Introduction

Charophytes, macroscopic green algae from the extant family Characeae (Charales, Charophyta), occur in different water environments all over the world except for Antarctic regions (e.g., Hutchinson, 1975; Wade, 1990). However, they prefer freshwater calcium-rich lakes with oligo- and mesotrophic waters (Krause, 1981) and inhabit both deep and shallow sites (e.g., Martin et al., 2003). Charophytes often form extensive underwater stands referred to as charophyte meadows. Covering large areas, charophytes considerably affect the properties of lake water (e.g., alkalinity and hardness, saturation with O2, and transparency). These macroalgae also affect the composition and structure of phyto- and zooplankton as well as other elements of water biocoenosis (Królikowska, 1997; Kuczyńska-Kippen, 2001; Kufel & Kufel, 2002; Blindow et al., 2014; Pełechata et al., 2016).

Dense charophyte meadows greatly intensify carbonate precipitation and contribute to the accumulation of marl sediments in lakes (Kufel & Kufel, 2002; Pentecost et al., 2006; Apolinarska et al., 2011; Pełechaty et al., 2013; Pukacz et al., 2016a). This capacity results from photosynthetic removal of CO2 from dissolved bicarbonates (McConnaughey, 1997) and leads to the precipitation of insoluble CaCO3 (mostly calcite) in the form of encrustations on the surface of charophyte thalli. Encrustation may account for 30–80% of the charophyte dry matter (Pentecost, 1984; Królikowska, 1997; Kufel & Kufel, 2002; Urbaniak, 2010; Pełechaty et al., 2013; Pukacz et al., 2016b). In temperate climate, the calcification process in charophytes is the most intensive in summer months (Pentecost et al., 2006; Pełechaty et al., 2010); thus, we can assume that in this season the majority of stem carbonates are precipitated. Charophyte remnants can be preserved in lacustrine sediments serving as indicators of past environmental conditions. While calcified oospores of charophytes (gyrogonites) are usually well preserved (Becker et al., 2002; Gałka & Sznel, 2013; Kołaczek et al., 2015), fossilized parts of thalli encrustations are found less frequently because they easily disaggregate after charophyte decay and as a fine-grained carbonates substantially contribute to sediment deposition (Pełechaty et al., 2013).

In studies of lake sediments, oospores and encrustations, among other aquatic plant macrofossils, are useful indicators of past ecological conditions at the site of sediment deposition (Croft, 1952; Anadón et al., 2000; Becker et al., 2002; Rutkowski et al., 2007; Apolinarska et al., 2011). Therefore, analysis of plant macrofossils is commonly applied in paleolimnological reconstructions (Hannon & Gaillard, 1997; Bešta et al., 2009; Gałka & Apolinarska, 2014; Kowalewski et al., 2016).

In addition to the above-mentioned direct application, carbonates precipitated and deposited in lake sediments by charophytes can also be applied in paleoreconstructions based on their carbon (δ13C) and oxygen (δ18O) stable isotope compositions (Anadón et al., 2000; von Grafenstein et al., 2000; Hammarlund et al., 2003; Apolinarska & Hammarlund, 2009). Carbon and oxygen stable isotopes have long been recognized as tracers of organic and inorganic processes in lakes. δ13C values of DIC (dissolved inorganic carbon) are used as indicators of the trophic status of lakes (de Kluijver et al., 2014), whereas δ18O values of lake waters are among others used to estimate the evaporative loss in lakes (Skrzypek et al., 2015). Application of carbon isotopes in the studies of lake sediments includes the reconstruction of changes in lake productivity (Lehmann et al., 2004) and shifts in lake water level (Woszczyk et al., 2014). Oxygen isotopes are used to reconstruct past climates (Słowiński et al., 2017) and changes in water retention in lakes (Hammarlund et al., 2003). Carbon and oxygen stable isotope composition was measured in various autochthonous carbonates occurring within sediments, i.e., mollusc shells (Stuiver 1970; Szymanek et al., 2016), ostracod carapaces (Lauterbach et al., 2011), and marl produced by phytoplankton (Słowiński et al., 2017). An attempt has also been made to use Chara encrustations and oospores in the studies of postglacial lacustrine deposits (e.g. Apolinarska & Hammarlund, 2009).

