You are here

Harvard Forest Data Archive

HF334

Hysteresis of the Sarracenia Purpurea Microecosystem in Northern Vermont 2015-2016

Related Publications

Data

Overview

  • Lead: Vanessa Avalone, Bryan Ballif, Aaron Ellison, Nicholas Gotelli, Amanda Northrop
  • Investigators:
  • Contact: Information Manager
  • Start date: 2015
  • End date: 2016
  • Status: completed
  • Location: Molly Bog (Morristown VT)
  • Latitude: +44.46 to +44.50
  • Longitude: -73.19 to -72.64
  • Elevation: 93 to 230 meter
  • Taxa: Sarracenia purpurea (northern pitcher plant)
  • Release date: 2020
  • Revisions:
  • EML file: knb-lter-hfr.334.2
  • DOI: digital object identifier
  • EDI: data package
  • DataONE: data package
  • Related links:
  • Study type: short-term measurement, modeling
  • Research topic: conservation and management; ecological informatics and modelling; watershed ecology
  • LTER core area: organic matter
  • Keywords: bacteria, carnivorous plants, community composition, detritus, organic matter, restoration, thresholds
  • Abstract:

    The restoration of plant and animal communities damaged by chronic detrital or nutrient enrichment is a long-standing environmental problem. In a replicated greenhouse experiment with the aquatic microbial community that inhabits the cup-shaped leaves of the carnivorous pitcher plant Sarracenia purpurea, we monitored O2 levels and the concentration of bovine serum album (BSA), an effective molecular substitute for detritus. Low BSA enrichment rates triggered a classic hysteresis response, a substantial lag in the return of O2 levels. At intermediate BSA enrichment rates, O2 levels closely tracked BSA concentrations during both the enrichment and recovery phases. High BSA enrichment rates induced an unusual "anti-clockwise" hysteresis in which O2 levels were higher during the recovery phase than during the enrichment phase. These experiments revealed complex dynamic behavior in response to enrichment rates of a single environmental driver. Our results suggest that the past trajectory of enrichment is critical for understanding how systems will respond to restoration initiatives. Specifically, systems that have been exposed to chronic low levels of enrichment may be the ones that are most resistant to restoration efforts that reduce nutrient or detrital inputs.

  • Methods:

    Experimental Setup

    Pitcher plants (Sarracenia purpurea) were housed at the University of Vermont’s Biological Research Complex in a temperature-controlled greenhouse. Recently fully-formed pitchers greater than 8 ml in volume were randomly chosen and assigned to control and enrichment treatments. Pitchers were randomly placed on the greenhouse bench in order to account for spatial variation in greenhouse conditions. All pitchers were rinsed twice with reverse osmosis water and allowed to dry overnight prior to the start of the experiment. Pitcher fluid was collected from Molly Bog (44.50N -72.64W), a poor fen in Morristown, VT. Fluid was collected randomly from multiple pitchers in the field on July 6, 2015 using a sterile pipette and was transported immediately to the greenhouse where it was passed through the 30 micrometer frit bed of a chromatography column (BioRad, Hercules, CA) to remove macrobes, homogenized, and added to experimental pitchers.

    Organic Matter Loading

    Pitchers were loaded with either 5.0 (high concentration) or 0.5 (low concentration) milligrams of organic matter per milliliter of pitcher fluid. A set of control pitchers received no organic matter. For the first four days of the experiment, pitchers were loaded with organic matter (between 9:00 am and 9:45 am) following pitcher fluid sampling and dissolved oxygen (DO) measurements (see below). Organic matter in previous experiments was in the form of dried and ground bald-faced hornet (Dolichovespula maculata), which has similar elemental composition to the most common prey item of pitcher plants. In this current experiment, we loaded the pitchers with OmniPur bovine serum albumin (BSA) fraction V (VWR). In addition to BSA, we included the trace elements potassium (potassium chloride), calcium (calcium chloride), sodium (sodium chloride), magnesium (magnesium sulphate), and manganese (manganese chloride) in the following ratio: 1:0.115:0.044:0.026:0.0001. These ratios reflect the elemental composition of royal jelly and adult honeybees and were the closest we could find to trace element ratios for bald-faced hornets. We also added 1.5 micrograms of DNA sodium salt from salmon testes (Sigma) per milligram of BSA based on DNA yields between 1.4 and 1.5 micrograms per milligram from Apis melifera. Solutions containing a combination of BSA, trace elements, and DNA were pre-made for each pitcher, stored in 1-ml microtubes, and frozen at -20 °C until loaded into pitchers. These solutions were introduced to pitchers using sterile 8-ml transfer pipettes and mixed into pitchers by drawing pitcher fluid in and out of the pipette three times. We were careful to limit the amount of air deposited by and introduced into the transfer pipettes during loadings so that DO measurements would be minimally affected. Control pitchers received sham loadings in which pitcher fluid was drawn into and deposited from a transfer pipette containing no solution to account for the effect of mixing.

