Methods

Protocol available in methods section of: http://msphere.asm.org/content/2/3/e00169-17
Prior to collection, water temperature and dissolved oxygen concentrations are measured using a YSI 550a. The ranges of the epilimnion and hypolimnion are determined based on the location of the thermocline (where temperature/oxygen is changing the fastest). The two layers are collected separately in 1 meter increments using an integrated water column sampler. Water samples are taken back to the lab, shaken thoroughly, and filtered via peristaltic pump through 0.22 micron filters (Pall Supor). Filters are temporarily stored at -20C after collection and then transferred to -80C after transport on dry ice from Trout Lake Station to UW-Madison. Nutrient samples are collected bi-weekly following standard LTER protocols. DNA is extracted from filters using a FASTDNA SpinKit for Soil with minor modifications. (In cases of low yield or specialized sequencing methods, a phenol-chloroform extraction is used instead). The protocol for sequencing and analysis of data varies by year and by sub-project.

Methods

Respiration of total plankton passing a 70 micron mesh, and bacteria passing a 1 micron mesh, calculated from loss of oxygen in lake water incubated at in situ temperatures. Oxygen concentration was determined using the Winkler reaction with azide modification. Final product concentration determined via spectrometry or titration with sodium thiosulfate. Titrations that may have overrun the endpoint were not included. The following equation for calculation of dissolved oxygen concentration from titration of Winkler end product with thiosulfate (from Wetzel, R. G. and G. E. Likens. 1991. Limnological Analyses, 2nd ed. Springer-Verlag, New York). Thiosulfate with a molarity of 0.20 N was used for all titrations. mg O2 L-1 = (ml titrant)*(molarity of thiosulfate)*(8000)/((ml of sample titrated)*((ml of bottle -3)/(ml of bottle))). The following equation is for calculation of dissolved oxygen calculation from spectophotometric analysis of Winkler reaction end product (from Roland, F., N. F. Caraco, J. J. Cole. 1999. Rapid and precise determination of dissolved organic oxygen by spectrophotometry: Evaluation of interference from color and turbidity. Limnol. Oceanogr. 44(4):1148-1154). mg O2 L-1 = absorbance at 430 nm (in units of cm-1) * 8.1-0.41 Sampling Frequency: fortnightly during ice-free season - every 6 weeks during ice-covered season Number of sites: 4