To reliably use the record of δ13C and δ18O in lacustrine carbonates, including charophyte carbonates, for paleoreconstructions, it is necessary to properly recognize the relations between the stable oxygen and carbon isotope composition of modern carbonates and δ18O in water and δ13C in DIC. The number of publications on the geochemistry of lake marl sediments formed by charophytes has been increasing in the past two decades. According to Coletta et al. (2001), oxygen and carbon stable isotope composition of carbonates precipitated on the charophyte thalli can record the environmental conditions of the time when the carbonates were precipitated. Moreover, the age gradient along the charophyte stem reflects seasonal changes in δ18OWATER and δ13CDIC (Pentecost et al., 2006 and references therein). Although carbon and oxygen stable isotope composition of carbonates precipitated on the charophyte thalli and oospores has already been discussed (Coletta et al., 2001; Andrews et al., 2004; Pentecost et al., 2006; Pełechaty et al., 2010; Rodrigo et al., 2016), studies comparing the isotopic values of different charophyte species are sparse (Apolinarska et al., 2016; Pronin et al., 2016). This had led us to the study of two common, morphologically different charophyte species: Chara tomentosa Linné 1753, which has a large thallus, and the small Chara globularis Thuillier 1799. Consequently, Chara tomentosa, developing numerous branches, forms large communities that are freely penetrated by ambient water. Chara globularis, a slender and densely growing species, forms very compact stands proximal to the lake bottom, which are less likely to be penetrated by water. As we have recently documented (Pronin et al., 2016), there exist sharp differences between these two species expressed in the δ13C values of their organic matter (δ13CORG) and carbonate encrustations (δ13CCARB) but not in DIC of ambient water (δ13CDIC). These differences can be attributed to different forms of growth and, thus, different ambient water layers utilized as a source of DIC for photosynthesis (Pronin et al., 2016). As a result, the species revealed contrasting trends in the shift between their δ13CCARB and δ13CDIC. In addition, in one of the charophytes studied, namely in Chara tomentosa, the values of δ13CDIC were positively correlated with those of δ13CORG and δ13CCARB.

Considering the above, the aim of this study was (i) to test whether C. tomentosa and C. globularis differ in the oxygen stable isotope composition of their carbonate encrustations (δ18OCARB) and the ambient water (δ18OWATER) and (ii) to investigate whether the shifts between the δ18OCARB and δ18OWATER values follow similar or different pattern in the studied charophytes. We hypothesized that the values of δ18OWATER, related to lake conditions, are reflected in the δ18OCARB values of the studied species, whereas the shifts between the δ18OCARB and δ18OWATER values are species-specific.

Study lakes

The stable isotope composition of C. tomentosa and C. globularis carbonates and ambient waters was studied in seven lakes located in western Poland, in Lubuskie Lake District: Złoty Potok, Niesłysz, Jasne, Męcko Duże, Malcz Południowy, and in Myśliborskie Lake District: Karskie Wielkie (Fig. 1, see also Pronin et al., 2016). In previous studies, the authors documented the presence of numerous lakes with well-developed charophyte meadows in the investigated area especially in Lubuskie and Myśliborskie Lake Districts (Pełechaty et al., 2007; Pukacz et al., 2014; Pełechaty et al., 2015). With the exception of slightly eutrophicated Lake Karskie Wielkie, the studied bodies of water are mesotrophic lakes with high water clarity and well-developed submerged vegetation, dominated by charophyte meadows. These features place the studied lakes in the Chara-lakes group (Pełechaty et al., 2007; Pukacz et al., 2014; Pełechaty et al., 2015; Pronin et al., 2016). They include both small and shallow polymictic lakes and large and deep stratified water bodies. The residence time of the water, strongly influencing its oxygen stable isotopic composition, varied from 0.5, 2.8, and 3.3 years in Lakes Malcz Południowy, Jasne, and Karskie Wielkie, respectively, to about 5 years in other lakes (no reliable data exist for Lake Męcko Duże) (Table 1; Pronin et al., 2016 and reference therein). For detailed description of the studied lakes and sites refer to Pronin et al. (2016).