    Data Collection

    Dissolved oxygen was measured twice a day each day from day 0 to day 20 at 8:30am and 5:00pm (plus or minus two hours), once per day from day 20 to day 28 at 8:30am (plus or minus two hours), and once on days 30, 31, 33, and 35 (8:30am, plus or minus two hours). Dissolved oxygen was measured using a D-166MT-1S microelectrode (Lazar Research Laboratories). The microelectrode was calibrated prior to sampling according to the manufacturer’s instructions. A single microelectrode was used to take readings from each pitcher and was rinsed twice with deionized water between readings. Readings were randomized so that changes in temperature and sunlight over the sampling period were not confounding factors. During readings, the microelectrode was placed 1 inch below the surface of the pitcher fluid and swirled so that the more oxygen-rich pitcher fluid at the top of the pitcher was mixed and readings reflected average DO concentrations. Due to the sensitivity of the microelectrode, readings were taken as soon as the reader settled on a value for more than 10 seconds.

    Pitcher fluid was sampled for BSA following each DO measurement. Using sterile pipette tips, a 300-microliter aliquot was taken from 2.5 cm below the surface of each pitcher and placed in a sterile 1-ml microfuge tube. Sample tubes were immediately transported to the lab where they were centrifuged at 13,000 × g. The supernatant containing soluble BSA was removed, placed in a sterile 1-ml microfuge tube, and stored at -80 °C until analyzed.

    Dissolved oxygen was measured and pitcher fluid samples were taken once or twice a day for a total of 36 days (day 0 – day 35). Pitchers were all removed from the experiment on Day 35, regardless of whether or not their dissolved oxygen profiles had returned to control conditions.

    BSA Loading validation via SDS-PAGE

    In order to validate that the majority of protein in extracted pitcher fluid was BSA, we ran a time series of pitcher fluid from two replicates of the high- and low-concentration loading treatments on gels using SDS-PAGE next to known concentrations of BSA (0.1, 0.5, and 5.0 mg/ml) and a BenchMark Pre-stained Protein Ladder (Invitrogen). A 10-microliter aliquot of pitcher fluid from days 0, 2, 4, 6, 9, 12, 15, 18, 21, 24, 27, and 30 was each added to 90 microliters of bromophenol blue sample buffer (150mM Tris pH 6.8, 2% SDS, 5% beta-mercaptoethanol, 7.8% glycerol) and boiled at 95 °C for five minutes. After centrifugation at 13,000 × g for 2 minutes, 10 microliters of each sample was mixed with 10 microliters of sample buffer and loaded into separate lanes of a 10% polyacrylamide (37.5:1 acrylamide:bis-acrylamide). Gels were subjected to SDS-PAGE and stained with Coomassie.

    Bradford Assay Procedure

    We used a Bradford assay to determine the concentration of BSA in our pitcher fluid samples. We created a standard curve with BSA at the following concentrations: 48, 32, 24, 16, 12, 8, 4, 2, 1 and 0 micrograms per milliliter. A standard curve was created each day that we conducted an assay, with a maximum of two assays per day. Due to the consistency of the standard curves, we chose to analyze all replicates using an average standard curve.

    Pitcher fluid samples were prepared for the assay by first centrifuging the sample tubes at 13,000 × g for 30 seconds to remove anything but soluble protein from the fluid. A 10-microliter aliquot was drawn from the top of the sample and added to 30 microliters of deionized water in a sterile 1-ml microfuge tube (except samples from pitcher 75, which were added to 10 microliters of water). The diluted samples were then spun again at 13,000 × g for 30 seconds. Five microliters of the diluted samples were then drawn from the top of the micofuge tube and added to another sterile 1-ml microfuge tube. One milliliter of Bradford reagent (VWR) was added to each tube in a random order so as not to introduce time as a confounding variable in the assay results. Absorbance was measured using a Biophotometer Plus (Eppendorf) at an optical density of 600 nm. Using R software, we calculated BSA concentrations using a dilution factor of 4 (except for pitcher 75, which had a dilution factor of 2).

    2016 Experiment

    A similar experiment was done in the summer of 2016 using identical field and greenhouse methods. This experiment was started on July 3rd, 2016 in an attempt to control for seasonal variation in the initial microbial community composition and prey capture of field-collected pitcher fluid. The only difference in field/greenhouse methods from 2015 to 2016 is that pitchers were enriched with 2 mg/ml BSA/day and pitchers were not removed from the experiment until they had reached control conditions.

