoxygenic phototrophic community (Figs. 3 and 4 ). To assess the ecological sensitivity of the system to changes in weather, we analyzed data from selected samplings conducted after rainfall events of varying intensity, using 2020 as the reference year (Fig. 5 and Table S1 ). We did, however, include some points for 2019 as a comparison to underscore the effect of the most intense rainstorm event in 2020. These events were accounted for by evaluating the precipitation observed seven days before the BL sampling (Fig. 5 , gray bars). We also considered total wind and net radiation, but these factors did not directly correlate with BL stability and are therefore not displayed in Fig. 5 (see Table S1 for more information). The turbidity profile shows a gradual increase until the end of August, with a peak in growth after the most intense rainfall observed in 2020 (August 29 and 30), followed by a decrease (Fig. 5 , pink graph). This situation is also found in the previous section, where the lack of reduction in the biovolume of the BL is emphasized due to the exceptional population growth of PSB C. okenii , which did not occur in the other monitoring years where instead, there was a marked decline in it (Fig. 4 ). Turbidity data measured in September 2019 were included to emphasize the difference from 2020 data (Fig. 5 , blurred pink rhombuses). The unusual peak of PSB C. okenii population growth (Fig. 4 A, 4 C and 5 , pink graph) corresponds with an extreme rainstorm detected on August 29 and 30, 2020 (Fig. 5 , gray bars). To unveil what changed after the intense rainstorm at the end of August, we first evaluated the position of the BL in the water column and found it to be around 12.0 m depth, i.e., about 1.0 m less in depth (Fig. 5 , black round plot). In addition, we noticed a sharp reduction in the presence of the BGAs community that is usually present about 1.0 m above the BL (Fig. 3 , fifth row; and Fig. 5 , green squares plot). This uplift in the position of the BL and the reduction in the concentration of BGAs community above it allowed for a sharp increase in light intensity in the APZ (Fig. 5 , yellow circles). Looking at the complete time window in Fig. 5 , the presence of the BGA community above the BL strongly influences the amount of light reaching the APZ. The inverse relationship between BGAs concentration and light intensity filtering down to the BL is further shown by the 2019 data, where high concentrations of the BGAs community correspond to low light intensities in the APZ (Fig. 5 , blurred green squares and blurred yellow circles). Finally, not all rainstorm events have a noticeable effect on the ecological dynamics of the BL. Excluding the exceptional event in late August 2020, no changes in the amount of light at the top of the BL are observed in the other cases. Discussion In this study, we analyze the influence of weather on the intra- and inter-seasonal stability of meromixis, as well as the ecological dynamics of the community of anoxygenic phototrophs living in the BL. The permanent stratification, aka meromixis, which makes Lake Cadagno a unique ecosystem for the study of anoxygenic microorganisms, does not seem to be threatened by any external meteorological event. Conversely, the ecological dynamics of the BL's three main phenotypic groups, carefully monitored between June and October in the years 2019, 2020, and 2021, allowed us to observe unexpected variability both intra- and inter-seasonally, which was constantly attributable to weather events different from the standard. Previous studies, often conducted over one or a few days of sampling, have generally reported a certain regularity in the shift from a strong presence of large cells PSB C. okenii in early summer to an increase in the other phenotypic groups, namely small cells PSB and two GSB species in September 22 , 23 , 34 , 35 . While this pattern was evident in 2019, distinct ecological dynamics were observed in 2020 and 2021 (Fig. 4 ). Weather events that were out of the ordinary, such as the case of August 29–30, 2020, with an intense rainstorm or prolonged rain periods as in July 2021, led to changes in the ecological dynamics of the BL's anoxygenic phototroph community. In both situations shown in this study, a change was observed in the amount of light reaching the BL (Table 1 and S1). Variations in light intensity are particularly significant for the ecology of anoxygenic phototrophs, as it is well known that GSB have an advantage over PSB at low intensities due to their superior antenna system 36 . In the case of the intense rainstorm event (Figs. 2 and 5 ), we saw how increased light intensity in the APZ allowed the PSB C. okenii cells to remain the dominant population of the BL (Fig. 4 C), contrary to what is usually observed (Fig. 4 B) 22 , 34 . This situation also has an effect on the whole trophic network of Lake Cadagno, given the higher biovolume (Fig. 4 A), intended as biomass (Fig. 3 , fourth row, turbidity), available to the rest of the heterotrophic microorganisms in the lake 9 , 10 . In fact, considering the total biovolume, PSB C. okenii emerges as the most relevant population of BL due to the significant size difference among the three microorganisms considered, with C. okenii (63.3 µm 3 ) 15 and 76 times more voluminous than T. syntrophicum (4.2 µm 3 ) and C. phaeobacteroides (0.8 µm 3 ), respectively. Interestingly, the reduction in light observed in July 2021 due to abundant rainfall (Fig. 2 and Table 1 ) did not result in an increase in GSB (Fig. 4 D). This is probably because, other than the reduction in light, a higher mixolimnion variability was also observed, as shown in Fig. 3 , where the temperature profile appears very heterogeneous, indicating frequent surface layer mixing. This stirring has the effect of increasing the oxygen concentration in the proximities above the BL (Fig. 3 , third row), which is more toxic to GSB 19 , 37 , 38 , compared with PSB, which are also often able to draw energy from it in the absence of light 27 , 39 – 41 . Moreover, during July, PSB C. okenii produced an additional mixing process in the BL – bioconvection 24 , 25 , 42 . The resulting (biogenic) turbulent mixing also displaces small PSB and GSB cells out of the APZ 34 , which are incapable of resisting the transport caused by the stirring as they move passively through gas vacuoles 8 , 43 . To summarize, weather conditions are essential for the ecological dynamics of BL anoxygenic phototroph organisms, as they determine the quantity and quality of light available to them in the APZ and, consequently, their growth rate. The development of the BL, along with other anoxygenic microorganisms in the monimolimnion, hinges on the lake maintaining its meromictic nature. A key question is thereby to evaluate how the stability of the meromixis and the structure of the lake can be impacted by external forcing. One way to estimate the stability of the water column of a lake is through the Schmidt stability index, which determines the energy required to mix the water column fully 31 , 32 . During the 3 years of monitoring, the value remained constant at 175 ± 0.02 kJ m − 2 , about 10 times higher than for lakes of similar size 44 . As long as the two-layer structure is maintained, this lake will remain a unique hotspot for studying anoxic life. Anoxygenic microorganisms found in the depths of the lake are potentially very similar to the primordial life forms that appeared on our planet and gave rise to the evolutionary process that is still ongoing. In the sediment and deepest dark zones, the SRB’s anoxygenic sulfur-based chemoheterotrophic metabolism allows them to thrive 45 . At the same time, in the lower part of the chemocline in the APZ, phototrophic sulfur bacteria rely on anoxygenic photosynthesis, a precursor to the more modern oxygenic photosynthesis observed in the BGA community at the upper part of the chemocline, where little oxygen is present 46 – 48 . Redox-stratified environments, such as euxinic or ferruginous systems 49 , 50 , are essential for implementing the biogeochemical knowledge of the primordial oceans of the Proterozoic era starting 2.5 billion years ago 51 , 52 . Given the pressing concern about climate change 53 , 54 , as average temperatures rise and extreme weather events become more frequent, is there a potential risk to the meromixis of Lake Cadagno? Historical evidence spanning more than 10,000 years, analyzed through sediment studies 15 , 55 , 56 , suggest the persistence of physical stratification and anoxygenic microorganisms 16 . Data collected over the past 30 years, from 1985 to 2021 (Fig. 6 ), shows relatively stable temperature (Fig. 6 A) and conductivity (Fig. 6 C) values in the water column, reflected in the elevated and constant Schmidt stability index (Fig. 6 B). Typically, PSB and GSB communities depend strongly on euxinic conditions and light availability. In this regard, it is interesting to note that the hydrogen sulfide (H 2 S) concentration profile underwent a major change at the beginning of the century (Fig. 6 D). In 2000, a violent hurricane named Lothar caused a partial mixing of the lake that altered the turbidity and light profiles, with the latter reduced by 10 times at the level of the BL 57 . This change in the water column led to a substantial increase in the number of cells in the BL due to