Fig. 1
figure 1

Studied lakes and distribution of sampling sites. CT1–CT3—stands dominated by C. tomentosa, CG1–CG3—stands dominated by C. globularis, and P—vegetation-free pelagic sites. Contour lines are at 5-m intervals

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Table 1 Selected habitat characteristics of investigated lakes and the values of δ18O of pelagic water
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Field sampling

Each species was studied in five lakes and at three sites in each lake in July 2012 (Pronin et al., 2016). This gives 15 study sites for C. tomentosa and 15 sites for C. globularis. The species co-occurred in three lakes (Męcko Duże, Karskie Wielkie, and Jasne) and each of them occurred separately in two other lakes (C. tomentosa—in lakes Niesłysz and Złoty Potok, C. globularis—in lakes Malcz Południowy and Pierwsze). Altogether, the study was performed in seven lakes (Fig. 1, Table 1). In all the lakes studied, the species formed extensive and compact charophyte stands. Slender and densely growing C. globularis formed compact meadows near the bottom sediments, at the depth of 3–4 m, while taller, thicker, and branched C. tomentosa formed less compact stands at depths between 2 and 3 m, allowing waters from above the stand to penetrate into it more easily compared to C. globularis. At each site, 10 individual charophyte thalli from an area of 4 m2 were collected by diving. Prior to charophyte collection, water for isotopic analyses was sampled directly from above the charophyte stands using a bathometer, poured to a 10-ml glass septa test tube, and preserved with two drops of HgCl2 (Li & Liu, 2011; Apolinarska et al., 2015; Pronin et al. 2016). Additionally, as a control sample, water for isotopic analyses was also sampled from one vegetation-free pelagic site in each lake.

Laboratory work and analyses

Air-dried thalli of ten charophyte individuals with calcite encrustations were homogenized using a mortar and pestle and constituted a single sample for each studied site. The samples were transferred to Eppendorf test tubes and, together with water samples, sent to the Isotope Dating and Environment Research Laboratory in Warsaw, Poland, for stable isotope analyses. In the laboratory, carbonates (on average 400 µg of each sample) were dissolved in 100% phosphoric acid (density 1.9) at 75°C (McCrea, 1950), using a Kiel IV online carbonate preparation line connected to a ThermoFinnigan Delta + mass spectrometer. All values are reported as δ values in per mil relative to V-PDB by assigning a δ18O value of − 2.20‰ to NBS19. The reproducibility was tested by replicate analysis of laboratory standards and was found to be better than ± 0.07‰.

The δ 18O values of the water were measured using a GasBench-II headspace autosampler connected to a Finnigan MAT 253 isotope ratio mass spectrometer (IRMS). To ensure the precision of the values reported as δ values in per mil relative to V-SMOW, three international standards were measured: GISP (24.76‰ to V-SMOW), USGS W6444 (− 51.4‰ to V-SMOW), and USGS W 67400 (− 1.97‰ to V-SMOW) (Coplen et al., 2006). The reproducibility was tested by replicate analysis of laboratory standards and was found to be better than ± 0.25‰. Analytical procedures were described in detail in Apolinarska et al. (2015) and Pronin et al. (2016). The values of δ18OWATER and δ18OCARB, reported in this study for each studied species and each single site, are mean values of ten individuals collected in the field.

Statistical analyses

Since the empirical data distribution was inconsistent with the normal one, for most of the variables nonparametric tests were applied for the statistical data analyses. Therefore, the significance of differences between the tested species was checked using the Mann–Whitney U test. The relationship between δ18OCARB and δ18OWATER of the studied charophytes was tested using the Spearman rank R correlation. For the statistics applied, P < 0.05 was regarded as statistically significant. Statistica 10 software (StatSoft Inc., Tulsa, OK, USA) was applied to the Mann–Whitney U test and Spearman correlation. To illustrate the relations between the values of stable oxygen and (comparatively) carbon isotope composition of charophyte carbonates and water sampled from above the studied Chara stands (separately for C. tomentosa and C. globularis), scatter diagrams were plotted. Furthermore, for the lakes where both charophyte species co-occurred (Lake Męcko Duże, Lake Karskie Wielkie, Lake Jasne), scatter diagrams were plotted to illustrate the differences between the δ13CCARB and δ18OCARB values and those of δ13CDIC and δ18OWATER above the studied stands and of pelagic water.

Results

Mean values of δ18OWATER in lakes varied from about − 1.50‰ (lakes: Męcko Duże, Malcz Południowy, Pierwsze) to about − 4.00‰ (Lake Karskie Wielkie) and about − 4.50‰ (Lake Niesłysz and Złoty Potok, Online Resource 1). Mean oxygen stable isotope values of C. tomentosa encrustations changed between − 6.11‰ in Lake Złoty Potok and − 3.14‰ in Lake Męcko Duże (Online Resource 1). The values measured in encrustations of C. globularis varied between − 6.28‰ in Lake Jasne and − 3.62‰ in Lake Męcko Duże (Online Resource 1).

