Methods

Conducted weekly data sampling (9 boat trips in June-August 2019) using an EXO multi-parameter sonde to collect temperature, conductivity, chlorophyll (ug/L), phycocyanin (ug/L), turbidity, dissolved organic material, ODO, pH, and pressure at 35 locations based on GPS guided stratified random sampling. 35 grid boxes were generated over Lake Mendota using qGIS. Point locations within each grid box were randomized before each sampling effort. At each point, variables were recorded continuously with the EXO sonde for a two-minute period. Continuous data was overaged over the two-minute period for each sample point.

Methods

Primary publications that provide more information about taxa, methods, and data are:
Carpenter, S.R., J.J. Cole, M.L. Pace, R.D. Batt, W.A. Brock, T. Cline, J. Coloso, J.R. Hodgson, J.F. Kitchell, D.A. Seekell, L. Smith and B. Weidel. 2011. Early warnings of regime shifts: A whole-ecosystem experiment. Science 332: 1079-1082.
Cline, T.J., D. A. Seekell, S. R. Carpenter, M. L. Pace, J. R. Hodgson, J. F. Kitchell, and B. C. Weidel 2014. Early warnings of regime shifts: evaluation of spatial indicators from a whole-ecosystem experiment. Ecosphere 5:art102. http://dx.doi.org/10.1890/ES13-00398.1
Pace, M.L., S.R. Carpenter, R.A. Johnson and J. T. Kurzweil. 2013. Zooplankton provide early warnings of a regime shift in a whole-lake manipulation. Limnology and Oceanography 58: 525-532.
For an explanation of our rationale and expected results see:
Carpenter, S. R., Brock, W. A., Cole, J. J., Kitchell, J. F., & Pace, M. L. 2008. Leading indicators of trophic cascades. Ecology Letters, 11(2), 128-138. doi:DOI 10.1111/j.1461-0248.2007.01131.x

Methods

Methods are described in Wilkinson et al. 2018 (Ecological Monographs 88:188-203) and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.

Methods

Methods are described in Wilkinson et al. 2018 (Ecological Monographs 88:188-203) and Pace et al. 2017 (Proceedings of the National Academy of Sciences USA 114: 352-357). These publications including supplements should be consulted for details.
In Paul, Peter and Tuesday lakes two sondes were deployed at 0.75 meters near lake center. One sonde was a Hydrolab (model DS5X) with temperature, oxygen, pH, phycocyanin, and chlorophyll a sensors. One sonde was a Yellow Springs Instruments (YSI) 6600-V2-4 with temperature, dissolved oxygen, pH, phycocyanin, and chlorophyll a sensors. Measurements were made every five minutes. Brief gaps in the data record due to calibration or sensor malfunction were interpolated using a bivariate autoregressive state-space model with the MARSS package in R version 3.9 to create a continuous daily time series.

