Oxygen dynamics and plant-sediment interactions in isoetid ...

Paper 4ensure mixing and air saturated conditions.Water was changed weekly.Pore-water content and O 2 penetrationMinute pore-water samples were extracted after135 days of experiments by small glass tubesinserted in 4 cm depth in the sediments (Møller& Sand-Jensen 2011) and analyzed fordissolved inorganic carbon (DIC), reduced Fe 2+ ,ortho-phosphate (O-P) and NH + 4 . DIC wasdetermined by injecting 5-50 µl samples into3% HNO 3 in a bubble chamber purged with N 2gas carrying evolved CO 2 into an Infrared gasanalyzer (IRGA, ADC-225-MK3, Hoddesdon,UK; Vermaat & Sand-Jensen, 1987). Fe 2+ wasmeasured on 100 µl samples diluted in 900 µl0.1 M HCL according to Eaton et al. (1995). O-P was measured according to a modified versionof Strickland & Parsons (1968) as previouslydescribed in Møller & Sand-Jensen (2011).NH +4 was measured on 100 µl pore-waterdiluted in 900 µl distilled water added 100 µlphenol and 100µl hypochlorite reagentsaccording to Solórzano (1969). O 2 penetrationdepth was measured by a mini O 2 electrodes(Ox 500, Unisense, Århus, Denmark) movedand positioned by a micromanipulator between0 and 40 mm depth in the sediment. O 2penetration was measured within the last twohours of the light period to ensure nearmaximum O 2 penetration.Plant morphology and myzorrhizal colonizationPlants were gently removed from the sedimentand rinsed in water after the 135-days longcolonization experiment. Number of leaves wasrecorded and plants were split into above-(leaves) and below-ground (stem and roots)biomass. Freeze-dried leaves were analyzed forTP, TN and chlorophyll content. TP wasmeasured according to Andersen (1976), TN bya CHN EA1108-elemental analyzer (Carlo ErbaInstruments, Milan, Italy) and chlorophyll byextraction in ethanol for 24 hours andspectrophotometrical analysis (Christoffersenand Jespersen (1986). Total root length of eachplant was determined by the grid interceptmethod (Newman (1966). Roots were cleared in10% KOH and stained for AMF with tryphanblue(Kormanik & Mosse 1980) and stored inlactoglycerol. AMF colonization frequency wasdetermined at 20 x magnification as the averageoccurrence of hyphae, arbuscles or vesicles in 2x 100 intercepts between grit and roots(Giovannetti & McGraw 1982). Supplied AMFfreeplants had been grown emerged with rootsin an agar medium exposed to light resulting inlateral root systems with chlorophyll. This madeit possible to determine that all roots had beenreplaced at the end of the experiment since onlyvertically oriented roots without chlorophyllremained. Similarly, the initial air leaves (thinand long) had been shed and renewed by aquaticleaves (shorter and thick) at the end of theexperiment.Experiment with indigenous AMF associationsSix intact sediment turfs (15 cm long, 17 cmwide and 13 cm deep) inhabited by densepopulations of Lobelia dortmanna and six withLittorella uniflora with indigenous AMFcolonization and normal hyphal density werecollected at a sandy site in Lake Värsjö. Turfswere brought back to the laboratory submergedin lake water and subjected to organicenrichments of 0, 0.1, 0.2, 0.4, 0.8, 1.6%organic matter per sediment DW by insertingfeeding pellets of variable length at 4 cm depth74