Recycling and deposition of inorganic carbon from calcium carbonate encrustations of charophytes

Many aquatic primary producers can use bicarbonates as a carbon source for photosynthesis. Charophytes of the two genera: Chara and Nitellopsis are quite efficient in this process. Some species of these macroalgae produce carbonate encrustations, mainly calcium carbonate, constituting up to 86% of the summer maximum dry weight of the standing crop. In this study, we analyzed the fate of inorganic carbon accumulated this way in Chara spp. and Nitellopsis obtusa from six Polish lakes located in two regions (warmer W Poland and cooler NE Poland). Our study distinguished two groups of charophyte species that differed in the way of CaCO3 release from their summer standing crops. On average, the corticate Chara rudis and C. tomentosa belonging to the first group were less efficient in depositing CaCO3 from summer to autumn than the less corticate C. contraria and ecorticate N. obtusa of the second group. The latter two species were more efficient in inorganic carbon burial in sediments. On the contrary, dissolution of encrustation was more typical of the first species group and was facilitated by decreasing the pH and saturation index of calcite in lake water. The final output of CaCO3 loss mainly resulted from combined species‐specific features, lake water properties and overwintering patterns. Our study revealed that inorganic carbon cycling through charophytes involves burial and dissolution and is more complex than previously thought.

. This way, charophytes become a sink for an extra amount of carbon apart from biomass production during photosynthesis.
Since the late 1990s, interest in carbon cycling has gained momentum with the global debate on climate change caused by excessive emissions of greenhouse gases-CO 2 and CH 4 (Falkowski et al. 2000;Larmola et al. 2004).Aquatic biota has been studied given their possible role as permanent stores of carbon (Tranvik et al. 2009;Brothers et al. 2013;Sobek et al. 2014).For example, the differences in carbon cycling between pelagic and littoral zones were studied by Larmola et al. (2004) and Spafford and Risk (2018).Charophytes and other aquatic bicarbonate users in littoral zones might appear promising in carbon burial since they accumulate redoxinsensitive carbonate encrustation, which, contrary to organic carbon, does not undergo microbial degradation.Once accumulated, calcium carbonate may be deposited in sediments or dissolved from charophyte standing crops and recycled into lake water (Sand-Jensen et al. 2019).The relative share of encrusted calcium carbonate losses may be affected by habitat conditions and species-specific traits.
The amount of CaCO 3 encrustations formed by charophytes depends mainly on calcium concentrations and pH in lake water (Herbst et al. 2018b).Charophytes generally prefer hardwater lakes.However, they can grow in lakes with a calcium concentration as low as 8.2 mg L À1 (Kufel et al. 2016).The production of charophyte biomass and accompanying CaCO 3 encrustation is affected by light attenuation in water.Thus, plants in shallower sites can be more encrusted than those in deeper sites (Pukacz et al. 2014a).Moreover, the CaCO 3 content in charophyte DW may be characteristic of different species (Kufel et al. 2013;Pukacz et al. 2014a).
The species-specific differences in calcium carbonate encrustation may arise from different plant traits, e.g., the presence or absence of cortex on charophyte thalli (Urbaniak and Gąbka 2014).Cortication affects the mode of thallus encrustation through the enlargement of the surface area of thallus.Thus, corticate charophytes can be more heavily encrusted than ecorticate ones (Kawahata et al. 2013).It is unclear whether the encrustation is formed parallel to biomass increments in the growing season.In the month-to-month study of CaCO 3 concentration, Pukacz et al. (2016) and Herbst et al. (2018b) evidenced an increasing percentage of calcium carbonate in charophyte DW from February till its maximum in July-August.However, temporal changes in CaCO 3 accumulation may differ among charophyte species even in the same lake (Herbst et al. 2018b).
In contrast to the abundant literature on the formation of encrustations, studies on the fate of accumulated calcium carbonate have seldom been undertaken.The evidence of permanent calcium carbonate burial comes from charophyte gyrogonites (calcified oogonia) commonly found in lake littoral sediments (Soulié-Märsche and Garcia 2015) as well as from tubular encrustations (see e.g., Pełechaty et al. 2013: fig.9).Quantitative analyses of CaCO 3 deposition in bottom sediments are, however, rare.When exploring the role of charophytes in carbon cycling, the main question is how much CaCO 3 produced in summer undergoes dissolution (recycling) from plant standing crops compared with the amount of calcium carbonate deposited in littoral sediments.The detailed and sophisticated set of field and experimental studies (reviewed in Sand-Jensen et al. 2019) showed that dark respiration resulted in CaCO 3 dissolution from plant standing crops.Kragh et al. (2017) and Andersen et al. (2019) considered dissolution important for primary production in lakes deficient in dissolved inorganic carbon.The described process of inorganic carbon recycling was possible in very specific water bodies under study.The ponds were small (71-10,000 m 2 ), shallow (0.2-1.5 m), and formed in limestone quarries abandoned about 30 yr prior.Cited authors analyzed four species of Chara but did not consider species-specific differences.Sand-Jensen et al. (2019) admit that in large, wind-exposed lakes, the dissolution of encrustations proceeds according to the same mechanisms as in small and shallow ponds but should be considered on seasonal and not daily time scales.
Inspired by these conclusions, we have undertaken the study of the fate of CaCO 3 from various charophyte species in six lakes according to the following questions: (1) is the calcium carbonate loss from charophyte standing crop site-or species-specific, (2) what are the seasonal rates of encrustation loss from the charophyte standing crops from summer till autumn and from autumn till spring, and (3) which environmental parameters and charophyte species-specific traits facilitate permanent burial of calcium carbonate from encrustations?