Methods

Spectrophotometer:A. Chlorophyll Extraction (using tissue grinder at DNR Research Station) 1. Dim the lights and keep the sample tubes in the freezer: Because chlorophyll degrades when exposed to light and heat, this procedure and all others associated with analyzing chlorophyll should be carried out in dim light conditions. Only one sample tube should be out of the freezer at any one time while the pre-grinding or grinding procedure is occurring. Return each tube to the freezer as soon as its filter has been ground. 2. Pre-grind filters: Use the sharpened stainless steel probe to chop up the filter into small pieces. This should take approximately 2 minutes. 3. Grind filters: The teflon tip on the tissue grinder should be sanded after grinding approximately 5 filters. Grind each filter for 2 minutes. Do not lift the teflon tip out of the test tube while the grinder is rotating. Grind the filters by attempting to keep the teflon tip in the acetone solution and pressing the tip against the filter and the tube. 4. Return the sample tubes to the freezer for 24 hours: Most protocols call for extracting the samples in the refrigerator (at 4 degrees C). However, after extracting duplicate samples in the freezer and refrigerator (after grinding) there was no significant difference in the chlorophyll results. Because past samples have been extracted in the freezer, this is the current procedure being used. B. Centrifuging the Samples: The samples should be centrifuged as close as possible to 24 hours after extraction. Before centrifuging the samples, turn on the spectrophotometer and enter the correct program number to be sure that it is working properly. Perform the procedures below in dim light. 1. Checking acetone volume: In dim light, use an identical tube as those used for the samples but with mL marked on it, to measure the volume of the acetone in the samples. Measure to the nearest 0.5 mL. If the sample has any other volume than 5 mL, write the volume on the sample label and remember to enter the volume later into the spreadsheet. 2. Loading the centrifuge: Making sure that the rubber stoppers are on tight, put tubes with equal acetone volumes opposite each other in the centrifuge. If there is an odd tube remaining or a tube with a different volume, put a spare tube opposite the sample with the same volume of water to counterbalance the centrifuge. 4. Running the centrifuge: Turn the speed dial below 40. Turn the timer past 15 minutes. Slowly turn up the speed allowing time for the centrifuge to increase in speed. If there is an imbalance in the centrifuge (or any other problem), the centrifuge will run much louder than normal. In this case, stop the centrifuge and attempt to locate the imbalance. If the centrifuge is running smoothly, set the speed at 90 and the timer at 15 minutes. Previously, the numbers on the dial were believed to correspond to revolutions per second; however, this is not the case, for the centrifuge will only reach rpms of approximately 2500. 5. Unloading the centrifuge: Allow the centrifuge to come to a stop on its own. Carefully take each sample tube out of the centrifuge with minimal mixing. If the filter paper is mixed with the liquid, it will be necessary to re-centrifuge the sample. Transport the samples to the spectrophotometer in a rack that has tinfoil on the sides in order to block out the light. C. Running a Sample: 1. Select the test: Allow the spectrophotometer to warm up for at least 15 minutes. Select the proper program by pressing the test number followed by Select. 2. Rinse the cuvettes 3 times with acetone. It is most efficient to rotate 4 matching 1 cm cuvettes. Try to touch the cuvettes only on the opaque sides avoiding touching the clear sides especially on the lower half of the cuvette. 3. Run a blank and check that all cuvettes read near 0: Add acetone to the 4 matching cuvettes (at least half full), wipe them clean with a tissue, and insert them into the spectrophotometer with the labeled sides all facing the same direction (always put the tops on the cuvettes when they are in the spec). Press Run and the spec. will ask for a blank. Use one of the cuvettes filled with acetone as the blank. Once the blank is run, run all of the cuvettes (the cuvette position is changed by pulling out the metal rod to the next notched position). All of the readings at all wavelengths should be within .001 of 0. If this is not the case, remove the suspect cuvette and rinse, wipe, add acetone, and rerun it. Make sure that the correct program is being run by checking the wavelengths. The LTER samples should be run at 750, 665, 664, 647, and 630 nm. 4. Rinse the pipette tip: Before adding sample to a cuvette, the pipette tip should be rinsed with acetone. You should have 2 different sized beakers, one for waste and one for acetone rinse. Set the 10-1000uL pipette to 1000 uL (1 mL) and pipette 1mL of acetone from the rinse beaker and dispose of it in the waste beaker. Be sure that the pipette tip is firmly on the pipette (press it on the bottom of the rinse beaker). 5. Add sample to a cuvette: Before bringing the samples into the spectrophotometer room, turn off the overhead light and turn on the desk light in the corner. Carefully remove a sample from the rack and pipette approximately 2 mL of sample into a cuvette. Use caution not to suck up any filter paper into the pipette; tilt the sample to the side and submerge the pipette tip only just below the fluid level. If the pipette tip is getting close to the filter paper when removing the second mL of sample, stop pipetting and add the partial mL to the cuvette (it is possible to read approximately 1.5 mL of sample). 6. Check the 750 nm reading and run the sample: Insert the cuvette into the spec. (making sure that the labeled side is always facing in the same direction). The default reading on the spec is 750 nm. Check to make sure that this reading is less than 0.010 A. If the reading is higher, remove the cuvette and re-wipe it with a tissue. If the reading is still high, pour the sample back into the tube and re-centrifuge it. To run the sample press Run. 7. Acidify the sample: Once the sample has been run, remove it from the spec and add 60 uL of 0.1 N HCl (30 uL per 1 mL of sample). Gently shake the sample and wait 90 seconds to run it. 8. Check the acidification ratio: The before acidorafter acid ratio of the LTER samples is usually between 1.3 and 1.7. Compare the two readings to make sure the ratio fits in this range. If the ratio is higher than 1.7, re-acidify the sample and run it again (the acid probably did not make contact with the sample). 9. Rinse the cuvette: After checking the acidification ratio, dispose of the sample in the waste beaker and rinse the cuvette 3 times with acetone. Be sure to fill the cuvette to the top with acetone during each rinse to be sure that there is not any trace of acid left. Running Multiple Samples: 1. It may be more efficient to run 2 samples before acidification and then run them both after acidification. If this is done, take caution to add the correct sample to the correct cuvette and not to mix up the samples when they are removed from the spec. for acidification. Recording the Results: 1. Write the spec. id number located on the left of the printout onto the label of the corresponding sample. Each sample should have a before and an after acidification spec. id number written on its label. After all of the samples have been run, enter the date of analysis onto the spec. printout. This date will be used to identify the spec. printout when the data is proofread (after which proofed from spec. printout should be written on the spreadsheet). Clean-up: 1. Rinse the cuvettes 3 times with acetone, allow them to dry for several minutes in the cuvette rack, and return them to their box. 2. Solutions of less than 20percent Acetone can be disposed of down the drain followed by at least 10 volumes of water. Fill the waste beaker with water and pour the waste down the sink with the water running. Leave the water running for several minutes 3. Rinse the beakers and pipette tips 3 times with tap water followed by 3 rinses with distilled water. Hang the beakers on the drying rack.  

