Methods
Field sample collection and processingAt each location, temperature, dissolved oxygen (DO), and pH were collected at 1 m increments from the surface to the maximum depth (YSI 556MPS). Photic zone depth was defined at 1percent of photosynthetically active radiation (PAR) as measured using a PAR sensor (LiCor 192SA). Integrated photic zone samples were then collected using a weighted 2-inch diameter polypropylene tube. Samples for DNA, nutrients, toxins, and pigment analyses were collected in acid-washed, sterile bottles, (rinsed three times with in situ water before collection) and stored on ice until further processing.Once transported back to the lab, samples were immediately processed. For dissolved reactive phosphorus (DRP), total dissolved phosphorus (TDP), total dissolved nitrogen (TDN), nitrate, and nitrite, 100 mL of water was filtered through a Whatman glass fiber filter (GForF) and frozen at -20 degreeC. For TP and total nitrogen (TN), HCl was added to 100mL of sample to a final concentration of 0.1percent and stored at -20 degreeC. Ammonium samples were immediately measured to avoid oxidation during freezing. Chlorophyll-a and phycocyanin samples were collected onto GForF filters and stored in black tubes at -20 degreeC. For community analysis (DNA), samples were filtered onto 0.2 µm polyethersulfone membrane filters (Supor-200; Pall Corporation) and frozen at -20 degreeC until extraction. 20 mL of unfiltered water was preserved in formalin (3percent final concentration) and stored at room temperature in the dark for microscopy. An additional 50 mL of unfiltered water was stored at -20 degreeC for toxin analysis.Analytical measurementsDRP was measured by the ascorbic acid-molybdenum blue method 4500 P E (Greenberg et al. 1992). Ammonium was measured spectrophotometrically (Solórzano 1969). Nitrate and nitrite were measured individually using high-performance liquid chromatography (HPLC) (Flowers et al. 2009). TPorTDP and TNorTDN were digested as previously described (White et al. 2010), prior to analysis as for DRP and nitrate. For TDN and TN, the resulting solution was oxidized completely to nitrate and was measured via HPLC as above. Nitrate, nitrite, and ammonium were summed and reported as dissolved inorganic nitrogen (DIN).Phycocyanin was extracted in 20 mM sodium acetate buffer (pH 5.5) following three freeze-thaw cycles at -20 ºC and on ice, respectively. The extract was centrifuged and then measured spectrophotometrically at 620 nm with correction at 650 nm (Demarsac and Houmard 1988). Chlorophyll-a (Chl-a) was extracted overnight at -20 ºC in 90percent acetone and then measured spectrophotometrically with acid correction (Tett et al. 1975).For toxin analysis, whole water samples were lyophilized, resuspended in 5percent acetic acid, separated by solid phase extraction (SPE; Bond Elut C18 column, Varian), and eluted in 50percent methanol as previously described (Harada et al. 1988). Microcystin (MC) variants of leucine (L), arginine (R), and tyrosine (Y) were detected and quantified at the Wisconsin State Lab of Hygiene (SLOH) using liquid chromatography electrospray ionization tandem mass spectroscopy (API 3200, MSorMS) after separation by HPLC (Eaglesham et al. 1999). We report only MCLR concentrations since MCYR and MCRR were near the limit of quantification for the sampling period (0.01 µg L-1).In situ N2 fixation measurementsN2 fixation rates were measured, with some modifications, following the acetylene reduction assay (Stewart et al. 1967). A fresh batch of acetylene was generated each day before sampling by combing 1 g of calcium carbide (Sigma Aldrich 270296) with 100 mL ddH2O. Following sample collection, 1 L of water was concentrated by gentle filtration onto a 47 mm GForF filter in the field. The filter was then gently washed into a 25 mL serum bottle using the lake water filtrate (final volumes 10 mL aqueous, 15 mL gas). Samples were spiked with 1 mL of acetylene