Study sites and sampling
We selected six lakes dominated by common charophyte species based on our earlier surveys.Three lakes were situated in the Lubuskie Lakeland (W Poland) and three in the Masurian Lakeland (NE Poland), two regions with different climatic conditions (Table 1; for further differences see Pełechata et al. 2023).Sites within a lake (one to three per lake-Table 2) were not <50 m away from each other, and charophytes grew at a depth of 1.5-3 m.Lake water and plant samples were collected once per season: in summer (the middle of July), autumn (the middle of November) and spring (2-3 weeks after ice-out, usually at the end of March or at the beginning of April) between summer 2017 and spring 2020.Additionally, sediment cores (50 cm long and 8 cm in diameter) were taken from each site in autumn.Plant samples were collected from each site with a 40 Â 40 cm grab sampler in duplicate (pooled after sampling).In the laboratory, plant samples were gently rinsed with tap water so as not to remove encrustations, divided into species, and air-dried.Separated subsamples were dried at 105 C, weighed, and powdered before chemical analyses.Sediment cores were cut into 2-cm thick slices, dried at 105 C, weighed and pulverized.The top two slices (total of 4 cm) from each core were taken for further analysis.Prior to macrophyte sampling, lake water was taken for physical and chemical analyses from about 1 m above plant cover with a Bernatowicz-type 5-L water sampler (UWITEC GmbH, Mondsee, Austria) or a Patalas-type 1-L water sampler (Mikołajki, Poland).Lake water pH was measured in situ with a portable pH-meter CP-215 in the NE lakes or a CX-401 integrated meter (Elmetron GP, Zabrze, Poland) in the W lakes.

Chemical analyses
In the laboratory, alkalinity was determined by titration of lake water with 0.1 M HCl against methyl orange (Golterman 1969) and calcium concentration by 0.01 M EDTA titration against murexide indicator (Williams and Moser 1953).Prior to analyses, lake waters were filtered through Whatman GF/C glass fiber filters with a pore size of 1.2 μm.
Concentrations of organic matter (OM) and calcium carbonate in plant material were determined by sequential combustion at 550 C and 925 C (Heiri et al. 2001) of pre-weighed plant samples (about 0.2 g weighed to the fourth decimal point).The loss on ignition at 550 C represented OM and the loss between 550 C and 925 C was equivalent to CO 2 released from calcium carbonate thermal decomposition.Concentrations of total carbon (TC) in plant material were analyzed with the elemental CHNS analyzer PerkinElmer equipped with the thermal conductivity detector (PerkinElmer, Waltham, Massachusetts, USA) for the samples from NE Poland and CNS elemental analyzer (VarioMax, Elementar, Germany) for the samples from W Poland. Acetanilide (C = 71.09%)(PerkinElmer), sulfadiazine and the Chalky Soil certified reference material (Elementar) were used to check the quality of analyses.Organic carbon (OC) was calculated as the difference between TC and inorganic carbon (IC) calculated from CO 2 .Concentrations of OM, TC, OC, and IC in sediment samples were determined with the same methods as those used to analyze plant material.