the development of a "new" previously absent species of GSB, Chlorobium chlatratiforme 20 , 23 . The reduction in hydrogen sulfide (H 2 S) concentration observed in Fig. 6 D is probably related to the increase of one order of magnitude in the number of cells in the BL 19 . However, it is so far not clear how climate change, induced hydrological variations, will affect salt-rich groundwater inflow and sulfate (SO 4 2− ) discharge in the deep water 13 , which is then converted to hydrogen sulfide (H 2 S) by the SRB, an essential for the development of the BL community. Such changes in groundwater flow would profoundly affect both the stability of the water column as well as all anoxic microorganisms. Thus, further research is needed to assess the effect of climate change on the supply of salt-rich water to Lake Cadagno. Conclusions In conclusion, this study emphasizes the role of abiotic and biotic factors in the development of a specific ecosystem. Although the background stratification of this meromictic lake is barely affected by short-lived summer rainstorms, external meteorological factors modify the dynamic and composition of the BL anoxic phototrophic community. Ongoing climate change will continue to alter the long-term seasonal composition of the BL community, mainly through extreme wind-related phenomena 20 , 23 , 57 , yet without changing the stability of the stratification. Lake Cadagno has maintained its meromictic state over the past three decades, featuring a persistent anoxygenic monimolimnion while recording only minor changes in the Schmidt stability index. Although the change in heat content should not modify the mixing regime, the supply of salt-rich water from sublacustrine springs might decrease under climate change, leading to a modification of the supply of deep-water ions. To avoid further speculations, we recommend measuring the yearly dynamics of hydrogen sulfide (H 2 S) in the deep waters, its interaction with salt-rich groundwater recharge and how changes in rainfall and snowpack will affect groundwater flow into Lake Cadagno. Abbreviations BL: bacterial layer PSB: purple sulfur bacteria GSB: green sulfur bacteria SRB: sulfate-reducing bacteria ORP: oxide reductive potential FTU: Formazine Turbidity Unit BGA-PC: blue-green algae CBA: Alpine Biology Center m a.s.l.: meters above sea level CTD: Conductivity-Temperature-Depth APZ: anoxygenic photosynthetic zone Declarations Acknowledgments The project was funded by the Swiss National Fund (SNF) for Scientific Research (BIOCAD: no. 179264). The quality of the scientific equipment used during monitoring was made possible by cantonal funding for the mandate “Indagini, perizie e consulenza in Microbiologia ambientale” of the Department of “socialità e sanità” (DSS). We are grateful to the Alpine Biology Center Foundation (Switzerland) for the use of its research facilities. We also thank David Janssen, Aquatic Geochemistry group leader at Eawag, for his valuable contribution to revising the manuscript. Author contributions (names must be given as initials) N.S., O.S.S. and D.B. conceived, designed, conducted the study, organized and followed the sampling network, analyzed the data and wrote the manuscript. S.R., F.D.N. and A.B.D coordinated sample collection during the campaign period (2019-21) and conducted laboratory analysis and data interpretation. N.S. wrote the main manuscript text and N.S., O.S.S. and F. D. N. prepared figures. All authors reviewed the manuscript. Data availability statement (mandatory) All the data analyzed and used for making the figures are included in the article published as Excel sheets in the supplementary material section. 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Lake stratification in the Carpathian basin and its interesting biological consequences. Inland Waters 5 , 173–186 (2015). Barton, L. L. & Fauque, G. D. Chapter 2 Biochemistry, Physiology and Biotechnology of Sulfate‐Reducing Bacteria. Adv Appl Microbiol 68 , 41–98 (2009). Blankenship, R. E. Early Evolution of Photosynthesis. Plant Physiol 154 , 434–438 (2010). Sánchez-Baracaldo, P., Bianchini, G., Wilson, J. D. & Knoll, A. H. Cyanobacteria and biogeochemical cycles through Earth history. Trends Microbiol 30 , 143–157 (2022). Chen, G. et al. Reconstructing Earth’s atmospheric oxygenation history using machine learning. Nature Communications 2022 13:1 13 , 1–13 (2022). Swanner, E. D. et al. The biogeochemistry of ferruginous lakes and past ferruginous oceans. Earth Sci Rev 211 , 103430 (2020). Janssen, D. J. et al. Chromium Cycling in Redox-Stratified Basins Challenges δ53Cr Paleoredox Proxy Applications. Geophys Res Lett 49 , e2022GL099154 (2022). Poulton, S. W., Fralick, P. W. & Canfield, D. E. The transition to a sulphidic ocean ∼ 1.84 billion years ago. Nature 2004 431:7005 431 , 173–177 (2004). Scott, C. T. et al. Late Archean euxinic conditions before the rise of atmospheric oxygen. Geology 39 , 119–122 (2011). Gilarranz, L. J., Narwani, A., Odermatt, D., Siber, R. & Dakos, V. Regime shifts, trends, and variability of lake productivity at a global scale. Proc Natl Acad Sci U S A 119 , e2116413119 (2022). Kraemer, B. M. et al. Climate change drives widespread shifts in lake thermal habitat. Nature Climate Change 2021 11:6 11 , 521–529 (2021). Berg, J. S. et al. Ancient and Modern Geochemical Signatures in the 13,500-Year Sedimentary Record of Lake Cadagno. Front Earth Sci (Lausanne) 9 , 754888 (2022). Zander, P. D., Wirth, S. B., Gilli, A., Peduzzi, S. & Grosjean, M. Hyperspectral imaging sediment core scanning tracks high-resolution Holocene variations in (an)oxygenic phototrophic communities at Lake Cadagno, Swiss Alps. Biogeosciences 20 , 2221–2235 (2023). Tonolla, M., Peduzzi, R. & Hahn, D. Long-term population dynamics of phototrophic sulfur bacteria in the chemocline of Lake Cadagno, Switzerland. Appl Environ Microbiol 71 , 3544–50 (2005). Fernández Castro, B. et al. Inhibited vertical mixing and seasonal persistence of a thin cyanobacterial layer in a stratified lake. Aquat Sci 83 , 1–22 (2021). Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files 2020meteoBL.xlsx BiocadMeteodailyweekly.xlsx supplementarymaterialv231127.docx Table1.jpg Table 1. Monthly sum of rainfall [mm], net radiation [J m -2 ] and wind speed [m s -1 ] for the 3 years of monitoring. N/A (not applicable). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3744815","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":259476098,"identity":"91903f44-089e-458d-bd9b-84d933c78c49","order_by":0,"name":"Nicola Storelli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYDACZiB+AOM8KJCQAzMSwAiPFpAsD1ilgYQxYS0MqFoYEhuQBLECc3bmhw8Sahjs7dl7gQwDi/T5M3IPf3jAYJeHS4tlM5uxQcIxhsQenuNAhoFE7oYbeWkSCQzJxbi0GBzmYZNIYGNI4JFIAzJAWiRyzICuOgB3IXYt/xjsgVrYfwC1pMvPyDH+QFBLYhsDYw/QFlCIJTDcyAGR+LQA/ZLYJ5HYc+YYM8hhhhvOvAP6xSAZt5bzhx8++PDNxp69vY3xw4eKOnn59tzDH39U2OHUAgUSyBxQFBngV48OeEhTPgpGwSgYBcMeAADdZU7lhWYeoAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Applied Sciences and Arts of Southern Switzerland (SUPSI)","correspondingAuthor":true,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Storelli","suffix":""},{"id":259476099,"identity":"5ec84bf5-e223-4ee0-a185-4891b3cb8def","order_by":1,"name":"Oscar Sepúlveda Steiner","email":"","orcid":"","institution":"Swiss Federal Institute of Aquatic Science and Technology, Surface Waters – Research and Management","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"Sepúlveda","lastName":"Steiner","suffix":""},{"id":259476100,"identity":"0654479a-49c7-4360-b6a9-d0115a61bba2","order_by":2,"name":"Francesco Di Nezio","email":"","orcid":"","institution":"University of Applied Sciences and Arts of Southern Switzerland (SUPSI)","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"Di","lastName":"Nezio","suffix":""},{"id":259476101,"identity":"5a1f5189-259b-4f8e-bf4e-bc51256cf5b2","order_by":3,"name":"Samuele Roman","email":"","orcid":"","institution":"University of Applied Sciences and Arts of Southern Switzerland (SUPSI)","correspondingAuthor":false,"prefix":"","firstName":"Samuele","middleName":"","lastName":"Roman","suffix":""},{"id":259476102,"identity":"7d0727ed-e0cc-4d0a-8f5b-03a55dbd3beb","order_by":4,"name":"Antoine Buetti-Dinh","email":"","orcid":"","institution":"University of Applied Sciences and Arts of Southern Switzerland (SUPSI)","correspondingAuthor":false,"prefix":"","firstName":"Antoine","middleName":"","lastName":"Buetti-Dinh","suffix":""},{"id":259476103,"identity":"646bb9c4-b53b-4977-a876-f7be5d022557","order_by":5,"name":"Damien Bouffard","email":"","orcid":"","institution":"Swiss Federal Institute of Aquatic Science and Technology, Surface Waters – Research and Management","correspondingAuthor":false,"prefix":"","firstName":"Damien","middleName":"","lastName":"Bouffard","suffix":""}],"badges":[],"createdAt":"2023-12-12 16:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3744815/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3744815/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51664404,"identity":"e24c321a-221e-40e9-8e88-6b3542e01417","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":162976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVertical profile of the Lake Cadagno water column\u003c/strong\u003e. Physicochemical profile of the water column of meromictic Lake Cadagno on 09 July 2019 measured with a YSI 6000 profiler (Yellow Springs, Inc., USA). In black is the profile of oxide reductive potential (ORP; mV), in blue that of dissolved oxygen (O\u003csub\u003e2\u003c/sub\u003e; mg l\u003csup\u003e-1\u003c/sup\u003e), in green that of sulfide (H\u003csub\u003e2\u003c/sub\u003eS; mg l\u003csup\u003e-1\u003c/sup\u003e), and in red that of dissolved particles (turbidity; Formazine Turbidity Unit FTU).