Uncoupled phytoplankton-bacterioplankton relationship by multiple drivers interacting at different temporal scales in a high-mountain Mediterranean lake

Global-change stressors act under different timing, implying complexity and uncertainty in the study of interactive effects of multiple factors on planktonic communities. We manipulated three types of stressors acting in different time frames in an in situ experiment: ultraviolet radiation (UVR); phosphorus (P) concentration; temperature (T) in an oligotrophic Mediterranean high-mountain lake. The aim was to examine how the sensitivity of phytoplankton and bacterioplankton to UVR and their trophic relationship change under nutrient acclimation and abrupt temperature shifts. Phytoplankton and bacteria showed a common pattern of metabolic response to UVR × P addition interaction, with an increase in their production rates, although evidencing an inhibitory UVR effect on primary production (PP) but stimulatory on bacterial production (HBP). An abrupt T shift in plankton acclimated to UVR and P addition decreased the values of PP, evidencing an inhibitory UVR effect, whereas warming increased HBP and eliminated the UVR effect. The weakening of commensalistic and predatory relationship between phyto- and bacterioplankton under all experimental conditions denotes the negative effects of present and future global-change conditions on planktonic food webs towards impairing C flux within the microbial loop.

HBP (µg C L -1 h -1 ) 0.067 ± 0.005 Table 1S. Mean values (± standard deviation) of the main chemical and biological variables studied in the water column under the initial conditions of the experiment. TN: total nitrogen; TP: total phosphorus; SRP: soluble reactive phosphorus; DIN: dissolved inorganic nitrogen; DOC: dissolved organic carbon; Chl a: chlorophyll a; N:P sestonic : nitrogen to phosphorus ratio of the seston on a molar basis; PA: phytoplanktonic abundance; PP: primary production; BA: bacterial abundance; HBP: heterotrophic bacterial production.

Supplementary text 1S
A large sample size (pairs irradiance-depth values, n >160) was used, and a good fit (R 2 > 0.95) was found for all regressions.

Supplementary text 2S
DIN was considered the sum of nitrate (NO 3 -), nitrite (NO 2 -), and ammonium (NH 4 + ). NO 3 -was analysed by UV spectrophotometric screening, NO 2was determined using the sulphanilamide method, and NH 4 + by the phenol-hypoclorite method 1 . Total nitrogen (TN) and TP were determined by analysing 50-mL aliquots after digestion with a mixture of potassium persulfate, boric acid, and sodium hydroxide at 120ºC for 30 min 1 . NO 3and phosphate concentrations in the digested samples were measured following Grasshoff et al. 2 .

Supplementary text 3S
A Perkin-Elmer model 2400 CHN elemental analyser (Perkin-Elmer Corporation, Waltham, Massachusetts, USA) was used for samples analysis.

Supplementary text 4S
Samples were thawed and placed in centrifuge tubes (15 mL) with 5 mL of acetone (90%) for 24 h in the dark at 4ºC. Next, the samples were centrifuged, and the fluorescence of the At least 600 cells of the most abundant algal species were counted in each sample.

Supplementary text 6S
Water samples were fixed with neutralized formaldehyde (2%), stained with DAPI to a final concentration of 2.5 µg mL -1 , and then filtered through a 0.2-µm pore-size black polycarbonate Nucleopore Filter. At least 400 cells per sample were counted by epifluorescence microscopy at 1000 magnification (Karl Zeiss AX10).

Supplementary text 7S
Aliquots of 1.5 ml were taken from each replicate and placed in microcentrifuge vials. TCA cold extraction was performed by keeping the vials in ice for 20 min, after which the precipitate was collected by centrifugation (at 16,000g for 10 min). Then, vials were rinsed twice with 1.5 mL of 5% TCA to remove any residual unincorporated radioactivity, and scintillation liquid (Ecoscint A) was added for subsequent measurement in an autocalibrated scintillation counter (Beckman LS 6000TA) 3 . The conversion factor 1×10 18 cell mol -1 4 was used to estimate the number of bacteria produced per mol of incorporated thymidine. The factor 2×10 -14 g C cell -1 5 was applied to estimate the amount of carbon.

Supplementary text 8S
The 14 C measured on the 1μm filters represents the 14 C incorporated into phytoplankton cells CARB may be an upper estimate of the actual bacterial demand for photosynthetic carbon because respired carbon is not included in the 14 C-Bact variable and steady state and 14 Cisotopic equilibrium with the autochthonous pool is assumed; therefore, the denominator (i.e. 14 C-Bact × EOC -1 ) can be underestimated 6 .