Data processing and statistical analyses
Based on analyzed concentrations of calcium, alkalinity and pH in lake water, we calculated the calcite saturation index (SI), which is a measure of water saturation with calcium carbonate (Stumm and Morgan 1981;Nõges et al. 2003).SI values > 1 indicate supersaturation of lake water with calcium carbonate.
The observed seasonal losses of charophyte encrustations were the sum of deposition and dissolution.To distinguish between the two processes, we adopted two assumptions.First, we assumed that CaCO 3 loss parallel to the decrease of OM content in charophyte standing crop equals deposition due to the detachment of encrustation from decaying plant material.The parallel course of both processes manifested itself in the similarity of the OC : IC ratios of standing crops from summer to autumn, and from autumn to spring.Second, any loss of calcium carbonate faster than that described above was assumed to represent the dissolution of CaCO 3 in lake water.In practice, based on the OC : IC ratio, OM content and the percent of OC in OM, we calculated a hypothetical trajectory of CaCO 3 loss parallel to OM decomposition (Fig. 1).In four sites of Chara tomentosa L., the maximum content of OM was noted between summer and autumn with accompanying loss of calcium carbonate.We calculated a hypothetical calcium carbonate increment parallel to the OM increase in these cases.This way, we estimated calcium carbonate losses from summer to autumn, and from autumn to spring.
Data were tested for normality (Shapiro-Wilk test) and homogeneity (Brown-Forsythe or Bartlett test).Linear regression was used to model the relationships between the OC : IC ratio in plant standing crops and water parameters such as pH and SI, between water alkalinity and Ca 2+ concentrations in waters, and between CaCO 3 concentrations in the top sediment layers and the OC : IC ratios in the overgrowing standing crop of charophytes.Principle component analysis (PCA) was employed to summarize the relationship between the calcium carbonate loss, OC : IC ratio in plant standing crops and water parameters such as pH and SI for different seasons.PCA as an exploratory tool needs no distributional assumptions and can be used on numerical data of various types (Jolliffe and Cadima 2016).PCA was performed using FactoMineR (L e et al. 2008) R's package (R Development Core Team 2023).
The data were scaled to unit variance prior to analysis.The results of PCA were visualized as a biplot using factoextra R's package (Kassambara and Mundt 2020).Then Mann-Whitney U test was used to compare the species groups distinguished by PCA in terms of the percent of carbonate losses.Non-parametric Kruskal-Wallis ANOVA and post-hoc Dunn test were applied to test seasonal differences among OC : IC ratios in the plant standing crop of two species groups or regions.The same tests were used to describe differences among seasonal carbonate lossesdeposition or dissolution and two groups of species.A significance level was set to 5%.The statistical analyses, except PCA, were performed using Statistica 13.3 (TIBCO Software, Palo Alto, California, USA).

