ANPP of annual and herbaceous perennial crops is determined by harvesting all the aboveground portion of plants that are rooted within the bounds of a sampling quadrat. The area sampled is typically 1 m2 but the quadrat dimensions vary depending on the spacing of crop rows (see Table 1). For sampling, orient the quadrat with the long side placed in an east – west direction, which is perpendicular to the planted rows and allows for assessment of both the row and inter-row plant communities. Clip all plants rooted within the quadrat at ground level, separate by species, if needed, and place into paper bags labeled with the date of harvest, field replicate, treatment, sampling station and the species code according to the standardized plant species codes listed at http://lter.kbs.msu.edu/datatables/36. When samples are not separated by species or when live plant fragments cannot be identified to species, combine all plants and/or fragments into a single sample and label as “unsorted”. After all standing plant material is removed from within the quadrat, collect the dead plant material remaining on the soil surface and label as “surface litter”. Surface litter likely represents previous year’s growth and is not included in the current year’s ANPP. Dry bags at 60C for a minimum of 48 hours, weigh and record dry plant weights. For some treatments, plants are separated into crop and non-crop samples and the seeds of grain-producing crops may be threshed and weighed separately.

For Poplar (G8) ANPP, estimate woody biomass from annual measurements of basal diameter using an allometric relationship between diameter and biomass of harvested trees. The herbaceous poplar understory is sampled as above but litter traps are used as sampling quadrats and then are left in place to collect falling leaves as estimates of poplar leaf production.

Table 1. Quadrat sizes and time period of biomass sampling. Note that crops planted in some BCSE treatments have changed over the experiment’s history, particularly in G1-G4. Consult the archived plot maps and/or the aglog for specific changes.

*Time of sampling may vary with location.

ANPP protocols are developed for each cropping system type: annuals (Corn, Soybean, Canola, and Sorghum); monoculture grasses (Switchgrass and Miscanthus); multispecies communities (Native Grasses, Early Successional and Restored Prairie); and woody plants (Poplars). The protocol for each system type is provided below.

Materials

1. PVC/Wooden Quadrats
2. Paper bags
3. Weighing Balance
4. Drying oven
5. Plant grinder
6. Labels and pens
7. Meter stick
8. Calipers for poplar diameters
9. Thresher for separating grain

Details for ANPP Estimates:

Annuals: Corn, Soybean, Canola and Sorghum (G1-G4 depending on year, L1, M1)

Annual crops are sampled at or near physiological maturity. For corn, physiological maturity is signified by a “black layer” located within the base of a kernel, which normally forms about 60 days after silking or 20 days after denting. For soybean, peak biomass sampling should be done prior to leaf drop or when only a few leaves and petioles have dropped. At this stage pods on all positions of the stem are losing green color and at least one normal pod on the main stem has turned yellow. For canola, biomass samples should be taken before swathing. At this stage, seeds in lower pods look green-brown or green-yellow mottled. The canola crop should be monitored carefully as these stages change very rapidly during the ripening period if conditions are warm and dry.

For treatments and/or years without cover crops, ANPP is estimated as peak crop biomass. Clip all plants rooted within the quadrat to ground level, separate plants by species (crop vs. non-crop or weedy species) and consolidate surface litter, and place into labeled bags. Cut corn plants in small pieces before putting into bags to facilitate drying. Place bags into an oven at 60C for 48 h, weigh each bag and record the dried mass of plant material. Thresh dried corn, soybean and canola crops using an Almaco corn or small grain thresher to separate the grain (seeds) from stover and record grain dry mass. Combine grain, stover, and vegetative samples by tissue type for each plot (composited over sampling stations); finely grind samples (or representative subsamples) and store for CN analysis and archival.

Calculations:

• For crops, express whole crop biomass and grain biomass as g/m2. Subtract grain biomass from the whole crop biomass to estimate stover biomass.
• For noncrops, express the biomass of each species and/or the total biomass of all noncrop species in the quadrat as g/m2. Surface litter is likely from previous years’ growth and is not included in the current year’s ANPP.
• For ANPP, first find the average total biomass (whole crop + noncrop biomass) for each treatment replicate (n=3 sampling stations). Then determine the mean total biomass accumulated for the year (g/m2/yr) and the standard error for the treatment (n= 5 replicates).

For treatments and/or years with cover crops, cover crop biomass must be also measured. Sampling occurs at cover crop peak biomass, usually just prior to killing (and possibly harvesting) the cover crop in order to plant the main crop. At each sampling station, place a quadrat (0.5m x 2 m) so that the long side is perpendicular to the rows; ensure that this location will not overlap with the peak biomass samples taken later in that year. Clip all aboveground biomass within the quadrat; separate species as rye (SECCE), Austrian pea (PISSA; when included in the rotation), and other (UNSRT); and process samples as directed above.

Calculations:

• Determine crop and noncrop biomasses as directed above.
• For cover crop biomass, express the biomass of each species and/or the total biomass of all cover crop species in the quadrat, including Unsorted, as g/m2.
• For ANPP, first find the average total biomass (whole crop + noncrop + cover crop) for each treatment replicate (n=3 sampling stations). Then determine the mean total biomass accumulated for the year (g/m2/yr) and the standard error for the treatment (n= 5 replicates).