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/c223ccd17d39c7dcae0c6fee.png"},{"id":51664864,"identity":"9729320a-d990-4c42-a3d5-55256f5f6a83","added_by":"auto","created_at":"2024-02-26 21:11:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeteorological data from 2019 to 2021\u003c/strong\u003e. On the left, air temperature (blue) and net radiation (red) were measured throughout the year; the blue shadow indicates the presence of ice on the surface of Lake Cadagno. On the right, the daily precipitation (blue bars) was measured only during the sampling period, delimited by the dashed lines from June to October.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/fe3b34a90c2d4a2ea1692dc1.png"},{"id":51664409,"identity":"3e99d354-c4dc-48e7-9170-340a5c9d5d1b","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":538585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWater column profiles for years 2019, 2020 and 2021\u003c/strong\u003e. The profiles of water temperature (first row, Temp. °C), conductivity (second row, C\u003csub\u003e20\u003c/sub\u003e µS cm\u003csup\u003e-1\u003c/sup\u003e), dissolved oxygen (third row, DO ml l\u003csup\u003e-1\u003c/sup\u003e), turbidity (fourth row, Tu FTU) and Blue Green Algae Sensors: Phycocyanin (fifth row, BGA-PC ppb) are given for the year 2019 (first column), 2020 (second column) and 2021 (third column). The black lines in the various plots refer to the temperature of 10 °C (highest line) and 5 °C (lowest line). White bars on the upper part of each plot indicate the sampling date.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/5ae45ca2b8262778bd3d5d60.png"},{"id":51664408,"identity":"e38f07b1-8094-4127-9d6d-59a483e79afb","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":960547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonitoring of the bacterial layer (BL) main parameters\u003c/strong\u003e. Depth and width of the BL during the sampling period from June to October (A, pink shadow), and anoxygenic phototrophic community composition in terms of lake-wide biovolume for the 3 dominant phenotypic groups, i.e., the PSB \u003cem\u003eC. okenii\u003c/em\u003e (A, blue bar), other small cells PSB (A, yellow bar), and the two GSB species (A, orange bar) were reported in panel A for the 3 years of monitoring, upper 2019, middle 2020 and lower 2021. The hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) concentration represented by the gray-square graph (figure A) corresponds to the measured value at the bottom of the BL (\u0026gt; 10 FTU). The right panels depict the cell count (cells ml\u003csup\u003e-1\u003c/sup\u003e) of every dominant phenotypic group (\u003cem\u003eC. okenii\u003c/em\u003e = blue, small cells PSB = yellow and two GSB species = orange) at the top of the BL (\u0026gt; 10 FTU) for the 2019 (B), 2020 (C) and 2021 (D) field campaigns.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/03220adf220afc1940d54361.png"},{"id":51664413,"identity":"971232ad-6aca-4c8c-ae45-fb471a20209c","added_by":"auto","created_at":"2024-02-26 21:03:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":896699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfluence of rainstorm events on the chemocline microbial community\u003c/strong\u003e. The gray bars correspond to the amount of rainfall (mm total) measured in the seven days preceding the sampling day. Color lines with symbols indicate light values at the top of the BL (µE, yellow), turbidity values integrated through the entire BL (FTU, pink), and integrated concentration of blue-green algae up to 1.0 m above the BL (ppb, green) measured at the date indicated by the symbol. The position of the BL in the water column was defined by the depth where the turbidity exceeds 10 FTU (Top BL, black-dotted line). We included the values measured in September 2019 as control values to highlight the effect caused by an extreme rainstorm event (single blurred dots). All values are listed in Table S1 in the supplementary materials.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/7bb8e52cbf2059a32eda09a0.png"},{"id":51664412,"identity":"85de14f4-06cc-49ff-98d5-96a579ca6368","added_by":"auto","created_at":"2024-02-26 21:03:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":324776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecent historical trends in Lake Cadagno\u003c/strong\u003e. (A) Temperature (°C) of water at the surface (red dots) and bottom (blue squares) recorded in July, accompanied by the air temperature obtained from ERA5-land weather reanalysis (gray line). (B) Schmidt stability (Sc; kJ \u003csup\u003em-2\u003c/sup\u003e) obtained from the CTD profiles and following Eq. 3 in Fernández Castro et al. (2021)\u003csup\u003e58\u003c/sup\u003e. (C) Conductivity normalized to 20°C (𝜅20; µS cm\u003csup\u003e-1\u003c/sup\u003e) at the surface (red dots) and bottom (blue squares). (D) Hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS; mg l\u003csup\u003e-1\u003c/sup\u003e) concentration at the lake bottom.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/aa37cf0f648f08fddaf85357.png"},{"id":57284967,"identity":"361deb47-abd2-4e09-9930-0763a70b95f5","added_by":"auto","created_at":"2024-05-28 15:59:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4193238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/d6fb337d-4428-4b0f-9fcf-2864bd9a622d.pdf"},{"id":51664406,"identity":"40135446-a2fe-4d37-9ae2-c89b7b9a1eb3","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101064,"visible":true,"origin":"","legend":"","description":"","filename":"2020meteoBL.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/805cdfa8beb7db65e1828612.xlsx"},{"id":51664407,"identity":"f88106a6-8608-47a2-8628-c906178aa254","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":119415,"visible":true,"origin":"","legend":"","description":"","filename":"BiocadMeteodailyweekly.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/a757711ef20e3aaeb9875162.xlsx"},{"id":51664411,"identity":"f29ef643-5bd9-4f01-946f-77e9d1a5ef07","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":379604,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialv231127.docx","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/7c9532d0f768004af346732e.docx"},{"id":51664410,"identity":"3703ed86-8233-42ab-811f-44fe2445f58e","added_by":"auto","created_at":"2024-02-26 21:03:19","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":64486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Monthly sum of rainfall [mm], net radiation [J m\u003csup\u003e-2\u003c/sup\u003e] and wind speed [m s\u003csup\u003e-1\u003c/sup\u003e] for the 3 years of monitoring. N/A (not applicable).\u003c/p\u003e","description":"","filename":"Table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3744815/v1/dabcd7d74fbe75e685ed75d4.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ecological dynamics of anoxygenic phototrophs in stably redox-stratified waters: Intra and inter-seasonal variability of Lake Cadagno","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe structure of the water column in lakes is primarily influenced by the distribution of temperature and concentration of dissolved ions, which control the water density and, thereby, stratification\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The type of stratification is either temporary (holomictic) or permanent (meromictic). Most lakes are holomictic and are divided into three subcategories, according to the water overturning tendency: monomictic lakes, dimictic lakes and polymictic lakes, all related to atmospheric temperature seasonality. In contrast, the stratification of meromictic lakes is mainly controlled by ion content in the water column. There are less than 200 documented meromictic lakes worldwide, although there are probably more, but they are not monitored\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Permanently stratified lakes are usually divided into three parts: 1) the lower layer, called monimolimnion, is anoxic and rich in dissolved ions with poor hydrodynamic circulation; 2) the upper layer, known as mixolimnion, behaves essentially as a holomictic lake directly influenced by the meteorological forcings (mainly air temperature); and 3) the region in between referred as chemocline, which hosts a steep chemical gradient with inversion of the redox potential. Due to the lack of mixing, the permanent stratification leads to a stable anoxic environment in the monimolimnion. This is an ideal spot to study biogeochemical processes mediated by microorganisms, such as the sulfur and nitrogen cycles that usually occur in anaerobiosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the few cases of meromictic lakes where light can penetrate the anoxic depths of the monimolimnion, dense bacterial layers (BLs) are formed and primarily composed of anoxygenic phototrophic sulfur bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These anoxygenic phototrophs are also ecologically vital, as they