Although differences in δ18OWATER between the lakes studied were evidenced, differences between δ18OCARB and δ18OWATER were comparable at all the sites and in all the lakes from which samples were collected for isotopic analyses (Online Resource 1). Carbonate encrustations of both charophyte species were 18O-depleted relative to δ18O values of ambient waters; however, the extent of 18O-depletion in C. globularis was greater compared to C. tomentosa (Online Resource 1, Fig. 2, Fig. 3).

The differences between oxygen stable isotope composition of carbonates of the charophyte encrustations (δ18OCARB) and ambient waters (δ18OWATER) (Fig. 2) were statistically significant (Mann–Whitney U test, P < 0.0006) in both studied species. Importantly, the differences in the δ18OCARB values between C. tomentosa and C. globularis were insignificant (Mann–Whitney U test, P > 0.05).

Fig. 2
figure 2

Differences in oxygen stable isotope values between carbonates and water in a C. tomentosa and b C. globularis stands. For each species N = 15; mean, 25–75% mean + SE and mean + 2 SD values are presented. For both species P < 0.001 (Mann–Whitney U test)

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In contrast to δ18OCARB values, the values of δ18OWATER significantly differed between the investigated species when all investigated lakes were considered together (Mann–Whitney U test, P < 0.005). However, when the two species co-occurred in the same lake, no significant difference in δ18OWATER between them was noted. Figures 3 and 4 show the differences between stable oxygen and carbon isotope composition of carbonates and ambient waters in all investigated lakes (Fig. 3) and in lakes where C. tomentosa and C. globularis co-occurred (Fig. 4). It is noteworthy that conversely to δ13CDIC and δ13CCARB values (Pronin et al., 2016), the difference between δ18OWATER and δ18OCARB values revealed a similar trend for the two studied species (Figs. 2, 3, 4). Therefore, in each lake and at each sampling site, the water from above the charophyte stands was richer in 18O compared to the carbonate encrustations of each of the studied species (Fig. 3a, b). The tendency described above is particularly evident in lakes in which both species co-occurred under similar conditions (Fig. 4a–c). In C. globularis, the differences between δ18OWATER and δ18OCARB values were greater than in C. tomentosa (mean ± SD; for C. globularis, δ18OCARB = − 5.41 ± 1.19‰; δ18OWATER = − 2.19 ± 0.91‰, while for C. tomentosa δ18OCARB = − 5.01 ± 1.13‰; δ18OWATER = − 3.36 ± 1.15‰). The values of δ18OWATER and δ18OCARB were highly and significantly correlated in both species (Fig. 5a, b) but the Spearman rank R correlation values were higher in the case of C. tomentosa stands.

Fig. 3
figure 3

The differences between stable isotope composition of carbonates and ambient waters in a Chara tomentosa and b Chara globularis stands. Both species are characterized by similar trends in δ18O values, whereas the opposite trend was evidenced for δ13C, here presented only for comparison (for details please refer to Pronin et al., 2016). Lakes: LMD Lake Męcko Duże, LJ Lake Jasne, LN Lake Niesłysz, LZP Lake Złoty Potok, LKW Lake Karskie Wielkie, LMP Lake Malcz Południowy, LP Lake Pierwsze; for each species N = 15

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Fig. 4
figure 4

The differences between stable isotope composition of carbonates and ambient waters in Chara tomentosa and Chara globularis stands co-occurring under similar conditions in the same lakes: a Lake Męcko Duże, b Lake Jasne, and c Lake Karskie Wielkie. Additionally, pelagic sites were marked in each lake. For each species, N = 15 in each studied lake. Comment to δ13C values as in Fig. 3

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Fig. 5
figure 5

Spearman R rank correlations between δ18OCARB and δ18OWATER values for a Chara tomentosa stands and b Chara globularis stands. For each species, N = 15

