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(Deer) X (Invasives)² : Interactive Effects in the Herb Layer of Suburban Forests – Protocols

Morrison Lab Protocols:

Plant Identification:
Plant Identification Guide
List of Species Name

Herb Layer Census Data:

Herb Layer Census data is taken twice during the growing season, once in the spring and once in the fall.
16 subplots are sampled within each plot and percent cover is calculated for each species observed.
Percent cover is calculated as “+, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10”
Then we use midpoints and average across all 16 0.25m² subplots
All species were identified and recorded

Shrub Cover:

Person 1 holds the 1m2 Secchi board (divided into 16 squares) at deer browse height (0.5 – 1.5 m), right along the plot border
Person 2 stands across the plot on the plot border and crouches to have their eye at about the board’s center
Person 2 counts the number of squares that ANY green vegetation obscures, even by a little bit.
Record that number, for:

  1. All woody plants
  2. Native woody plants
  3. Non-native woody plants

Positioning of Secchi boards – two samples per plot; the two samples have intersecting midpoints across the plot.

The first measurement of shrub layer density is indicated by the red line, with Person 2 holding the Secchi board at the top of the plot and Person 1 standing at the bottom.  The second measurement is indicated by the blue line with Person 2 standing on the right side of the plot with the Secchi board and the Person 1 standing on the left side of the plot.

shrub

 

Woody Heights:

 

Measure the height and record the species of each woody plant in a strip within each plot, as shown below.  Measure all individuals in the strip, up to 3m in height.

woody height

Woody Browse Data:

All woody plant species located within a half meter of the bottom and right edges of the plot (shaded portion depicted in Figure 3) were included in the deer browse measurements.  We walked on the edge of the plot from the lower left corner to the lower right corner with a meter stick and recorded each individual of each species and the presence or absence of deer browse evidence that fell within 1/2m of the edge, and then from the lower right corner to the upper right corner, repeating the same procedure.

The shaded portion of the plot depicted in the figure shows the ½m radius in side of the plot in which woody plants were examined for the presence or absence of evidence of deer herbivory.

 woody browse

 

Leaf Litter:

Four samples of leaf litter per plot, obtained by tossing (without looking) four circular hoops – (24cm diameter) into the plot, then placing all leaf litter from the circle into a brown paper lunch bags (one bag per sample), and drying at 60°C for three days before recording the mass. The average of the four samples is used for analysis.

 

ALPE Counts:

The entire plot is divided into four equal strips of 1m x 4m, with a pole or rope dividing the strips. The researcher walks along each strip slowly, carefully traversing each strip by eye, counting the number of ALPE plants present, using a hand-held counter.

 

ALPE MIVI Browse:

We are doing timed visual scans for tell-tale “shreddy ends” – deer browsed stems – on ALPE and MIVI in each plot that has ALPE, MIVI, or both. Walk along one side of the plot’s perimeter, looking into the plot approximately 1m in, and scan for deer browse for 30 seconds. Record the number of stems of ALPE or MIVI with shreddy ends (it may well be zero). Repeat for the opposite perimeter side. Repeat for the inner meter on one side of the central pathway, then repeat for the other inner meter on the other side of the pathway. In the both plots, do the protocol once for ALPE and then once for MIVI.
If both ALPE and MIVI have been added to the plot, there will be 8 sets of 30 second surveys (if there is only either ALPE or MIVI added, there will be 4 sets of 30 second surveys). Do this for only the plots that have ALPE, MIVI or both added.

 

Soil PFLA and S1-A test package:

We chose a subset of plots for soil sampling for the PLFA and S1-A soil test package from Ward’s lab. The plots are the same as those chosen for the earthworm sampling in Summer 2014: 16 plots per forest, including 4 of each treatment combination.