Methods

See abstract for methods description

Methods

Reading Temperature and Dissolved Oxygen1. Before leaving to sample a lake, check to make sure that there are no air bubbles under the probe membrane of the YSI TemperatureorDissolved Oxygen meter. If there are air bubbles or if it has been several months since changing the membrane (or if the instrument does not calibrate well or the oxygen readings wander), change the membrane as explained in the manual. Note: We have always used the Standard membranes. If adding water to new membrane fluid bottle (KCl), make sure to add Milli-Q water and not the CFL distilled water.2. Be sure to always store the probe in 100percent humidity surrounded by a wet sponge or paper towel.3. Turn on the temperatureordissolved oxygen meter at least 30 minutes before using it. It is best to turn it on before leaving to sample a lake as it uses up batteries slowly.4. Calibrate the meter using the chart on the back of the instrument (adjusted to the Madison altitude - 97percent oxygen saturation). Leave the plastic cap on the probe (at 100percent humidity). The temperature should not be changing during the calibration. Zero the instrument. When the temperature equilibrates, adjust the oxygen to correspond to the chart. After calibrating the instrument, switch the knob to percent oxygen saturation to make sure it is close to 97percent.5. Take readings at 1 meter intervals making sure to gently jiggle the cord when taking the oxygen readings (to avoid oxygen depletion). Jiggling the cord is not necessary if using a cable with a stirrer. Take half meter readings in the metalimnion (when temperature andoror oxygen readings exhibit a greater change with depth). A change of temperature greater than 1degreeC warrants half-meter intervals.6. Record the bottom depth using the markings on the temp.oroxygen meter cord and take a temperature and dissolved oxygen reading with the probe lying on the lake bottom. Dont forget to jiggle the probe to remove any sediment.7. If any readings seem suspicious, check them again when bringing the probe back up to the surface. You can also double check the calibration after bringing the probe out of the water (and putting the cap back on). Light (PAR) extinction coefficient is calculated by linearly regressing ln (FRLIGHT (z)) on depth z where the intercept is not constrained. FRLIGHT(z) = LIGHT(z) or DECK(z) where LIGHT(z) is light measured at depth z and DECK(z) is light measured on deck (above water) at the same time.For open water light profiles, the surface light measurement (depth z = 0) is excluded from the regression.For winter light profiles taken beneath the ice, the first light data are taken at the bottom of the ice cover and are included in the regression. The depth of uppermost light value is equal to the depth of the ice adjusted by the water level in the sample hole, i.e., the depth below the surface of the water. The water level can be at, above or below the surface of the ice. If the water level was not recorded, it is assumed to be 0.0 and the calculated light extinction coefficient is flagged. If ice thickness was not recorded, a light extinction coefficient is not calculated.For light data collected prior to March, 2007, light values less than 3.0 (micromolesPerMeterSquaredPerSec) are excluded. For light data collected starting in March 2007, light values less than 1.0 (micromolesPerMeterSquaredPerSec) are excluded. Except for bog lakes before August 1989, a light extinction coefficient is not calculated if there are less than three FRLIGHT values to be regressed. For bog lakes before August 1989, a light extinction coefficient is calculated if there are least two FRLIGHT values to be regressed. In these cases, the light extinction coefficient is flagged as non-standard.FRLIGHT values should be monotonically decreasing with depth. For light profiles where this is not true, a light extinction coefficient is not calculated.For samples for which light data at depth are present, but the corresponding deck light are missing, a light extinction coefficient is calculated by regressing ln (LIGHT (z)) on depth z. Note that if actual deck light had remained constant during the recording of the light profile, the resulting light extinction coefficient is the same as from regressing ln(FRLIGHT(z)). In these cases, the light extinction coefficient is flagged as non-standard.