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Discussion

Oxygen stable isotope composition of lake water is dependent on the δ18O values of the waters supplying the lake (precipitation, surface, and groundwater) and the rate of water exchange in the basin (Leng & Marshall, 2004 and references therein). The latter factor controls the degree to which the lake becomes 18O-enriched by evaporation. All the studied lakes were considerably 18O-enriched relative to local groundwater (− 9.2‰, d’Obryn et al., 1997), indicating strong influence of evaporation on δ18OWATER. The degree of the evaporative enrichment in 18O was lake-specific. Lakes Jasne, Pierwsze, and Męcko Duże, characterized by the highest δ18O values in waters (− 1.71, − 1.56, and − 1.57‰, respectively), are small and/or closed with long water retention (Table 1), and thus are prone to strong evaporation. Lake Malcz Południowy, although it has a throughflow character and thus δ18O values of its waters should be closer to δ18O values in local groundwater, is also relatively small and shallow and is characterized by intermediate δ18O values (− 2.31‰). Lakes Karskie Wielkie and Niesłysz, both big, deep, and throughflow lakes, are less 18O-enriched (− 3.53‰ and − 4.48‰, respectively), as expected (based on Leng & Marshall, 2004). Interestingly, δ18O values of waters in Lake Złoty Potok (− 4.25‰), a closed lake with long water retention, are comparable to δ18OWATER in the big and throughflow Lake Niesłysz. Considering the above data, we suggest that the supply of groundwater is important in the water budget of Lake Złoty Potok, which lowers δ18OWATER values in the lake.

It seems important that the differences between δ18OWATER and δ18OCARB values were concurrent in each studied lake and at each studied site, similarly to those of δ13CDIC and δ13CCARB (compare Pronin et al. 2016). The factors controlling oxygen stable isotope composition of encrustations are thus considered to be species-specific, which we attempt to evidence below.

The differences between the values of δ18OCARB and δ18OWATER for C. tomentosa in four out of the five studied lakes were less than 2‰ and only in Lake Jasne did they slightly exceed that value (Online Resource 1). Apolinarska et al. (2016) observed a comparable difference between the mean values of δ18OCARB for C. tomentosa and δ18OWATER in Lake Lednica, − 5.20 and − 3.98‰, respectively. As assumed by Andrews et al. (2004), the shift in values between δ18OWATER and δ18OCARB, proving the precipitation of carbonate encrustation of charophytes in isotopic equilibrium with the surrounding water, is 1.5‰. However, due to the fact that our study did not cover the whole period when encrustations could have been precipitated, it is impossible to assert whether carbonate precipitation occurred in the condition of equilibrium with the water surrounding charophytes.

In the case of C. globularis (Fig. 4b), differences in values between δ18OCARB and δ18OWATER were much greater (mean: 3.2‰) and not as evenly distributed as in C. tomentosa. This may explain why a positive correlation between δ18OCARB and δ18OWATER, found for both the studied species (Fig. 5a, b), was considerably stronger for C. tomentosa (Fig. 5a). Because C. globularis was more 18O-depleted in all the studied lakes relative to ambient water than C. tomentosa, a common factor is considered to be responsible for the difference observed. Even greater difference between δ18OWATER and δ18OCARB of C. globularis encrustations was reported by Huon & Mojon (1994). In mid-July, the above values were − 2.83‰ and − 10.4‰, respectively. However, the great 7.75‰ difference between the water and encrustation δ18O values may to some degree result from the partial drying up of the pond in July and strong evaporative 18O-enrichment in water observed by Huon & Mojon (1994). Moreover, due to decreased water pH in mid-July, carbonates were not precipitated at this final stage of the pond existence. Considering the oxygen stable isotope composition of water measured in mid-June, i.e., − 4.74‰ at pH 8.5, the above discussed difference in δ18O values between water and encrustations decreases to 5.66‰, which is still greater than the value of − 3.2‰ observed in the present study. The influence of pH on the δ18OCARB values was addressed in Pentecost et al. (2006) study. However in that study, charophytes investigated by Pentecost et al. (2006) were derived also from a small pond which desiccated during the vegetative season. Progressive lowering of the water level in this study and continuous photosynthesis by charophytes resulted in strong increase of pH. As a consequence, water pH reached values higher than 9 and the CO3 2− species that was present in water has influenced the δ18O values.

Although we performed our study at the peak of growing season when pH is expected to be the highest, the pH values were always lower than 9. Additionally, the differences of pH were negligible between the sites in each lake as well as between investigated lakes (see Table 3 in Pronin et al., 2016). Therefore, we assumed that the presence of CO3 2− species of carbon was negligible (for details please refer to Pronin et al., 2016). To justify our standpoint, we attach the model of carbon speciation according to pH (MINEQL 4.6 software, Environmental Research Software, Hallowell, ME, USA; Online Resource 2). It clearly points to bicarbonate ions as a dominant DIC form in the studied lake. All the above allowed us to assume that in case of our study the pH influence on δ18O values was minor.