Results
Four charophyte species were found in the study sites: two fully corticate Chara rudis (A.Braun) Leonhardi (Chara subspinosa Ruprecht) and C. tomentosa, one less corticate Chara contraria A. Braun ex Küting and ecorticate Nitellopsis obtusa (Desvaux) J. Groves (Table 2).Three species are medium large or large plants up to 120 cm high (N.obtusa) with fairly thick axis up to 6 mm in diameter (C.rudis and C. tomentosa), while the fourth species, C. contraria is a slender, small to mediumsized alga up to 50 cm high and with axis diameter up to 2 mm (Urbaniak and Gąbka 2014).
Theoretically, when calcification is coupled with photosynthesis and bicarbonates are the sole carbon source for plants, the OC : IC ratio in plants equals 1 (McConnaughey and Whelan 1997).Our study noted such a ratio only in the summer standing crop of C. contraria from Lake Kołowin (Table 2).In other cases, the summer OC : IC ratio was higher (up to 2.8 in C. tomentosa).Lake water parameters significantly affected the OC : IC ratio.The linear regression equation describing the relationship between the OC : IC ratio in the charophyte standing crops and lake water pH calculated across all sites and seasons was OC : IC = -16.05+ 2.45 pH (F 1,52 = 4.89, p = 0.032, R 2 = 0.07).The saturation index combines three water parameters namely pH, alkalinity and Ca concentration.That is probably why the similar regression of OC : IC on SI was OC : IC = 1.29 + 0.52 SI (F 1,52 = 33.53,p < 0.001), and its explanatory power was higher than that for pH alone (R 2 = 0.38).
Tracking the seasonal changes of OC : IC ratios allowed us to assume the relative rate of calcium carbonate and OM losses.The negligible change of OC : IC ratio from summer to autumn (set arbitrarily at no more than 0.3 units and given in bold in Table 2) indicates parallel OM decomposition and CaCO 3 loss rates.PCA showed that the overall variability of the CaCO 3 losses and OC : IC ratios in plant standing crops are associated with water parameters, mainly autumn and spring pH as well as spring SI (Fig. 2).Variables such as OC : IC ratios for the two seasons (positive correlation) together with the autumn pH (negative correlation) contributed in over 77% to the creation of the PC1 that explained 31% of the overall data variance.All this indicates strong relations between the OC : IC ratio and the autumn pH and SI.The first gradient is orthogonal to the second one created mainly by carbonate loss, spring pH, and SI (a total contribution of almost 70%).The PC2 explained 29% of the variance and distinguished the two main groups of charophytes: one consisting of C. rudis and C. tomentosa and the second one of C. contraria and N. obtusa.The groups significantly differed in the calcium carbonate loss from summer till autumn (Z = -3.24,n = 18, p = 0.001) and till spring (Z = -2.44,n = 18, p = 0.015).Species from the first group lost 18.8-61.1% of carbonates (Me = 31.2%)till autumn, while the species from the second group lost 54.6-94.5% of CaCO 3 (Me = 71.8%)at that time.
A comparison of the OC : IC ratio for all charophytes shown in Table 2 revealed that the ratio varied seasonally (H 2,54 = 23.80,p < 0.001) and increased from summer to the following spring, reaching median values of 1.6 for summer, 2.4 for autumn and 3.7 for spring.When the regions (W and NE Poland) were included in the analysis, the OC : IC ratio increased from summer to spring in both regions (H 5,54 = 25.36,p < 0.001), i.e., from 1.6 in summer to 4.2 in spring in W Poland and from 2 to 3.5, respectively, in NE Poland.A comparison between two charophyte groups (H 5,54 = 26.71,p < 0.001) showed a significant increase in the OC : IC ratios between summer and spring only for the species aggregated in the top panel of Fig. 2, i.e., C. contraria and N. obtusa (p < 0.001).Contrary to clear differences between species and sites, no interregional differences emerged from the PCA (Fig. 2).
As seen from seasonally varying OC : IC ratios (Table 2; Fig. 2), the mutual relationship between OM decomposition and calcium carbonate loss differed among lakes and plant species.We found that the analyzed charophytes 2. Principal component analysis biplot was created using calcium carbonate losses and seasonal OC : IC ratios in charophyte standing crop and seasonal lake water parameters pH and SI.The studied lakes of W and NE Poland: J-Lake Jasne (W), Ko-Lake Kołowin (NE), Mo-Lake Mojtyny (NE), MM-Lake Majcz Mały (NE), Lu-Lake Lubikowskie (W), ZP-Lake Złoty Potok (W).Au, autumn; Sp, spring; Su, summer; Carbonates lost Su-Au, a percent loss of CaCO 3 from the charophyte standing crop from summer