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.