To sample the soil, we used a 3/4 “ soil corer to remove 5 cores per plot. The cores were taken from haphazardly selected spots in the plot, with an attempt to keep them spaced as far apart as possible. Spots with rocks, woody stems, or other obstacles had to be avoided. A few plots had only 4 cores. Leaf litter was removed prior to pulling the core. Cores were made at a depth of  15-20cm, but some were less due to  unavoidable rocks in the soil. All samples from a plot were mixed together and stored in a Ziploc bag in a standard (-20°C)  freezer prior to shipping to the lab. Sampling was done in five forests over 2 days, on 7/24/15, which was 5-6 days after a rainfall of 0.25 inches and in the past 10 days there had been about 1 inch of rain total, and high temps in the high 80s. Because of time constraints, the final forest (Eames) was done five weeks later, on 8/28/15, which was the 7th day after a 0.25 in rain, with about 1.5 inches rain in the past 10 days, and high temps in the mid eighties. The samples were shipped to Ward’s overnight on ice packs, in Styrofoam coolers, on Sept. 2, 2015.

 

PAR:

We measure PAR with the AccuPAR from Decagon Devices. There are two methods.

  1. If two people can work together, then one person measures full sun PAR in a nearby clear area:

Keep ACCUPAR level. Record full-sun PAR and the time every five minutes,
alternating between the ground (but above any plants) and  1.4m.
Be careful to NOT block the sun with your body.   Do not record if any clouds block the sun.
The other person measures in-plot PAR at the positions shown here, at the same time as the full-sun measurements:

ss                                                                                                               

Be careful to NOT block the sun with your body.   Do not record if any clouds block the sun. Keep AccuPAR level.
Do the 5 positions at ground-level and have the instrument average them; record that average and the time.
The data point is the proportion of full sun that is measured in the plot, using the full sun measurement that is closest in time to the in-plot measurement.

  1. If one person is doing it, the same procedure is followed, but the full-sun measurements are taken once every 20 minutes. Then, the full-sun measurement used for the proportion is obtained from the regression equation of PAR on time, at the time point that matches the recorded time from the in-plot sample.

 

Worms:

We designed and built an electrical apparatus for earthworm extraction from soil, in collaboration with an electrical engineer, and followed the plans in Weyers et al. (2008). We extracted in three periods in June and July of 2014. All extractions were completed when the soil was at an optimal level of moisture; extractions were avoided if it had rained the day before or the day of or if the ground was deemed to be too dry for the water in the soil to be able to conduct an electrical current. Eight metal rods were inserted into the ground in a circular fashion to create an area of 0.22m2 and attached to an electrical device powered by a 12-volt battery. The apparatus was used in sixteen plots of all six suburban forests (approximately ninety-six plots). The plots were selected based on the aforementioned eight possible treatments. The plots with the most abundance of ALPE, MIVI, both, or other plant species were selected for the extractions. Two extractions were done for each treatment. The apparatus was turned on for two one-minute sets of electrocution. In between each set earthworms were collected and stored in an isopropanol (rubbing alcohol) solution and then transferred to a 4% formaldehyde solution and left overnight for preservation. The earthworms were then stored for long term in isopropanol. The earthworms were then sent to Dr. Chih-Han Chan at Johns Hopkins University for identification, and then lab members in this study were also chosen to identify the earthworms due to their cross-sectional shapes, the amount of setae, and the positioning of the clitella in relation to the different ridges.

 

Soil water potential

Sampling:

Done when soil is moist; not water logged and not bone dry, all on the same day from all plots.
Two samples per plot, taken from 0.5m in from the bottom right and top left corners, or the closest to there as possible.
Samples taken from PVC circle with screw threads (approx. 5cm in diam and 3cm deep.
Leaf litter cleared from soil surface, then the sampler is screwed into the soil, the resulting soil volume is mixed in situ and spooned into the WP4 plastic sample cups, filling them no more than 1/3 full; then sealed with Parafilm.

Stored in refrigerator until measurement.

Measurement:

WP4 Soil Water Potential Meter Protocol

  1. Turn on machine to warm up for 30 min.
  2. Take samples from fridge to warm to room temperature for 30 min.
  3. After 30 min., check calibration with KCl standard.

**** This should be done at first use of the day and once more if the machine is having heavy use across the day. Make a note on the form near the machine when you have done a calibration check ****

– Empty vial into clean cup
– Carefully slide into drawer
– Push lower right button. Wait until Ts – Tb is between 0 and 1.
– Turn to “Read”
– Make two readings. The second one should be with + 0.1 of the correct reading for the KCl at that temp (-2.19 at 20C, -2.22 at 25C). If not, stop, do not use the WP4, and inform me.
– Remove cup, rinse and let dry.