provide an additional food source for the lake's trophic chain\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCadagno is a crenogenic meromictic lake whose stratification is controlled by deep, ion-rich, subaquatic inflows \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The lake owes this characteristic to the peculiar geological conformation of the Piora Valley (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This formation comprises Triassic carbonate rocks, including tectonized dolomitic limestones and gypsum deposits with Karstic hydrology. As a result, the water flowing into the lake is enriched with salts, giving it its unique properties\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The water that seeps in through the karst dolomite and re-emerges from sub-lacustrine springs in the southern region of the lake has a high ionic content (9\u0026ndash;10 mM; Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e, SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and supplies the anoxic lower part (monimolimnion) of the water column\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The deep water of the monimolimnion remains isolated from the rest of the lake, resulting in the development of a stable anoxic environment that, according to the sediment records, dates back to 10,000 years\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The primary source of the mixolimnion surface water is a small stream flowing from Lake Stabbio, a small lake located 2351 m above sea level (a.s.l.). The crystalline rocks (gneiss) in its catchment area are chemical-resistant, so the water in the mixolimnion has a low ionic strength. The difference in density between the two water layers in Lake Cadagno results in the development of a very stable stratification with a chemocline around 10\u0026ndash;12 m depth characterized by a rapid change in the concentrations of chemical components, producing a redox stratification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe high concentrations of sulfate (\u0026gt;\u0026thinsp;80 mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) coming from the sublacustrine springs promoted the development of chemoheterotrophic anoxygenic sulfate-reducing bacteria (SRB). The metabolism of SRB leads to the production of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) in the deep layers of the lake (e.g., monimolimnion and sediment). When H\u003csub\u003e2\u003c/sub\u003eS reaches the illuminated part of the anoxic layer, it is used by phototrophic sulfur bacteria as an electron donor for anoxygenic photosynthesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This leads to the development of a highly concentrated bacterial layer (BL; turbidity\u0026thinsp;\u0026gt;\u0026thinsp;10 FTU) composed of purple (PSB) and green (GSB) sulfur bacteria species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, red line)\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This light-dependent community is dynamic and concentrated in the photic zone of the anoxic layer. During the year when the lake is not covered by snow and ice, the PSB and GSB communities form a pink-colored BL that can exceed 1 m in thickness. This ecological niche hosts a distinctly heterogeneous community of anoxygenic phototrophic sulfur bacteria utilizing different evolutionary strategies. The BL of Lake Cadagno harbors rich biodiversity, hosting at least 7 species of purple sulfur bacteria (PSB) and 2 species of green sulfur bacteria (GSB)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. One important species is the PSB \u003cem\u003eChromatium okenii\u003c/em\u003e, which accounts for most of the BL biovolume due to its large cell size (8\u0026ndash;10 \u0026micro;m main rod axis length). Moreover, the microorganism's flagella-driven motility towards light (positive phototaxis) and repulsion to oxygen (negative aerotaxis) results in the accumulation of a high cell concentration zone at the top of the BL. This leads to an increase in the local (physical) density of the water-bacteria mixture that exceeds the ambient water density, causing the heavier fluid to sink and drag the microorganisms down \u0026ndash; a phenomenon known as bioconvection\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Among the other PSB species, all sharing a similar spherical cell shape with a diameter of 3\u0026ndash;4 \u0026micro;m, \u003cem\u003eThiodictyon syntrophicum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e is known for its ability to fix CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e27\u003c/sup\u003e and to form aggregates with SRB\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. On the purely numerical level, GSB are the most abundant members of the BL, but because of their small size (\u0026lt;\u0026thinsp;1 \u0026micro;m), their contribution to the total biovolume is minor\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we analyzed the intra- and inter-seasonal influence of external meteorological factors such as air temperature, rainfall, net radiation and wind on the stability of stratification in the lake water column and the development of the BL anoxygenic phototroph community. The stability of the stratification was analyzed by regular measurements of the main physicochemical parameters of the water column using a multiparametric probe. At the same time, the anoxygenic phototrophs community of the BL was monitored by flow cytometry in combination with turbidity data recorded by the multiparametric probe. This intensive monitoring campaign (2019\u0026ndash;2021) first enabled us to assess the stability of the water column and then determine the dynamics of the BL bacterial community. The variety of data collected allowed us to describe the dynamics on a seasonal scale, i.e., the time scale relevant to BL growth and stabilization. Moreover, the three consecutive years of monitoring provided an extended time scale to correlate the influence of abiotic factors, such as weather, on the BL development. Finally, access to more than 30 years of measurement data gave us valuable insights into the effect of climate change on the meromixis and the anoxygenic phototrophs community of the peculiar and intriguing Lake Cadagno.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy site, sampling and meteorological station\u003c/h2\u003e \u003cp\u003eLake Cadagno is a crenogenic meromictic lake located in the Piora Valley at 1921 m a.s.l. in the southern Swiss Alps (46\u0026deg;33\u0026rsquo;N, 8\u0026deg;43\u0026rsquo;E and depth approximately 21m). The vertical profile of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, showing the redox stratification of Lake Cadagno was determined using a YSI 6000 profiler (Yellow Springs, Inc., USA) especially equipped with dissolved oxygen (mg l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), oxide reductive potential (ORP, mV) and turbidity (FTU, formazine turbidity unit). All other Physical parameters of the water column were determined using a multiparameter probe (CTD115M, Sea\u0026amp;Sun Technology, Germany) equipped with several sensors such as pressure, temperature, conductivity, dissolved oxygen, turbidity, Blue Green Algae Sensors: Phycocyanin (BGA-PC), in combination with a Tygon tube (20 m long, inner diameter 6.5 mm and volume 0.66 L) and a peristaltic pump (KNF Neuberger Inc., USA) for the BL sampling, as described in Di Nezio et al.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTurbidity served as a proxy for determining the position of the BL in the water column. Specifically, a consistent peak (\u0026gt;\u0026thinsp;10 FTU) in the turbidity profile was used as a physical signature of the BL and sampling depths were determined accordingly. Water samples were collected one meter above, at the top, 50 cm within, at the bottom and one meter below the BL. Samples were stored in 1.5 ml Eppendorf tubes for the flow cytometry analysis, in 50 mL Falcon tubes for the chemical analysis and in 12.0 ml glass vials containing zinc solution (4.0% ZnCl\u003csub\u003e2\u003c/sub\u003e) to avoid the oxidation of the hydrogen sulfide. All samples were kept in the dark and analyzed within a few hours at the CBA facilities directly in Piora. Chemical analyses were done using Merck colorimetric kits following the user's manual and quantified by Spectroquant spectrophotometer.\u003c/p\u003e \u003cp\u003eAtmospheric radiation data at 10 min resolution were retrieved from a meteorological station (istSOS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hydromet.supsi.ch/\u003c/span\u003e\u003cspan address=\"https://hydromet.supsi.