till autumn; Carbonates lost Au-Sp, a percent loss of CaCO 3 from the charophyte standing crop from autumn till spring; con, C. contraria; obt, N. obtusa; rud, C. rudis; tom, C. tomentosa.demonstrated two different patterns of CaCO 3 loss.The first, species-specific pattern could be seen in the seasonal biomass changes in two or three species of charophytes growing in the same lake, hence being affected by similar lake water conditions.Species-specific differences in calcium carbonate loss can be observed in C. tomentosa and C. rudis from Lake Mojtyny, C. tomentosa and N. obtusa from Lake Majcz Mały and C. contraria, C. tomentosa, and N. obtusa from Lake Kołowin (Fig. 3, Supporting Information Fig. S1).In the latter, the loss of CaCO 3 from C. contraria proceeded faster from summer to autumn and prolonged at a lower rate from autumn to spring.Chara tomentosa lost its carbonate encrustation at the same rate from summer through autumn till spring.Noteworthy, the loss of calcium carbonate was parallel to OM decomposition from summer to autumn but faster than OM loss from autumn to spring (Fig. 3).
The second pattern of calcium carbonate loss was site-specific-the same species growing in different lakes released CaCO 3 differently as shown for N. obtusa in two lakes (Fig. 4, the other examples of this pattern are shown in Supporting Information Figs.S2 and S3).A common feature of N. obtusa was an intensive decomposition of OM and CaCO 3 loss from  summer to autumn compared with losses from autumn to spring.In most cases, the loss of calcium carbonate was faster than the decomposition of OM.Consequently, the OC : IC ratio increased in the standing crop from summer through autumn till the following spring (Table 2; Fig. 2, Supporting Information Fig. S2).Changes in the content of OM and calcium carbonate during wintertime were negligible.The marked divergence between OM decomposition and CaCO 3 loss in N. obtusa was observed only in Lake Lubikowskie (Fig. 4, Supporting Information Fig. S2).
Different ways of calcium carbonate losses raise the following questions: what part of once-encrusted calcium carbonate is deposited in littoral sediments, and what part is recycled to lake water as bicarbonate ions.When CaCO 3 losses proceed parallel to OM decomposition, one may expect physical detachment of encrustation and its deposition in sediments (Fig. 1).Such co-occurrence of both processes manifests itself by minimum seasonal changes of the OC : IC ratios in the charophyte standing crop.The more rapid loss of calcium carbonate than OM decomposition indicates the CaCO 3 dissolution (Fig. 1).This was confirmed by the regression of calcium carbonate concentrations in the top 4 cm of the sediment on the OC : IC ratio in the autumn standing crop of charophytes (Fig. 5).The higher OC : IC ratio of plant standing crop is the manifestation of faster loss of calcium carbonate than OM decomposition.The ratio close to 1 indicates parallel OM decomposition and CaCO 3 deposition in littoral sediments.Following this line of reasoning, we calculated the percentage of  calcium carbonate deposited in sediments or dissolved and recycled into lake water in particular seasons.Using the amount of decomposed OM, the percent of OC in OM and the OC : IC ratios, we calculated the amount of CaCO 3 that would represent the deposition parallel to OM decomposition.In the case of negligible seasonal changes in OC : IC (gray cells in Table 2), the whole loss of calcium carbonate was ascribed to deposition.The difference between seasonal calcium carbonate loss and that calculated from OM decomposition was assumed to represent dissolution (Table 3).The regression of alkalinity on calcium (expressed in mval L À1 ) in autumn (alkalinity = 0.05 + 1.18 [Ca], F 1,13 = 16.23,p = 0.014, R 2 = 0.56) and in spring (alkalinity = 0.79 + 0.93 [Ca], F 1,13 = 49.64,p < 0.001, R 2 = 0.79) had the slope values close to 1, which is characteristic for the stoichiometry of CaCO 3 dissolution.Thus, the dissolution of charophyte-derived CaCO 3 affected lake water composition in autumn and spring (Supporting Information Table S1).Noteworthy, the same regression calculated for summer data was not significant.However, calcium carbonate deposition contributed most to CaCO 3 loss from the standing crop of charophytes, particularly of C. contraria and N. obtusa.The two species buried the biggest amount of carbonates from summer till autumn and recycled no more than 30% of CaCO 3 accumulated in summer.On the contrary, C. rudis and C. tomentosa were on average seven times less effective in the autumn carbonate deposition (Table 4).