Take sample readings:
– Use samples that are at room T.
– On data sheet, fill in Date, Forest, Plot, and Sample A or B
– Remove Parafilm and cap. (work over paper on the bench top)
– Make sure cup is not filled more than halfway; if it is, remove some soil.
– Use tweezers to remove rootlets and debris, and spread soil evenly across bottom of cup.
– IMPORTANT: make sure that soil is not sticking to the inside of the cup, up its wall to the rim. You may need to wipe off the upper part of the inside wall with your finger.
– IMPORTANT: carefully wipe off the rim of the cup so there is NO SOIL on it, and make sure there are no soil particles on the outside of the cup.
– Put cup in drawer and slide it in.
– Push lower right button. Wait until Ts – Tb is between 0 and 1.
– Turn knob to “Read” position.
– WP4 will beep once and then go through various screens.
– In about 5 min it will beep four times and the green LED will flash. Record on the data sheet the value for MPa.
– Remove sample, put its lid on, and set aside in neat stacks.
– Inspect the drawer for any soil particles and remove them.
– Turn off machine when done unless someone else is coming soon.

Soil Compaction 

Done when the soil is relatively dry– contingent upon weather. Five samples are taken per plot. One at every corner 10-20 cm from the flags and one in the center of the plot (that’s not in the walkway). The soil compaction meters, attached with the adapter foot, are used to collect data. Prior to using the meter, the white ring around the meter needs to be set at the highest point it an go. Leaf litter is cleared from the soil surface to take readings and is placed back after finishing. Insert the soil compaction meter until the soil surface reaches the height of the adapter foot. The white ring is pushed down as the meter is pushed into the soil and will land on the appropriate measurement. When using the adapter foot, soil measurements need to be divided by 16. Use these five measurements and calculate an average for the plot.

Leaf Sampling Protocol:

Leaf Sample Protocol – Fagus grandifolia

ID Characteristics:

  • Alternate Leaves
  • Simple leaves that are waxy and smoothed with a toothed margin
  • Buds are long and pointy, light brown with overlapping scales

Size Classification:

  • Class 1: 0-10cm in height
  • Class 2: 20-40cm in height
  • Class 3: 50-140cm in height

Leaf Classification:

  • Class 1: Normal
  • Class 2: Deer Herbivory
  • Class 3: Insect Herbivory
  • Class 4: Diseased (discoloration)
  • Class 5: Other

Sample Number:

The sample number is the Site, Plot #, Quadrant #, Species, Class Size, Leaf Class

Sampling Design:

  1. Open “Random.org” on phone/tablet
  2. Each plot is broken into 4 quadrants.
    1. The bottom right corner is quadrant 1, bottom left is quadrant 4
  3. Use the random number generator (“1” is the minimum and “4” is the maximum”) to determine which plot to sample
    1. If there is no Fagus in the plot, then move to the next sequential plot
    2. If there is more than one Fagus that meets the criteria for sampling, then assign a number to all of the Fagus in that class and adjust the maximum number in “Randon.org” to be the total number of Fagus that can be sampled.
      1. Whatever number is chosen by “Random.org”, you will sample from that tree
    3. Repeat these steps for all three size classes

Physical sampling:

  • If the plant has only one stem (no branching) remove the two most distal leaves from the top of the plant
  • If the plant is branched:
    • Step 1:
      • Start at the base of the plant and move along the stem to the first branching point.
        • Follow the first branch to terminal bud and remove the most distal leaf
      • Step 2:
        • Find the terminal bud on the main stem and remove the most distal leaf
      • Insert leaves into the coin envelope and write the sample number on the envelope
        • Make sure to have the same sample number on the data sheet!
      • Add identification marker to tree sampled

Exceptions:

  • If the most distal leaf is missing, please select the next most distal leaf on the stem and note whether you sampled from the right or the left. Please alternate which side you sample from each time this event occurs

Leaf Sample Protocol – Fraxinus pennsylvanica

ID Characteristics:

  • Opposite leaves
  • Usually compound à except when young, then can be simple
  • Entire leaf margin
  • Terminal bud dark brown
  • Lateral buds sit on top of petiole

Size Classification:

Class 1: One set of leaves that are simple

  • Disregard plants that have simple leaves but more than one set

Class 2: Compound leaves with a stem height of up to 20cm

Class 3: Compound leaves with a stem height of over 25cm

Leaf Classification:

Class 1: Normal

Class 2: Deer Herbivory

Class 3: Insect Herbivory

Class 4: Diseased (discoloration)

Class 5: Other

Sample Number:

The sample number is the Site, Plot #, Quadrant #, Species, Class Size, Leaf Class

Sampling Design:

  1. Open “Random.org” on phone/tablet
  2. Each plot is broken into 4 quadrants.
    1. The bottom right corner is quadrant 1, bottom left is quadrant 4
  3. Use the random number generator (“1” is the minimum and “4” is the maximum”) to determine which plot to sample
    1. If there is no Fraxinus in the plot, then move to the next sequential plot
    2. If there is more than one Fraxinus that meets the criteria for sampling, then assign a number to all of the Fraxinus in that class and adjust the maximum number in “Randon.org” to be the total number of Fraxinus that can be sampled.
      1. Whatever number is chosen by “Random.org”, you will sample from that tree
    3. Repeat these steps for all three size classes

Physical sampling:

  • Remove the two (opposite) distal leafleats from the uppermost whorl
    • Exceptions: if distal leaflet is not present, remove the leaf that is closest to the distal end
    • Make sure to alternate sides (right or left of the petiole) if this needs to be done on multiple plants
  • Insert leaves into the coin envelope and write the sample number on the envelope
    • Make sure to have the same sample number on the data sheet!
  • Add identification marker to tree sampled

Exceptions:

  • If the most distal leaf is missing, please select the next most distal leaf on the petiole and note whether you sampled from the right or the left.  Please alternate which side you sample from each time this event occurs

 

Second Wave of ALPE addition protocol

Seed collection date, location and weight:

Date Location Weight
No date Hopewell Park 56.23g
9/1/15 Baldpate 53.864g
9/1/15 Site 1, rosedale, curlis, kunkel park 43.38g
9/3/15 stony brook watersheed 65.3g
9/4/15 mountain rd, stony brook rd, baldpate, 123 lamb-hope 59.57g
9/11/15 Shiptauker woods 16.01g
9/22/15 ETS 36.679g
9/22/15 Mayer Park 51.096g
9/25/15 Eames 19.4g

 

3.8g of ALPE seeds were added to each bag.

Moist potting mix was added to each bag until the bag was filled (each bag was a snack sized bag (put in size here), holding approximately 200mL of moist soil.

ALPE seed stratification began on December 22 and 23, and were checked on periodically for germination.  Once germination began on March 2, ALPE was distributed at all field sites between March 2 and March 7.

Seed addition protocol:

One bag containing 3.8g of stratified ALPE seeds plus moistened soil was added to each of the additional ALPE treatment plots.  This bag was distributed evenly across the entire plot.  Using ¼ of the bag at a time, the researcher distributed seeds across ¼ of the plot.  Once seeds and soil were evenly distributed across the treatment plot, the leaf litter was disturbed to ensure the ALPE seeds made contact with the soil surface.  After the complete addition of ALPE seeds at all plots in the six forests, the researchers disturbed the leaf litter in plots that not receive the ALPE seed addition treatment to ensure consistent abiotic conditions across all treatments.

Seeds began the stratification process on December 22 and December 23, 2015 and ended on March 2-7, 2016

ALPE seeds were added to the sites on the following days:

Eames: 3/2/16

Curlis: 3/3/16

Rosedale: 3/3/16

Nayfield: plots 1-18 (3/1/16), plots 23-32, 40 (3/2/16), plots 34-38 (3/6/16)

Herronton: 3/6/16

Baldpate: 3/8/16

Non-ALPE plots were disturbed at the sites on the following days:

Nayfield: 3/8/16

Curlis: 3/8/16

Rosedale: 3/8/16

Herronton: 3/8/16

Baldpate: 3/9/16

Eames: 3/9/16

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