    In order to streamline 2016 BSA concentration data collection, we changed the Bradford Assay protocol. Whereas every time point sample in the 2015 data was analyzed in a separate microfuge tube, every time series/replicate was processed at once on a 96-well plate in 2016. All samples were subject to an initial 10× or 20× dilution and processed using the Thermo Scientific Coomassie Plus (Bradford) Assay Kit. For each plate, two standard curves were constructed and the average was used to fit a 4-parameter curve. Absorbance was measured at 545 nm with a Synergy HTX (Biotek).

    Data Analysis

    We used R software (version 3.4.3) to plot the hysteresis loops using the loess function to fit curves (span=.5) to each of the enrichment and recovery trajectories. Hysteresis indices (HI) were calculated for each replicate pitcher to determine the direction and magnitude of hysteresis in the relationship between BSA concentration and dissolved oxygen concentration. Mean HIs were compared among treatments using a one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test.

    The R script file HYS_vFinal.R is the main code file which normalizes data, calculates BSA concentrations using standard curve and absorbance values, plots publication figures, calculates hysteresis indices, calculates probability of interspecific encounter, and conducts contrast analysis. All statistical tests and figures for publication are produced with this code.

    The R script file hysteresis_functions.R is the function file to source in the main code file that allows multi plotting in ggplot and contains a function for calculating the concentration of BSA from absorbance values given a standard curve fit with a polynomial function using stat's "lm" function in R.

  • Use:

    This dataset is released to the public under Creative Commons CC0 1.0 (No Rights Reserved). Please keep the dataset creators informed of any plans to use the dataset. Consultation with the original investigators is strongly encouraged. Publications and data products that make use of the dataset should include proper acknowledgement.

  • Citation:

    Avalone V, Ballif B, Ellison A, Gotelli N, Northrop A. 2020. Hysteresis of the Sarracenia Purpurea Microecosystem in Northern Vermont 2015-2016. Harvard Forest Data Archive: HF334 (v.2).

Detailed Metadata

hf334-01: absorbance and dissolved oxygen values

  1. Year: year in which experiment was conducted - 2015 or 2016
  2. Day: day of the experiment - from 0 to 35
  3. Time.of.Day: time of day - am or pm
    • am: am
    • pm: pm
  4. Pitcher.Number: replicate pitcher number
  5. BSA_Concentration: mg/ml/day of BSA cocktail added - 0.5, 2.0, or 5.0 (unit: milligramPerMilliliterPerDay / missing value: NA)
  6. Absorbance: absorbance measured at 600 nm, unadjusted to standard curve (unit: dimensionless / missing value: NA)
  7. D.O.: dissolved oxygen concentration in pitcher fluid, measured as % (unit: dimensionless / missing value: NA)

hf334-02: absorbance values for curves

  1. Concentration_ugPERml: known concentration of the standard samples in micrograms per milliliter (unit: microgramPerMilliliter / missing value: NA)
  2. Curve_Number: identifier for each standard curve, 1 to 6
  3. Absorbance: absorbance measured at 600 nm (unit: dimensionless / missing value: NA)

hf334-03: bacterial class abundance values

  1. class: bacterial taxonomic class
  2. initial: total number of peptides in all initial pitcher fluid samples associated with each identified bacterial class (unit: number / missing value: NA)
  3. low: total number of peptides in all samples from pitchers enriched with 0.5 mg/ml/day BSA associated with each identified bacterial class (unit: number / missing value: NA)
  4. intermediate: total number of peptides in all samples from pitchers enriched with 2.0 mg/ml/day BSA associated with each identified bacterial class (unit: number / missing value: NA)
  5. high: total number of peptides in all samples from pitchers enriched with 5.0 mg/ml/day BSA associated with each identified bacterial class (unit: number / missing value: NA)

hf334-04: peptide counts

  1. class: bacterial taxonomic class
  2. pp00a: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  3. pp00b: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  4. pp00c: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  5. pp00d: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  6. pp004: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  7. pp072: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  8. pp145a: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  9. pp014: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  10. pp195: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  11. pp231: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  12. pp146: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  13. pp154: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  14. pp042: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  15. pp043: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)
  16. pp113: total number of peptides (proxy for abundance) for this replicate pitcher (unit: number / missing value: NA)

hf334-05: pilot experiment at Harvard Forest, May 2015

  1. pitcher.id: pitcher identification number
  2. day.0.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  3. day.0.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  4. day.1.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  5. day.1.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  6. day.2.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  7. day.2.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  8. day.3.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  9. day.3.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  10. day.4.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  11. day.4.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  12. day.5.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  13. day.5.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  14. day.6.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  15. day.6.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  16. day.7.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  17. day.7.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  18. day.8.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  19. day.8.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  20. day.9.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  21. day.9.pm: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)
  22. day.10.am: dissolved oxygen (%) at this day and time (unit: dimensionless / missing value: NA)

hf334-06: main code and function file R scripts

  • Compression: none
  • Format: R code
  • Type: R code