Discussion
Numerous studies were devoted to the formation of CaCO 3 encrustations on charophytes (e.g., Kufel et al. 2013;Pełechaty et al. 2013;Sand-Jensen et al. 2018, 2021).Our study focuses on the further fate of calcium carbonate once deposited on plants in the peak of the summer season.Presented results indicate that the amount and rate of encrustation losses depend on lake water properties, but various plant species release carbonates differently.In the subsections given below, we discuss the complexity of this process with particular attention paid to the permanent burial of inorganic carbon in littoral sediments.

Species-and site-specificity of charophyte carbonate loss
Many studies have addressed charophyte calcification, including its mechanisms (e.g., McConnaughey and Falk 1991;McConnaughey and Whelan 1997;Sand-Jensen et al. 2018, 2021), the composition and amount of encrustation specific to species, thallus age, site and season.They frequently concluded that the encrusted calcium carbonate was finally deposited in lake sediments (e.g., Pukacz et al. 2014aPukacz et al. , 2014bPukacz et al. , 2016;;Herbst and Schubert 2018;Herbst et al. 2018aHerbst et al. , 2018b)).
Our study shows that the fate of summer maximum encrustation is more complex than previously thought.First, some part of the calcium carbonate is dissolved and released into ambient water.Second, the loss of CaCO 3 is both species-and site-specific.
So far, studies on encrustation persistence have not addressed differences among charophyte species (Sand-Jensen et al. 2021;Martinsen et al. 2022).In our study, substantial differences in calcium carbonate losses were found between C. rudis and C. tomentosa, on one hand, and C. contraria and N. obtusa, on the other (Figs. 2, 3, Supporting Information Fig. S1).The differences in the rates of CaCO 3 loss between the two groups of charophyte species may be explained by morphological differences in their thalli.Nitellopsis obtusa vs. C. rudis and C. tomentosa represent extremes of charophyte morphological characteristics-a lack of cortex and welldeveloped cortex, respectively (Urbaniak and Gąbka 2014).We found that an ecorticate plant loses encrustation more easily and faster than a corticate species.Nitellopsis obtusa, similarly to other ecorticate charophytes (Kawahata et al. 2013;Eremin et al. 2019), may produce encrustation on the outer surface of cells.On the contrary, corticate species like C. rudis and C. tomentosa, similarly to C. globularis studied by Kawahata et al. (2013), may have uniform encrustation on the surface of internodal cells as well as under the cortex.Thus, we can assume that in corticate species, only surface calcium carbonate may dissolve under lower pH in ambient water.In contrast, CaCO 3 accumulated under the cortex is deposited parallel with OM decomposition.Moreover, such a multilayer encrustation may increase the resistance of the thalli to harsh environmental conditions, including low temperatures, which corresponds well with different overwintering patterns in C. contraria and N. obtusa vs. C. rudis and C. tomentosa.In NE Poland, N. obtusa overwintered mainly as colorless rhizoids and starch-filled bulbils with high OC : IC ratios of 5 up to 19 (Table 2).In warmer W Poland, it partially overwintered as a green plant (Supporting Information Fig. S1) and the calcium carbonate accumulated in the thalli was deposited in the Table 4. Medians and ranges of calcium carbonate seasonal losses from the standing crops of two charophyte groups distinguished in PCA (Fig. 2).

Species group
CaCO 3 deposited (% of the summer content) CaCO 3 dissolved (% of the summer content) Summer till autumn Autumn till spring Summer till autumn Autumn till spring sediment along with decomposing OM.The similar rate of calcium carbonate loss in N. obtusa and C. contraria observed in Lake Kołowin (NE Poland) suggests a relationship between thallus structure and the amount and persistence of encrustation because C. contraria can partially be ecorticate.In contrast, some parts of the standing crop of C. rudis and C. tomentosa overwintered as a calcified green plant even in the lakes of NE Poland (Supporting Information Fig. S1).The interregional differences in the mode of calcium carbonate release from charophytes overlapped with site-specific parameters.For example, summer pH and SI were higher in the water of the western lakes than in the northeastern lakes.Both environmental parameters decreased in autumn (Table 2 and Supporting Information Table S1), followed by increasing OC : IC ratios in plants (Fig. 2).
In dense charophyte beds, water exchange may be limited and the basal parts of thalli may be overshaded by an apical canopy.This may result in more intensive respiration, reducing water pH (Kragh et al. 2017;Pronin et al. 2018;Sand-Jensen et al. 2018, 2019, 2021).The similar rate of encrustation losses in Lake Jasne (dense beds of C. tomentosa) and in Lake Kołowin (much lower density of C. tomentosa) indicates a negligible effect of the mechanism cited above (Supporting Information Fig. S3).
To summarize, environmental and, to some extent, climatic conditions modified the species specificity of calcium carbonate loss in the studied lakes.