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) close to the lake shore. This station is equipped with temperature and humidity sensors (Rotronic), a rainfall meter (1518 H3, Lambrecht), a pyranometer (CNR-4, Kipp\u0026amp;Zonen), and a weathervane-oriented anemometer (L14512, Lambrecht). Despite some technical issues in the first year of monitoring (2019, pyranometer and anemometer), we collected regular air temperature, solar radiation, and rainfall data for 3 consecutive years (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, left, missing red graph in 2019). The meteorological data used for analysis for the past 30 years were taken from the Copernicus Climate Change Service (C3S) (2017): ERA5- Fifth generation of ECMWF atmospheric reanalyses of the global climate. Copernicus Climate Change Service Climate Data Store (CDS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eA BD Accuri C6 cytometer (Becton Dickinson, San Jose, CA) equipped with two lasers (488 and 680 nm), two scatter detectors and four fluorescence detectors (laser 488 nm: FL1\u0026thinsp;=\u0026thinsp;533/30, FL2\u0026thinsp;=\u0026thinsp;585/40, FL3\u0026thinsp;=\u0026thinsp;670; laser 640 nm: FL4\u0026thinsp;=\u0026thinsp;675/25) was used for samples analysis. A threshold of 2000 on FSC-H was applied to exclude most of the unwanted abiotic particles. Furthermore, an FL3-A\u0026thinsp;\u0026gt;\u0026thinsp;1100 threshold was applied to FL3 (red fluorescence) to discriminate cells emitting autofluorescence due to chlorophyll and bacteriochlorophyll. Phototrophic sulfur bacteria were enumerated by flowcytometry (FCM), measuring chlorophyll-like autofluorescence particle events as described by Danza et al. \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDefinition of the cellular biovolume\u003c/h2\u003e \u003cp\u003eCell volumes were calculated as in Tonolla et al. (2003)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Biovolumes of bacterial cells were analyzed on images captured with a Zeiss Axiocam 305 color camera connected to a Zeiss Axio Scope A1 epifluorescence microscope (Zeiss, Germany) using the ZEN 2.6 (blue edition) imaging software (Zeiss, Germany). For each species, between 30 and 40 cells in the exponential phase were considered to determine the mean size.\u003c/p\u003e \u003cp\u003eTo give bacterial biovolumes a lake-wide representation, we converted concentrations obtained from the analysis above into \u003cem\u003edimensional\u003c/em\u003e volumes (m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) by considering the BL thickness and the lake's surface area at its position.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhysical Calculations\u003c/h2\u003e \u003cp\u003eThe water density (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\rho }_{w}\\)\u003c/span\u003e\u003c/span\u003e; without bacteria) was calculated using the ionic water composition of Lake Cadagno\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e as implemented in Sep\u0026uacute;lveda Steiner et al. (2021)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, represented as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$${\\rho }_{w}\\left(T,S\\right)={\\rho }_{w}^{{\\prime }}\\left(T\\right)+\\beta S$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\rho {\\prime }}_{w}=999.84\\hspace{0.17em}+\\hspace{0.17em}6.55\\times {10}^{-2}\\hspace{0.17em}\\text{T}\\hspace{0.17em}-\\hspace{0.17em}8.56\\hspace{0.17em}\\times \\hspace{0.17em}{10}^{-3} {\\text{T}}^{2}\\hspace{0.17em}+ 5.94\\hspace{0.17em}\\times \\hspace{0.17em}{10}^{-5}{\\text{T}}^{3}\\)\u003c/span\u003e\u003c/span\u003e is the temperature (T) dependent water density and β\u0026thinsp;=\u0026thinsp;0.96 \u0026times; 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e kg g\u003csup\u003e\u0026minus;1\u003c/sup\u003e is Lake Cadagno\u0026rsquo;s water haline contraction coefficient. Salinity (S) is obtained using the expression S\u0026thinsp;=\u0026thinsp;ακ\u003csub\u003e20\u003c/sub\u003e, where α\u0026thinsp;=\u0026thinsp;0.72 \u0026times; 10\u003csup\u003e\u0026minus;3\u003c/sup\u003e kg m\u003csup\u003e\u0026minus;3\u003c/sup\u003e (\u0026micro;S cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003csup\u003e\u0026minus;1\u003c/sup\u003e is the ion-specific conductivity to salinity factor for Lake Cadagno\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and κ\u003csub\u003e20\u003c/sub\u003e (\u0026micro;S cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e) is conductivity normalized to 20\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo quantify the overall water column stability, we calculated the Schmidt stability index (Sc)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This quantity represents the amount of mechanical work per unit area required to vertically mix the water column of a density-stratified lake. Using the CTD data, Sc was calculated as\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$Sc=\\frac{1}{{A}_{o}}{\\int }_{0}^{{z}_{bot}}g{\\rho }_{w}\\left(z-{z}_{v}\\right)A\\left(z\\right) dz$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003eo\u003c/sub\u003e = 0.23 km\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e is the surface area, g\u0026thinsp;=\u0026thinsp;9.81 m s\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e is the gravitational acceleration, z\u003csub\u003ebot\u003c/sub\u003e is the lake bottom depth (21 m), A(z) is the lake\u0026rsquo;s hypsometric curve as a function of depth (z), and z\u003csub\u003ev\u003c/sub\u003e = 5.3 m is the depth of the lake\u0026rsquo;s center of volume.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eWeather during the 3 years of monitoring\u003c/h2\u003e\n\u003cp\u003eWeather readings were collected at a meteorological station near Lake Cadagno (\u0026lt;\u0026thinsp;100 m) at the Alpine Biology Center (CBA, coordinates 46\u0026deg;54\u0026rsquo;N, 8\u0026deg;71\u0026rsquo;E) foundation, equipped with different sensors such as air temperature and humidity, rainfall, wind, and irradiance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eDue to its geographical position in the Alps at 1921 m a.s.l., Lake Cadagno is covered with ice for almost half the year. In an average year, the lake freezes in early December and remains covered until mid-May (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, left, blue shadow) for about 5 months. Consequently, the sampling timeframe was constrained to the period between June and October. Access to the Piora Valley is only possible in snow-free conditions, precluding an extension of the sampling period into November or December.\u003c/p\u003e\n\u003cp\u003eThe meteorological conditions varied notably across the years 2019, 2020, and 2021, especially in spring 2020, which was extraordinarily warm, and in summer 2021, which recorded much more precipitation than usual in July (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel). The 2019 spring was icy and rainy, resulting in a late lake defrost (June 2, 2019). On the contrary, 2020 saw an early ice melt (April 27, 2020) due to warm and sunny weather. Lastly, 2021 featured a fairly cold springtime with a late defrost (May 27, 2021), followed by an unusually cold and wet summer, particularly in July. In any case, the air temperature measured during the sampling period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, left, blue graphs within the dashed lines) shows little difference between the three years considered.\u003c/p\u003e\n\u003cp\u003eIn this region of the Southern Alps, precipitation in July is usually low, under 100 mm per month, as shown by 2019 and 2020 data (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel). Therefore, the values recorded in 2021 should be considered relatively uncommon, with almost 3 times more mm of water falling (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel). Instead, August saw 3 times less precipitation than 2019 and 2020, and more generally, the situation found in August in the Piora Valley (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, left panel). One consequence of the high rainfall in July 2021 is the lower net radiation measured during that period, which was almost 40% inferior to that in 2020 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, central panel). Interestingly, there is no increase in the total energy reaching the lake in August 2021 despite low precipitation, which remains similar to what was measured in 2021 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, central panel). Note that measurements in 2019 from May to September are missing due to a pyranometer breakdown.\u003c/p\u003e\n\u003cp\u003eThe total sum of wind speed measured for each month does not appear to be correlated with either total rainfall or net radiation (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, right panel). Interestingly, however, the strong winds measured in August and September of 2019 were not found in the various months of 2020 and 2021. The wind sensor was installed in July 2019, so measurements for June and July are missing.\u003c/p\u003e\n\u003cp\u003eIn summary, we were confronted with three relatively different weather conditions during our monitoring. This allowed us to evaluate the effect of various abiotic factors on the physicochemical and microbiological stability of Lake Cadagno.