gas and incubated in situ for two hours. The assay was terminated with 5percent final concentration trichloracetic acid and serum bottles were transported back to the lab. For each sampling period, rates were controlled and corrected for using a series of the following incubated acetylene blanks: 1) 1 mL of acetylene in filtrate alone, 2) 1 mL of acetylene in a killed sample, and 3) 1 mL of acetylene in ddH2O. Ethylene formed was measured by a gas chromatograph (GC; Shimadzu GC-8A) equipped with a flame ionization detector (FID), Porapak N column (80or100 mesh, 1or8OD x 6), and integrator (Hewlett Packard 3396) with N2 as the carrier gas (25 mL min-1 flow rate). Molar N2 fixation rates were estimated using a 1:4 ratio of N2 fixed to ethylene formed (Jensen and Cox 1983). All N2 fixation values are reported as integrated photic zone rates of µg N L-1 hr-1.DNA extraction and processing of PC-IGS fragmentDNA was extracted from frozen filters using a xanthogenate-phenol-chloroform protocol previously described (Miller and McMahon 2011). For amplification of the phycocyanin intergenic spacer (PC-IGS) region, we used primers PCalphaR (5-CCAGTACCACCAGCAACTAA-3) and PCbetaF (5-GGCTGCTTGTTTACGCGACA-3, 6-FAM-labelled) and PCR conditions that were previously described (Neilan et al. 1995). Briefly, each 50 µl reaction mixture contained 5 µl of 10X buffer (Promega, Madison, WI), 2.5 µl of dNTPs (5 mM), 2 µl of forward and reverse primers (10 µM), 2 µl of template DNA, and 0.5 µl of Taq DNA polymerase (5 U µl-1). Following precipitation with ammonium acetate and isopropanol, the DNA pellet was resuspended in ddH2O and digested for 2 hrs at 37 ºC using the MspI restriction enzyme, BSA, and Buffer B (Promega, Madison, WI). The digested product was precipitated and then resuspended in 20 µL of ddH2O. 2 µL of final product was combined with 10 µL of formamide and 0.4 µL of a custom carboxy-x-rhodamine (ROX) size standard (BioVentures, Inc).Cyanobacterial PC-IGS community fingerprinting and cell countsWe analyzed the cyanobacterial community using an automated phycocyanin intergenic spacer analysis (APISA) similar to the automated ribosomal intergenic spacer analysis (ARISA) previously described (Yannarell et al. 2003). Briefly, this cyanobacterial-specific analysis exploits the variable PC-IGS region of the phycocyanin operon (Neilan et al. 1995). Following MspI digestion, the variable lengths of the PC-IGS fragment can be used to identify subgenus level taxonomic units of the larger cyanobacterial community (Miller and McMahon 2011). The MspI fragments were sized using denaturing capillary electrophoresis (ABI 3730xl DNA Analyzer; University of Wisconsin Biotechnology Center (UWBC)). For each sample, triplicate electropherogram profiles were analyzed using GeneMarker® (SoftGenetics) software v 1.5. In addition, a script developed in the R Statistics Environment was used to distinguish potential peaks from baseline noise (Jones and McMahon 2009, Jones et al. 2012). Relative abundance data output from this script were created using the relative proportion of fluorescence each peak height contributed per sample. Aligned, overlapping peaks were binned into subgenus taxonomic units (Miller and McMahon 2011). These taxa were named based on the genus and base pair length of the PC-IGS fragment identified (e.g. For Mic215, Mic = Microcystis and 215 = 215 base pair fragment). Fragment lengths were matched to an in silico digested database of PC-IGS sequences using the Phylogenetic Assignment Tool (https:ororsecure.limnology.wisc.eduortrflpor).The NTL-LTER program collects biweekly phytoplankton samples between April and September for cell counts and detailed descriptions of the field and laboratory protocols are available online at http:ororlter.limnology.wisc.edu. When indicated, biomass has been converted to mg L-1 using the biovolume calculated during the cell count process and assuming a density equivalent to water.