Methods

Sample Collection and Processing. For phytoplankton and planktonic protozoan community analyses, whole water samples of 500–1000 mL were collected from Crystal Bog in the fall of 1999 (September, October, November), the winter of 2000 (January and February), and biweekly throughout the ice-free period for 2000 (March 26 through November 17). Crystal Bog was sampled over the entire 2-m water column at the point of maximum depth using an integrated water column sampler consisting of a length of PVC pipe equipped with a ball-joint valve. A single sample from a station at the maximum depth of this small bog was collected on each sample date. Samples were preserved with 25percent glutaraldehyde to a final concentration of 2percent in each sample bottle. Samples were stored in the dark in a refrigerator until counted.On the same collection dates throughout the ice-free period of the year 2000, water samples for bacterial community fingerprint analysis were obtained from Crystal Bog and filtered through autoclaved 10-lm nylon mesh screening (Spectrum) to remove eukaryotic cells. Samples were cooled on ice for transport back to the Trout Lake field station. Water samples were filter-concentrated in aliquots of 250 or 500 mL onto sterile 0.2- lm filters (Supor-200, Gelman). Filters were then placed in cryovials, frozen in liquid nitrogen, and stored at 80 C until DNA could be extracted with a FastPrep DNA purification kit (BIO 101). In addition, 250 mL of unfiltered water was also preserved in 2percent glutaraldehyde for later enumeration of bacterial cells. Identification and Enumeration of Algae and Protozoa. Twenty-mL aliquots of preserved sample were J.M. GRAHAM ET AL.: DIVERSITY IN A HUMIC LAKE 529 settled in chambers for 48 h prior to counting. Counting was performed on an Olympus IX-50 inverted microscope at 200 and 400. Algae and protozoa were identified and counted in one half the surface area of the settling chamber, equivalent to 10 mL of sample. The remaining half of the chamber was scanned at 200· for additional counts of larger phytoplankton and protozoan species present at low densities. Identifications of phytoplankton were based on Smith [39] and Prescott [30] plus additional specialized texts for dinoflagellates [29], desmids [7, 31–34], and chrysophytes [4]. Identifications of protozoa were based on Kudo [21], Patterson [24], and particularly Foissner and Berger [10], together with their associated taxonomic volumes [11]. Identifications were made to species where possible. Abundance of each species was expressed as number of cells, colonies, or filaments per liter. For mean cell, colony, or filament volume estimates, at least 10 individuals (when available) were measured for size with a calibrated ocular micrometer on each sample date, and volumes were calculated based on standard geometric formulas [14]. Novel geometric formulas were devised for some taxa, for example, those shaped like a cone of elliptic cross section or a cylindrical filament wound into a coiled spring. Biovolume of each species was the product of the countorliter and the mean volume. Bacterial Abundance Analysis. Bacterial abundance was determined by staining 2 mL of unfiltered preserved water from Crystal Bog with 40, 60-diamidino- 2-phenylindole (DAPI) according to the procedures given in Porter and Feig [28]. The stained bacterial cells were filtered onto black 25-mm 0.2-lm pore size PCTE filters, mounted on slides, and examined under oil immersion with a Nikon Diaphot epifluorescence microscope. The numbers of bacterial cells were then counted in 10 random Whipple grids per slide on two perpendicular transects. Additional information on bacterial enumeration is available in the on-line methods manual for the Microbial Observatory for the NTLLTER site (http:orormicrobes.limnology.wisc.eduormethods. htm).Community Fingerprint Analysis of Bacteria. Bacterial DNA was extracted from 500 mL of filtered lake water using the FastPrep DNA purification kit (BIO101). Bacterioplankton diversity was assessed by automated ribosomal intergenic spacer analysis (ARISA). PCR for ARISA was performed following the method of Fisher and Triplett [8] with modifications. PCR reactions contained PCR buffer consisting of 50 mM Tris (pH 8.0), 250 lg of bovine serum albumin per mL and 3.0 mM MgCl2 (Idaho Tech), 250 lM of each dNTP, 10 pmol of each primer, 1.25 U of Tag polymerase (Promega), and 1 lL of lake-extracted DNA in a final volume of 25 lL. The primers used for ARISA were 1406f (universal 16S rRNA gene; 50-TGYACACACCGCCCGT-30) labeled with 6- FAM, and 23Sr (bacteria-specific, 23S rRNA gene; 50- GGGTTBCCCCATTCRG-30). All PCR was carried out in an Eppendorf MasterCycler Gradient (Eppendorf). The initial denaturation was performed at 94 C for 2 min, followed by 30 cycles of 94 C for 35 s, 55 C for 45 s, and 72 Cfor 2 min, with a final extension carried out at 72 C for 2 min.Denaturing capillary electrophoresis was carried out for each PCR reaction using an ABI 310 Genetic Analyzer (PE Biosystems). Electrophoresis conditions were 60 C and 15 kV with a run time of 50 min using the POP-4 polymer. A custom 200- to 2000-bp rhodamine X–labeled size standard (Bioventures) was used as the internal size standard for each sample. The data were analyzed using GeneScan 3.1 software (PerkinElmer). To include the maximum number of peaks while excluding background fluorescence, a fluorescence cutoff of 500 fluorescence units was used.