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eStratification stability: Water column monitoring\u003c/h2\u003e\n\u003cp\u003eWhen the lake was accessible for water column measurements (June to October), physicochemical parameters relevant to meromixis were monitored on a regular basis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Temperature (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, first row, Temp. \u0026deg;C) and conductivity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, second row, C\u003csub\u003e20\u003c/sub\u003e \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) profiles provide information on the density structure defined by two distinct layers. The chemocline of the lake is located at 10\u0026ndash;12 meters depth, with a transition marked by an increase in the concentration of dissolved ions, a water temperature nearly constant around 4\u0026deg;C, and oxygen dropping to anoxic conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, third row, DO ml l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). These three physical-chemical parameters define the permanent stratification of Lake Cadagno. Even when the temperature of the upper layer becomes similar to that of the lower layer, the lake remains stratified, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Even when the temperature profile shows homogeneous temperatures of about 4 degrees along the entire depth (0\u0026ndash;18 m), meromixis is persistent. In fact, despite the same temperature, the density gradient is maintained by the difference in ion concentration between mixolimnion and monimolimnion (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, second row, C\u003csub\u003e20\u003c/sub\u003e \u0026micro;S cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eDevelopment of anoxygenic phototrophs forming the characteristic bacterial layer (BL; turbidity\u0026thinsp;\u0026gt;\u0026thinsp;10 FTU) can be observed at about 12.0 m depth, where oxygen levels drop to zero (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, third row, DO ml l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The turbidity data (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, fourth row, FTU) can serve as a proxy of the intra and inter-seasonal variability of the BL, both in terms of spatial distribution and intensity. We note the presence of a blue-green algae (BGA) community containing phycocyanin (PC), a photosynthetic pigment typical of these microorganisms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, fifth row, BGA-PC ppb) about one meter above the BL, yet still in the chemocline zone with low concentration of oxygen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, third row, DO ml l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This population of aerobic photosynthetic microorganisms is constantly present throughout the summer season, exhibiting varying concentrations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, fifth row, BGA-PC ppb).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFine-scale monitoring of the anoxygenic phototrophs community in the BL.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne of the unique characteristics of the meromictic Lake Cadagno is the development of a BL with a high concentration of cells (up to 10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) mainly composed of a heterogeneous community of anoxygenic phototrophic sulfur bacteria. The BL is best localized by the turbidity profile and is defined when a value greater than 10 FTU is observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, fourth row, FTU). The increase in turbidity above 10 FTU is generated by the wide variety of anoxygenic phototrophs (7 PSB and 2 GSB) concentrated in the upper part of the anoxic zone where light penetrates, referred to as the anoxygenic photosynthetic zone (APZ)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. During the sampling season (June to October), the BL can be as thick as 1.5 meters (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, pink shadow).\u003c/p\u003e\n\u003cp\u003eDuring the three years of monitoring, the main characteristics of the BL, such as depth, thickness and composition of the main populations of anoxygenic phototrophs, both in biovolume and cell number, were collected (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). In addition, we measured the concentration of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS), which is not only necessary for anoxygenic photosynthesis but also defines the redox gradient of the chemocline, which, however, does not seem to show specific patterns (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, grey square graph).\u003c/p\u003e\n\u003cp\u003eThe biovolume of the whole BL is dominated by PSB \u003cem\u003eC. okenii\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, blue bars), mainly due to their large cell size (about 8.0\u0026ndash;10.0 \u0026micro;m). The biovolume values contrast with the purely numerical counts (cells ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), where the two GSB species (\u003cem\u003eChlorobium phaeobacteroides\u003c/em\u003e and \u003cem\u003echlatratiforme\u003c/em\u003e) dominate instead (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-C-D, orange graph), although with a much smaller cell size (about 0.8-1.0 \u0026micro;m). Small cells PSB populations have lower biovolumes (approx. 2.0\u0026ndash;4.0 \u0026micro;m) and numerical counts.\u003c/p\u003e\n\u003cp\u003eInterestingly, both intra- and inter-seasonal population dynamics in the BL show different profiles. In 2019, \u003cem\u003eC. okenii\u003c/em\u003e was the dominant population in the BL until the end of August, after which it appears that the GSB population increased in importance (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Notably, with the decline in the number of PSB \u003cem\u003eC. okenii\u003c/em\u003e and the increase in GSB, a substantial 50% reduction in the total biovolume was observed by the end of September compared to July. While less conspicuous, the small-cell PSB also exhibited an uptick in numbers during September (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, red graph).\u003c/p\u003e\n\u003cp\u003eThis shift of populations reported in 2019 and in previous years\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e is no longer observed in 2020 and 2021. Up until the end of August 2020, the situation was similar to what we had observed in the past and 2019. However, we then witnessed a second growing phase of the PSB \u003cem\u003eC. okenii\u003c/em\u003e population, which continued to dominate the BL at the expense of GSB (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). As a consequence, we did not observe a decrease in biovolume in September; instead, biovolume remains as high as in July. For their part, small cells PSB behaved similarly to 2019, with slight growth in September. However, the exact cause of this deviation from the typical ecological dynamics observed in September remains unknown.\u003c/p\u003e\n\u003cp\u003eThe lack of light associated with the exceptional rainfall in July 2021 (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) affected the phototrophic communities in the BL, with a marked reduction in both biovolume and total cell number (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and D). However, a significant increase in biovolume is observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), mainly related to PSB \u003cem\u003eC. okenii\u003c/em\u003e, which, in contrast, is not observable when considering cell numbers (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). The calculation of biovolume is based on the amplitude of the BL measured in the lake (see material and methods), which is then multiplied by the number of cells counted by the flow cytometry and then normalized to the different cell sizes of the 3 distinct populations. Sampling on July 14, 2021, was carried out under adverse weather conditions, i.e., with rain and wind (a situation also encountered in the days before), which destabilized the BL by showing a turbidity value greater than 10 FTU for more than 2 meters (from 11.25 to 13.33 m). As a result, an unlikely biovolume exceeding 3.0 m\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e was generated, more than twice the value typically observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntense rainstorm events can alter the ecological dynamics of the BL.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown before, weather conditions can influence the development of the BL anoxygenic phototrophic community (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). To assess the ecological sensitivity of the system to changes in weather, we analyzed data from selected samplings conducted after rainfall events of varying intensity, using 2020 as the reference year (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). We did, however, include some points for 2019 as a comparison to underscore the effect of the most intense rainstorm event in 2020. These events were accounted for by evaluating the precipitation observed seven days before the BL sampling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, gray bars). We also considered total wind and net radiation, but these factors did not directly correlate with BL stability and are therefore not displayed in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e (see Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e for more information).