Sampling processing

Plant material was collected at a depth of 0.5–1.5 m from the shore, or by snorkeling. Undamaged plants of C. canescens, C. hispida, C. subspinosa, and C. tomentosa were picked. In the laboratory, epibionts were removed gently with a brush. For further analyses, plants were analyzed in parts of thalli. First to third, forth to sixth, seventh to ninth whorls and internodes were used for analysis (Fig. 2). Side branches and other parts of thalli were removed.

Comparison of carbonate content based on DW (%) of C. canescens (can), C. hispida (his), C. subspinosa (sub), and C. tomentosa (tom) from sampling sites (Angersdorfer Teiche, Asche, Bruchwiesen, Krüselinsee, and Lützlower See). Plants were analyzed in first to third (white), forth to sixth (light grey), seventh to ninth (dark grey boxplots) whorls and internodes. Box plots include whiskers (5–95% of variability) and outliers (points). Different letters indicate significant difference within the same sampling site (Tukey HSD post hoc test, p < 0.05)

Carbonate proportion of plant dry weight was determined by the loss of ignition (LOI) method as described by Heiri et al. 2001. Sample size was n = 7 for the LOI analysis. The samples were dried at 60 °C (UM 400, Memmert) overnight. Plant DW was weighed and combusted in crucibles in a two-step process, at 550 °C (DW₅₅₀) and 925 °C (DW₉₂₅) for two hours, respectively (LE 6/11/B 150, Nabertherm). Carbonate content of DW (%) was calculated of the carbonate bounded CO₂ loss multiplied by 1.36 (fraction of CO₂ in CO₃²⁻) after Pełechaty et al. 2013 (Eq. 1).

Element contents of Ca, Fe, K, Mg and P were measured with inductively coupled plasma-optic emission spectroscopy (ICP-OES). Plant DW was ground and 0.1 g was weighted for the digestion. Samples were extracted in duplicate with 5 ml HNO₃ (65%) and 3 ml H₂O₂ (30%) for 1.5 h in CEM MARS 6 microwave. After extraction, samples were filled up to 25 ml with ultrapure water and were filtered (601P, Rotilabo). Samples were analyzed spectrometrically for Ca (317.9 nm), Fe (238.2 nm), K (766.5 nm), Mg (285.2 nm) and P (214.9 nm) with Optima 8300 (Perkin Elmer, Singapore).

Photosynthesis of C. canescens, C. hispida from Angersdorfer Teiche, C. hispida from Asche, and C. tomentosa from Bruchwiesen was measured by means of O₂ evolution with a Clark-type electrode (Microelectrodes, USA). Within a day after sampling, plants were dark adapted at least for 30 min and were set in a temperature-controlled 2.5 ml cuvette (15 °C). The cuvette was filled with habitat medium which was stirred during measurement. Photon flux densities were applied by a light dispenser system (MK2, Illuminova, Sweden) described by Wolfstein and Hartig (1998). Dark respiration was measured for 10 min, followed by increasing irradiation levels of 14, 18, 31, 102, 494, 912, 1462, 1811, 2120 µmol photons m⁻² s⁻¹. Each irradiation level was set for 3.3 min; measurement took 40 min in total.

Photosynthesis of C. tomentosa from Asche was measured with the addition of CaCl₂, KCl, MgCl₂ and NaCl. Plants were cultivated in habitat water and at a constant temperature (15 °C) and light (50–80 µmol photons m⁻² s⁻¹ on a 12 h/12 h light–dark cycle) to allow consistent conditions for measurements. Light saturation irradiance (I_s) of cultivated C. tomentosa was calculated in preliminary measurements (I_s = 120 µmol m⁻² s⁻¹). For 20 min measurements, 1.5 × I_s (1.5I_s = 180 µmol m⁻² s⁻¹) was used to gain light saturation. After a dark period of 3 min, constant irradiance level of 180 µmol m⁻² s⁻¹ was set. Concentration of 1.2 mmol L⁻¹ of chloride salt was added at minutes 5, 7, 9, 11, 13, 15, and 17 of measurement. The end concentration was 8.4 mmol L⁻¹ added chloride salt in the cuvette. Control measurements were conducted without addition of chloride salt.

Photosynthesis/irradiance (P/I) curve was calculated using the model of Walsby (1997) to determine photosynthesis (P), maximum photosynthesis (P_m), dark respiration (R_d), α (slope of P/I curve at limiting irradiance), light compensation point (I_c = R_d/α), and light saturation irradiance (I_s = P_m/α). Photosynthetic parameters were based on chlorophyll a (chl a). Pigment content of first to third whorls and internodes were extracted with 3 ml of N,N-dimethylformamide. Samples were incubated overnight at 4 °C. Extinction at 470, 630, 647, 664, and 750 nm was measured using a Lambda 2 spectrometer (Perkin Elmer, Germany). Chl a content was calculated after Wellburn (1994).

Water samples were taken in 50 ml tubes above plant meadows, closely beneath the water surface. pH value and conductivity were measured directly at sampling sites with HACH HQ40d (Hach-Lange, Germany). The water samples were analyzed at Helmholtz Centre for Environmental Research (UFZ)—Central Laboratory for Water Analytics and Chemometrics. The concentrations of Ca²⁺, Cl⁻, K⁺, Mg²⁺, Na⁺, and SO₄²⁻ were measured with an ion chromatograph (Dionex ICS 3000, Thermo Fisher, USA). Total inorganic carbon (TIC) was determined with a Dimatoc 2000 (DIMATEC, Germany). Water samples were measured in accordance with EN ISO 14911:1999 and EN ISO 10304:2009-1.