Methods

Bacterial respiration of bacteria passing a 70 micron mesh. Based on bacterial growth efficiency determined empirically on bacteria production and oxygen depletion on microbes passing a 1 micron mesh. All samples from integrated sample of epilimnion to thermocline or 12 meters, which ever was more shallow. Method based on Roland, F. and J. J. Cole. 1999. Regulation of bacterial growth efficiency in a large turbid estuary. Aquatic Microbial Ecology 20:31-38 Sampling Frequency: fortnightly during ice-free season - every 6 weeks during ice-covered season Number of sites: 1

Methods

Net production of bacteria passing a 70 micron mesh, or bacteria passing a 1 micron mesh, calculated incorporation of 3H labeled leucine into cell proteins. Method based on microcentrifuge method of Smith, David C., and Farooq Azam. 1992. A simple, economical method for measuring bacterial protein synthesis rates in seawater using 3H-leucine, Marine Microbial Food Webs 6(2):107-114. Nanomolar treatments refer to leucine concentration for calculation of isotopic dilution and necessary leucine concentration to saturate uptake into bacterial cells. For a good example on how to calculate isotopic dilution see Pace. Michael L., and Jonathan J. Cole. Primary and bacterial production in lakes: are they coupled over depth? Plankton Research 16(6):661-672. Sampling Frequency: fortnightly during ice-free season - every 6 weeks during ice-covered season Number of sites: 4

Methods

BEFOREHAND prepare MUF-P substrate. Dissolve 180.04 mg of MUF-P in 120.0 mL total fresh Nanopure water to get 5 mM solution. Aliquot 1 mL each into thirty microcentrifuge tubes. Store in freezer. These aliquots will be thawed as needed.BEFOREHAND prepare borate buffer by dissolving 4.7675 g in 250 mL Nanopure water.PREPARE MUF standards the day before and store in cold room. Standards are prepared as follows: Prepare 5 mM MUF by adding 158.56 mg MUF in 100 mL. Vortex. Dilute 0.120 mL of 5 mM MUF in 15.0 mL total to get 40 uM solution. Prepare solutions for working standards as follows. Vortex solutions before dilutions.MUF Concentration (nM) - Preparation:0 -- 0 mL of 200 nM in 10 mL.10 -- 0.5 mL of 200 nM in 10 mL.50 -- 2.5 mL of 200 nM in 10 mL.100 -- 5 mL of 200 nM in 10 mL.200 -- 75 mL of 200 nM in 10 mL.THAW MUF-P substrate the day before in cold room.LABEL scintillation vials day before sampling. For substrate need one vial labelled 100 uM. For lake water sample need 9 vials. Three replicates samples are run for each of three size fractions. The three size fractions are less than 0.2 um water, less than 1 um water and less than 70 um (field filtered) water.DAY of sampling, filter borate buffer solution through a 0.2 um syringe filter. Dilute 0.300 mL of 5 mM MUF-P solution in 15.0 mL total Nanopure water to get 100 uM solution. Vortex MUF-P before aliquoting to samples.TURN on fluorometer to warm up (10 minutes) and set filters to 365 nm excitation, 460 nm emmission. Aliquot 10 mL of lake water into appropriate 20 mL scintillation vials labelled the day before.PERFORM measurement in room with fluorometer in the dark. First run the standard curve. If the standard curve does not appear to be linear, make new standards before running samples. If standard curve is good, add 0.010 mL of 100 uM MUF-P substrate to each ten mL sample for a final concentration of 100 nM. Note time when you spiked the samples.BEGIN immediately aliquoting 3.0 mL of sample each to glass test tubes. Aliquot all nine test ubes before adding borate buffer. Add 1.0 mL borate buffer to test tubes and mix. Note fluorescence. Continue with next sample. Do less than70, less than1 and less than 0.2 um replicates together to make more comparable (i.e. less than70 um replicate 1, less than 1 um replicate 1, less than 0.2 um replicate 1, less than 70 um replicate 2, etc.). Note time when finisihed running all initial samples. Put rest of samples in incubator.REPEAT measurements of sample for final time point thirty to sixty minutes later. Note time when you begin aliquoting 3 mL of samples to test tubes. Note time when final samples have all be run.

Methods

this is a summary dataset based on knb-lter-ntl.45, knb-later-ntl.50, and knb-lter-ntl.51 where the methods are described in detail. in addition bacterial cell counts were optained from epifluorescence microscopy using DAPI cell stain.