\u003c/p\u003e\n\u003cp\u003eThe turbidity profile shows a gradual increase until the end of August, with a peak in growth after the most intense rainfall observed in 2020 (August 29 and 30), followed by a decrease (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, pink graph). This situation is also found in the previous section, where the lack of reduction in the biovolume of the BL is emphasized due to the exceptional population growth of PSB \u003cem\u003eC. okenii\u003c/em\u003e, which did not occur in the other monitoring years where instead, there was a marked decline in it (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Turbidity data measured in September 2019 were included to emphasize the difference from 2020 data (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, blurred pink rhombuses). The unusual peak of PSB \u003cem\u003eC. okenii\u003c/em\u003e population growth (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, pink graph) corresponds with an extreme rainstorm detected on August 29 and 30, 2020 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, gray bars).\u003c/p\u003e\n\u003cp\u003eTo unveil what changed after the intense rainstorm at the end of August, we first evaluated the position of the BL in the water column and found it to be around 12.0 m depth, i.e., about 1.0 m less in depth (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, black round plot). In addition, we noticed a sharp reduction in the presence of the BGAs community that is usually present about 1.0 m above the BL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, fifth row; and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, green squares plot). This uplift in the position of the BL and the reduction in the concentration of BGAs community above it allowed for a sharp increase in light intensity in the APZ (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, yellow circles). Looking at the complete time window in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, the presence of the BGA community above the BL strongly influences the amount of light reaching the APZ. The inverse relationship between BGAs concentration and light intensity filtering down to the BL is further shown by the 2019 data, where high concentrations of the BGAs community correspond to low light intensities in the APZ (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, blurred green squares and blurred yellow circles). Finally, not all rainstorm events have a noticeable effect on the ecological dynamics of the BL. Excluding the exceptional event in late August 2020, no changes in the amount of light at the top of the BL are observed in the other cases.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we analyze the influence of weather on the intra- and inter-seasonal stability of meromixis, as well as the ecological dynamics of the community of anoxygenic phototrophs living in the BL. The permanent stratification, aka meromixis, which makes Lake Cadagno a unique ecosystem for the study of anoxygenic microorganisms, does not seem to be threatened by any external meteorological event. Conversely, the ecological dynamics of the BL's three main phenotypic groups, carefully monitored between June and October in the years 2019, 2020, and 2021, allowed us to observe unexpected variability both intra- and inter-seasonally, which was constantly attributable to weather events different from the standard.\u003c/p\u003e \u003cp\u003ePrevious studies, often conducted over one or a few days of sampling, have generally reported a certain regularity in the shift from a strong presence of large cells PSB \u003cem\u003eC. okenii\u003c/em\u003e in early summer to an increase in the other phenotypic groups, namely small cells PSB and two GSB species in September\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. While this pattern was evident in 2019, distinct ecological dynamics were observed in 2020 and 2021 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Weather events that were out of the ordinary, such as the case of August 29\u0026ndash;30, 2020, with an intense rainstorm or prolonged rain periods as in July 2021, led to changes in the ecological dynamics of the BL's anoxygenic phototroph community. In both situations shown in this study, a change was observed in the amount of light reaching the BL (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1).\u003c/p\u003e \u003cp\u003eVariations in light intensity are particularly significant for the ecology of anoxygenic phototrophs, as it is well known that GSB have an advantage over PSB at low intensities due to their superior antenna system\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the case of the intense rainstorm event (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), we saw how increased light intensity in the APZ allowed the PSB \u003cem\u003eC. okenii\u003c/em\u003e cells to remain the dominant population of the BL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), contrary to what is usually observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This situation also has an effect on the whole trophic network of Lake Cadagno, given the higher biovolume (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), intended as biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, fourth row, turbidity), available to the rest of the heterotrophic microorganisms in the lake\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In fact, considering the total biovolume, PSB \u003cem\u003eC. okenii\u003c/em\u003e emerges as the most relevant population of BL due to the significant size difference among the three microorganisms considered, with \u003cem\u003eC. okenii\u003c/em\u003e (63.3 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) 15 and 76 times more voluminous than \u003cem\u003eT. syntrophicum\u003c/em\u003e (4.2 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) and \u003cem\u003eC. phaeobacteroides\u003c/em\u003e (0.8 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e), respectively. Interestingly, the reduction in light observed in July 2021 due to abundant rainfall (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) did not result in an increase in GSB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This is probably because, other than the reduction in light, a higher mixolimnion variability was also observed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, where the temperature profile appears very heterogeneous, indicating frequent surface layer mixing. This stirring has the effect of increasing the oxygen concentration in the proximities above the BL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, third row), which is more toxic to GSB \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, compared with PSB, which are also often able to draw energy from it in the absence of light\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Moreover, during July, PSB \u003cem\u003eC. okenii\u003c/em\u003e produced an additional mixing process in the BL \u0026ndash; bioconvection\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The resulting (biogenic) turbulent mixing also displaces small PSB and GSB cells out of the APZ\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, which are incapable of resisting the transport caused by the stirring as they move passively through gas vacuoles\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To summarize, weather conditions are essential for the ecological dynamics of BL anoxygenic phototroph organisms, as they determine the quantity and quality of light available to them in the APZ and, consequently, their growth rate.\u003c/p\u003e \u003cp\u003eThe development of the BL, along with other anoxygenic microorganisms in the monimolimnion, hinges on the lake maintaining its meromictic nature. A key question is thereby to evaluate how the stability of the meromixis and the structure of the lake can be impacted by external forcing. One way to estimate the stability of the water column of a lake is through the Schmidt stability index, which determines the energy required to mix the water column fully\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. During the 3 years of monitoring, the value remained constant at 175\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 kJ m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, about 10 times higher than for lakes of similar size\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. As long as the two-layer structure is maintained, this lake will remain a unique hotspot for studying anoxic life. Anoxygenic microorganisms found in the depths of the lake are potentially very similar to the primordial life forms that appeared on our planet and gave rise to the evolutionary process that is still ongoing. In the sediment and deepest dark zones, the SRB\u0026rsquo;s anoxygenic sulfur-based chemoheterotrophic metabolism allows them to thrive\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. At the same time, in the lower part of the chemocline in the APZ, phototrophic sulfur bacteria rely on anoxygenic photosynthesis, a precursor to the more modern oxygenic photosynthesis observed in the BGA community at the upper part of the chemocline, where little oxygen is present\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Redox-stratified environments, such as euxinic or ferruginous systems\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, are essential for implementing the biogeochemical knowledge of the primordial oceans of the Proterozoic era starting 2.5\u0026nbsp;billion years ago\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the pressing concern about climate change\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, as average temperatures rise and extreme weather events become