The seasonality of the calcium carbonate loss
Summer was the starting point in our study.We then followed the fate of the encrustation produced by charophytes in the subsequent seasons.In contrast, many earlier studies (e.g., Van den Berg et al. 2002;Pukacz et al. 2016;Herbst et al. 2018b) started in spring and ended in autumn of the same year.Therefore, it is difficult to compare their results with ours.
During our study, all sampled charophyte species were strongly encrusted, but the calcium carbonate content decreased from summer to spring, raising the OC : IC ratios in charophyte DW.The amount of seasonal losses of CaCO 3 through deposition or dissolution depended on the group of charophytes distinguished by the PCA (C.rudis and C. tomentosa vs. C. contraria and N. obtusa; Fig. 2).The maximum deposition of CaCO 3 realized by plants of the first group took place from autumn to spring, while plants of the second group deposited most CaCO 3 from summer to autumn.Dissolution of calcium carbonate was negligible in the second group compared with deposition in sediments (Table 4).Regardless of the group of plants and season, the dissolution of calcium carbonate affected lake water as confirmed by the regression of alkalinity on Ca 2+ concentration in both autumn and spring.The stoichiometric proportion of nearly 1 : 1 is typical of calcium carbonate dissolution.

The factors facilitating calcium carbonate burial in sediments
Between summer and spring, analyzed charophytes lost from 43.7% to 100% of CaCO 3 accumulated in the summer standing crop.Calcium carbonate was deposited in littoral sediments or dissolved in lake water in proportion depending on species and site.The first process was confirmed by the significant correlation between calcium carbonate content in the 4-cm-thick top layer of sediments and the autumn OC : IC ratio in overgrowing charophytes (Fig. 5).This finding agrees with the results of Kufel et al. (2020) demonstrating that sediments in charophyte-dominated littoral zones contain more IC than those overgrown with vascular plants.
C. contraria and N. obtusa performed a more efficient calcium carbonate deposition than C. rudis and C. tomentosa (Tables 3 and 4).One of the possible explanations for this phenomenon is the presence or lack of thallus cortication that affects the pattern and amount of CaCO 3 release.The ecorticate species tend to deposit relatively more calcium carbonate than the corticate ones.The opposite process, dissolution, is related to low water pH and SI in a charophyte bed.
Overall, the final output of CaCO 3 loss mainly results from interwoven species-specific features, lake water properties, and overwintering patterns.Our study indicates that carbon uptake and release by charophytes are more complex than previously thought and require further investigation.Calcium carbonate burial can be highly relevant under specific conditions (plant overwintering and lake water parameters).Site-and species-specific patterns of CaCO 3 fate should also be considered in discussions about the role of charophytes as a carbon sink in aquatic ecosystems.

Fig. 1 .
Fig. 1.Conceptual graph of seasonal changes of OMred line and calcium carbonate (CaCO 3green lines).The dashed green line shows hypothetical CaCO 3 losses parallel to OM decomposition, which is equivalent to unchanged OC : IC ratio in charophyte standing crop, i.e., CaCO 3 deposition.Steeper slope of the solid green line than that of the red line represents CaCO 3 dissolution and the increase of the OC : IC ratio.The black line represents a summer calcium carbonate standing crop.

Fig. 3 .
Fig. 3. Examples of species-specific trajectories of seasonal changes of DW, OM, and CaCO 3 of C. contraria and C. tomentosa in site 1 of Lake Kołowin (note the different scales on the y-axes).For the whole set of trajectories see Supporting Information Fig. S1.

Fig. 4 .
Fig. 4. Examples of site-specific trajectories of seasonal changes of DW, OM, and CaCO 3 of N. obtusa in two study lakes.For the whole set of trajectories see Supporting Information Fig. S2.

Table 1 .
Selected climatic characteristics of the regions (min-max) and lakes studied in west (W_PL) and northeastern Poland (NE_PL).

Table 2 .
The loss of calcium carbonate from charophyte encrustations given as a percent of its maximum summer content, the OC : IC ratio in plant standing crop and lake water pH in subsequent seasons.
Negligible seasonal changes of OC : IC ratio are given in bold in gray cells.

Table 3 .
The content of calcium carbonate encrustations on summer standing crop of charophytes and its loss by deposition or dissolution in subsequent seasons.
† OM content increased from summer till autumn.‡ Percent calculated with respect to summer CaCO 3 content enriched by hypothetical increment caused by the increase of OM.