Despite the greater depth of the occurrence of C. globularis stands (3–4 m) compared to C. tomentosa (2–3 m), δ18O values of water sampled from above the two species when they co-occurred in the same water bodies were very similar (Fig. 4). This indicates a well-mixed upper water column in all the lakes, at least to the water depth of 4 m. Although no difference in δ18O values of ambient water above the charophyte stands of the two species was observed, δ18O values of the water within the charophyte stands could have been different. Therefore, in future studies, it would be useful to compare oxygen stable isotope values of both charophyte encrustations and water from above the macroalgae with δ18O values of the water from within the charophyte stands.

We ascribe the difference in δ18O values between C. tomentosa and C. globularis to the morphology of the two species studied. The stands of C. tomentosa, a charophyte with a large thallus and numerous branches, are relatively loose and freely penetrated by ambient water. In contrast, the dense stands formed by C. globularis are more isolated; thus, the depletion in 18O of the water from within the charophytes may result from restricted mixing with the above waters, 18O-enriched by evaporation.

The second factor determining oxygen stable isotope composition of carbonates apart from δ18OWATER is water temperature. Applying the well-known relation between carbonate δ18O values and water temperature, i.e., a 0.24‰ decrease with each 1°C (Kim & O’Neil, 1997), the 1.62‰ difference in δ18O values between C. tomentosa and C. globularis means that water temperature is higher by 6.75°C in the encrustation precipitation of the latter species (δ18OCARB from Lakes Karskie Wielkie and Jasne was considered because of the co-occurrence of the two species and comparable δ18OWATER above the stands of the two species, Online Resource 1). Considering the greater depth of C. globularis occurrence and restricted mixing with overlying waters, the opposite trend is expected. Thus, differences in water temperature failed to explain the shift between δ18O values in C. tomentosa and C. globularis.

Another factor that must be considered when studying stable isotope composition of biogenic carbonates is the so-called vital effect referring to the organism precipitating carbonates. In charophytes, 18O-depletion in encrustations relative to δ18OWATER is linked with rapid CaCO3 precipitation on stems (McConnaughey, 1989; Andrews et al., 2004). δ18O values in C. tomentosa and C. globularis are in line with this observation (Fig. 3). The offset between δ18OWATER and δ18OCARB increases with a higher rate of calcification resulting from intensive photosynthesis. Of the two species studied, C. tomentosa, the larger charophyte found at shallower sites where photosynthesis is more intensive due to higher light availability, is expected to have encrustations more 18O-depleted compared to C. globularis. This theoretical assumption disagrees with the available data and does not explain lower δ18O values in C. globularis (Online Resource 1). However, in laboratory studies, Sendra et al. (2010) found out that the charophytes can be affected by UV-B radiation, especially in long-term treatment. The UV-B radiation may decrease the photosynthesis rate. Consequently, C. tomentosa growing at shallower sites should precipitate carbonates, which are less 18O-depleted compared to C. globularis. On the other hand, Schmidt et al. (2010) and Sendra et al. (2010) did not find any statistically significant differences between charophyte individuals exposed to UV-B radiation compared to treatments without UV-B radiation, which, as they suggested, may result from adaptation to radiation. This aspect, however, requires further study performed in situ.

Conclusions

Encrustations of Chara tomentosa and Chara globularis were found to be 18O-depleted relative to δ18O values of water in all the studied lakes, in agreement with the effect expected during fast calcification. However, the rate of CaCO3 precipitation failed to explain lower δ18O values in C. globularis compared to C. tomentosa. The morphology of the two species, determining the density of charophyte stands (loose in C. tomentosa and dense in C. globularis) and, respectively, allowing or restricting water penetration into the studied stands, is regarded to be most likely to explain greater 18O-depletion of the latter species.

Nevertheless, the constant shift in stable isotope composition between the mean δ18O values of C. tomentosa and C. globularis encrustations and waters, 2‰ and 3.2‰, respectively, indicates that δ18O values of water are recorded by charophytes. However, this constant 18O depletion in charophyte encrustations in relation to δ18O of water must be considered when applying charophyte δ18O values in paleolimnological studies. Problematic seems to be the different extent of 18O-depletion observed in the two species studied. Considering this outcome of our study stratigraphic change in δ18O values of the carbonates from a lake core can result from a change in the specific composition of charophytes and not from environmental factors, as expected.