more frequent, is there a potential risk to the meromixis of Lake Cadagno? Historical evidence spanning more than 10,000 years, analyzed through sediment studies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, suggest the persistence of physical stratification and anoxygenic microorganisms\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Data collected over the past 30 years, from 1985 to 2021 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), shows relatively stable temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and conductivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) values in the water column, reflected in the elevated and constant Schmidt stability index (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTypically, PSB and GSB communities depend strongly on euxinic conditions and light availability. In this regard, it is interesting to note that the hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) concentration profile underwent a major change at the beginning of the century (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In 2000, a violent hurricane named Lothar caused a partial mixing of the lake that altered the turbidity and light profiles, with the latter reduced by 10 times at the level of the BL\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This change in the water column led to a substantial increase in the number of cells in the BL due to the development of a \"new\" previously absent species of GSB, \u003cem\u003eChlorobium chlatratiforme\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The reduction in hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) concentration observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD is probably related to the increase of one order of magnitude in the number of cells in the BL\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, it is so far not clear how climate change, induced hydrological variations, will affect salt-rich groundwater inflow and sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) discharge in the deep water\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which is then converted to hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) by the SRB, an essential for the development of the BL community. Such changes in groundwater flow would profoundly affect both the stability of the water column as well as all anoxic microorganisms. Thus, further research is needed to assess the effect of climate change on the supply of salt-rich water to Lake Cadagno.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study emphasizes the role of abiotic and biotic factors in the development of a specific ecosystem. Although the background stratification of this meromictic lake is barely affected by short-lived summer rainstorms, external meteorological factors modify the dynamic and composition of the BL anoxic phototrophic community. Ongoing climate change will continue to alter the long-term seasonal composition of the BL community, mainly through extreme wind-related phenomena\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, yet without changing the stability of the stratification. Lake Cadagno has maintained its meromictic state over the past three decades, featuring a persistent anoxygenic monimolimnion while recording only minor changes in the Schmidt stability index. Although the change in heat content should not modify the mixing regime, the supply of salt-rich water from sublacustrine springs might decrease under climate change, leading to a modification of the supply of deep-water ions. To avoid further speculations, we recommend measuring the yearly dynamics of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) in the deep waters, its interaction with salt-rich groundwater recharge and how changes in rainfall and snowpack will affect groundwater flow into Lake Cadagno.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBL: bacterial layer\u003c/p\u003e\n\u003cp\u003ePSB: purple sulfur bacteria\u003c/p\u003e\n\u003cp\u003eGSB: green sulfur bacteria\u003c/p\u003e\n\u003cp\u003eSRB: sulfate-reducing bacteria\u003c/p\u003e\n\u003cp\u003eORP: oxide reductive potential\u003c/p\u003e\n\u003cp\u003eFTU: Formazine Turbidity Unit\u003c/p\u003e\n\u003cp\u003eBGA-PC: blue-green algae\u003c/p\u003e\n\u003cp\u003eCBA: Alpine Biology Center\u003c/p\u003e\n\u003cp\u003em a.s.l.: meters above sea level\u003c/p\u003e\n\u003cp\u003eCTD: Conductivity-Temperature-Depth\u003c/p\u003e\n\u003cp\u003eAPZ: anoxygenic photosynthetic zone\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe project was funded by the Swiss National Fund (SNF) for Scientific Research (BIOCAD: no. 179264). The quality of the scientific equipment used during monitoring was made possible by cantonal funding for the mandate \u0026ldquo;Indagini, perizie e consulenza in Microbiologia ambientale\u0026rdquo; of the Department of \u0026ldquo;socialit\u0026agrave; e sanit\u0026agrave;\u0026rdquo; (DSS). We are grateful to the Alpine Biology Center Foundation (Switzerland) for the use of its research facilities. We also thank David Janssen, Aquatic Geochemistry group leader at Eawag, for his valuable contribution to revising the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions (names must be given as initials)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.S., O.S.S. and D.B. conceived, designed, conducted the study, organized and followed the sampling network, analyzed the data and wrote the manuscript. S.R., F.D.N. and A.B.D coordinated sample collection during the campaign period (2019-21) and conducted laboratory analysis and data interpretation. N.S. wrote the main manuscript text and N.S., O.S.S. and F. D. N. prepared figures. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement (mandatory)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data analyzed and used for making the figures are included in the article published as Excel sheets in the supplementary material section. In addition, raw data, such as those from CTD measurements, cytometry, and the weather station, will be accessible via Dryad (https://datadryad.org/).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information (including a Competing Interests Statement)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBoehrer, B. \u0026amp; Schultze, M. 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Long-term population dynamics of phototrophic sulfur bacteria in the chemocline of Lake Cadagno, Switzerland. \u003cem\u003eAppl Environ Microbiol\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 3544\u0026ndash;50 (2005).\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez Castro, B. \u003cem\u003eet al.\u003c/em\u003e Inhibited vertical mixing and seasonal persistence of a thin cyanobacterial layer in a stratified lake. \u003cem\u003eAquat Sci\u003c/em\u003e \u003cstrong\u003e83\u003c/strong\u003e, 1\u0026ndash;22 (2021).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3744815/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3744815/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lake Cadagno differs from typical alpine lakes as it is stratified into two water layers that never vertically mix. This stratification creates a niche for the development of primordial anoxygenic phototrophs, which thrive in the chemocline of the lake, forming a characteristic bacterial layer (BL). Yet, the relationship between the temporal variation of meteorological factors that regulate stratification and the development of the BL remains unclear. Here, we explored the intra- and inter-seasonal stability of the water column stratification and ecological dynamics of the anoxygenic phototroph community of the BL over three years. Our continuous monitoring showed that the meromixis of the lake is highly stable, with density stratification seemingly unaffected by external meteorological factors. Further reanalysis of the lake’s recent history substantiated this remarkable stability. In contrast, the community of anoxygenic phototrophs showed significant intra- and inter-seasonal variability, modulated by weather events that primarily impacted light penetration. In fact, an exceptional intra-seasonal light increases in September 2020 led to an overgrowth of purple sulfur bacteria compared to commonly dominant, green ones. At the inter-seasonal level, there is a difference in BL development in July 2021, which was characterized by much precipitation and less light, compared with that in 2019/2020.","manuscriptTitle":"Ecological dynamics of anoxygenic phototrophs in stably redox-stratified waters: Intra and inter-seasonal variability of Lake Cadagno","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-26 21:03:14","doi":"10.21203/rs.3.rs-3744815/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8f517e4f-6673-49b9-856f-110802b14034","owner":[],"postedDate":"February 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27400072,"name":"Biological sciences/Ecology/Microbial ecology"},{"id":27400073,"name":"Earth and environmental sciences/Limnology"}],"tags":[],"updatedAt":"2024-05-28T15:51:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-26 21:03:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3744815","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3744815","identity":"rs-3744815","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}