Methods

Chlorophyll is measured by collecting separate integrated samples from the epilimnion, metalimnion, and hypolimnion

Methods

CHLOROPHYLL a ANALYSISEQUIPMENT: Film canistersTurner 450 Fluorometer fitted with:1. Quartz-halogen lamp2. Emission filter -SC6653. Excitation filter -NB44047mm Whatman GForF filters12 x 75 mm disposable glass culture cuvettes (Do not reuse cuvettes!)1-5 mL Oxford pipettorFinnpipette Stepper Pipetter with 5 mL tiptimestimesNOTEtimestimes-Change filters with fluorometer off! (Remember that chlorophyll analysis filters are different from APA analysis filters.)-Make sure Fluorometer has been calibrated for chlorophyll a (see Fluorometer Calibration for Chlorophyll a Analysis).REAGENTS: 100percent Methanol, spectrophotometric gradeCAUTION - wear gloves whenever you use methanol.0.1 N HCLEthidium Bromide Stock 3 standard (40microM solution)PROCEDURE:A. Filter water samples from each of the 6 light-depths onto a 47 mm GForF filter.1. Filters have a grid side and a smooth side. Place filter smooth side up.2. Shake sample bottle well before filtering (do this after the DIC sample has been taken from the same bottle.)3. For each depth, filter enough water so there is a faint color on the filter. For our lakes this ranges between 100-300ml. Record the volume filtered. Make sure you filer at less than 200 mm Hg pressure.4. Rinse filter towers and filters with DI water, place filters in labeled film canisters and place in freezer. Labels should include lake, date, and depth ID.5. If measuring edible chlorophyll as well, repeat steps 1-4 above, but first filter the sample through 35 microm mesh. (This has not been done since 2001, inclusive.)B. Extraction - DO IN DIM LIGHT and WEAR GLOVES!!1. Remove one tray of film canisters from the freezer. Extract chlorophyll by adding 25 mL 100percent MeOH to each film canister. If using re-pipettor, verify dispensed volume. (Record extraction volume if different from 25 mL.) Note the extraction time for each group of samples.2. Re-cap and place canisters in refrigerator to extract for exactly 24 hours (in the dark).3 Repeat steps 1 and 2 for all trays that have been in the freezer more than 24 hours.C. FluorometryCalibration of the fluorometer using a chlorophyll standard is typically performed at the beginning of the field season, or when a bulb is changed. Calibration using Ethidium Bromide is done at the beginning of each sample set.1. Insert correct filters in fluorometer while fluorometer is off. (Emission filter -SC665, Excitation filter -NB440), and warm it up for 1 hour .2. TURN LIGHTS OUT. Chlorophylls must be read in low light and samples must be kept cool. Do not remove film canisters from the refrigerator until you are ready to process the samples.3. Place clean cuvettes into a labeled rack (12 cuvettes per rack). Remove one lake-day of film canisters from the refrigerator.4. Place Ethidium Bromide Stock 3 standard into fluorometer and record reading on datasheet. Then, turn the span knob until the reading is 908. Record this on the datasheet.5. Shake film canister, remove the lid, and rinse the pipette tip with 2.5 mL of the sample. Then remove 2.5 mL of sample and place in cuvette.times Repeat for all film canisters.6. Pipette 2.5 mL of 100percent methanol into a cuvette for the blank and use it to zero the fluorometer. Choose a gain and turn the zero knob until the fluorometer reads 000. You must zero the machine every time you change gains.7. Remove the first sample cuvette from the rack, wipe with a Kimwipe, and place in fluorometer. Record the gain and the fluorescence before acidification, Fb. Repeat for all 12 cuvettes in the rack. Readings should be between about 200 and 1000. If not, adjust the gain and re-zero.8. Acidify each cuvette with 100 microL 0.0773 N HCl using the repeating pipetter and mix (hold the top of the cuvette securely, then "thump" the bottom several times). Check for condensation on the outside of the cuvettes, and wipe with a Kimwipe if necessary. Wait about 1 min from the acidification of the first cuvette.9. Record the fluorescence after acidification for all 12 cuvettes. VERY IMPORTANT: Make sure you read the Fb and Fa values for each sample on the same gain.10. Remove a new lake-day batch of film canisters from the refrigerator and repeat steps 3-9.times if particulate matter is present, centrifuge sample for 10 min. and use supernatant.D. Clean Up: DO THIS UNDER THE HOOD!1. Dump methanol solution from cuvettes and film canisters into a metal tray. Place the film canisters and lids in a separate tray. Position them in one layer on the tray with their openings facing up. Leave the trays under the hood overnight to evaporate the methanol.REFERENCES:Marker, A.F.H., C.A. Crowther, and R.J.M. Gunn. 1980. Methanol and acetone as solvents for estimating chlorophyll a and phaeopigments by spectrophotometry. Arch. Hydrobiol. Beih. Ergebn. Limnol 14: 52-69.Strickland, J.H. and T.R. Parsons. 1968. A practical handbook of seawater analysis. Fish. Res. Brd. Can. Bulletin 167.pp. 201-206.Holm-Hansen, O. 1978. Chlorophyll a determination: improvements in methodology. Oikos 30:438-447.

Methods

Chlorophyll is measured within the exclosure and at a nearby control site in the littoral zone. Spectrophotometric analysis and fluorometric analysis are done on integrated samples from surface to 1 meter.