Monoculture Perennials: Switchgrass (G4 depending on year, G5, L2, M3) and Miscanthus (G6)

At peak biomass, clip above-ground material within quadrats at ground level. Depending on year and research needs, either sort samples to species and bagged separately or leave samples unsorted. Gather the senesced biomass remaining on the soil surface, place in bag and label as surface litter. After drying and weighing samples as above, separate plant samples into two groups: the planted species (switchgrass or Miscanthus) and weeds (all other species). Finely grind samples (or representative subsamples) from each group and store for CN analysis and archival.

During establishment years, plots may be mowed to control weeds. Mown biomass is sampled after mowing and included in estimates of ANPP. After mowing, place 1m2 (2.0 m x 0.5 m) quadrats randomly at 3 spots near the sampling stations, gather up the mown biomass, and process as above.

At ARL, plants were partitioned into dead leaf, green leaf, pseudostem and flower/seed heads; leaves were detached from the pseudostem at the ligule. Initially ARL used a 1 m2 quadrat, but reduced that in subsequent years to 0.5 m2 due to increased stem density.

Calculations:

• Determine mean crop and noncrop biomasses (when samples are sorted) as directed above. Also determine mean mown biomass, if the sample plot was mowed.
• For ANPP, first find the average total biomass [crop + noncrop (when samples are sorted) + mown (when plots are mown)] for each treatment replicate (n=3 sampling stations). Then determine the mean total biomass accumulated for the year (g/m2/yr) and the standard error for the treatment (n= 5 replicates).

Multispecies Communities: Native Grasses (G7), Early Successional (G9), Restored Prairie (G10, L3, M2), and CRP Reference (M4)

Above ground productivity is estimated at peak biomass, usually in August. Peak biomass is a good estimate of net primary productivity in this system because frost kills aboveground biomass during the winter and there is no carryover of live biomass from year to year. Clip all plants within the quadrat at ground level and process samples as above for herbaceous perennials. Depending on year and/or treatment, sort samples and weigh by species or leave samples “unsorted”. Dry and record weights as above. For unsorted samples, finely grind the unsorted sample (or representative subsample) and store for CN analysis and archival. For samples sorted to species, finely grind samples for each of the 3 most dominant individual species, a pooled (aggregate) sample of the remaining species, and surface litter, if present, and store for CN analysis and archival.

Note that “unsorted” may also refer to unidentifiable plant fragments in samples that are sorted to species.

Calculations:

• If samples are sorted to species, first sum the biomass of all species. Then determine the average crop biomass for each treatment plot as the average total biomass of all species when samples are sorted or the average unsorted biomass when samples are not sorted (n= 3 sampling stations per plot).
• For ANPP, determine the mean total biomass accumulated for the year (g/m2/yr) and the standard error for the treatment (n= 5 replicates).

Woody: Poplars (G8)

ANPP of the poplar system is determined as the sum of annual estimates of woody biomass growth, leaf production, and understory production plus the biomass affected by occasional agronomic practices to treatment plots during establishment and/or after clear cut harvest. Different procedures are used to collect these samples.

Woody growth increment:

The annual woody growth increment is estimated from a regression equation based on stem diameter. The equation is variety-specific, so must be determined over a rotation interval if one is not otherwise available. To develop the equation, trees representing a range of stem diameters are destructively sampled each year and weighed. In late fall (Nov-Dec), choose five trees (1 tree/replicate plot) to represent the range in each diameter size class for that year; preferentially select trees from the plot edge. Measure the basal diameter at 15 cm above the soil surface using a caliper, then cut at ground level and separate into stem (main axis or trunk) and branches (connections to stem) and weigh. Also measure basal diameter where cut (mm) and total tree height (m) and take representative subsamples to determine a fresh weight to dry weight conversion as directed above. Develop separate regressions to relate tree height, tree diameter, and basal area to woody tree mass (branches plus stem mass) and compare relationships to determine the best equation to use to predict mass from diameter measurements.

Every year in the late fall to early winter (Nov-Jan), measure the basal tree diameter (mm) at 15 cm above the soil surface with a calipers on 10 randomly selected trees per plot at KBS. These annual diameter measurements are used to estimate the woody biomass of the poplar stand. At Arlington, annual basal measurements were made on the same nine trees per plot.

Note that for the first year or two after planting, poplar growth is bushy, with several stems growing from the trunk. As a result, slight modifications to the ANPP measurements are required. For diameter measurements, choose the largest stem (the leader) and record its diameter. When the largest stem is indistinguishable, measure all stems; the largest stem is considered the leader. For destructive harvests, clip all stems of the trees chosen for destructive harvest. Record height and diameter of each stem, the total mass of all stems, and mass of the leader.

• Calculate the mean woody tree dry biomass (kg/tree) and express on an areal basis (g/m2) by adjusting for plant density; (trees planted in 2008 and 2019 are on a 2.43 × 1.52 m spacing = 0.27 trees per m2).
• Calculate the average annual woody growth increment per replicate as the difference in areal woody dry biomass (g/m2) between successive growing seasons (g/m2/y) for each plot (n= 5 replicates).

Leaf biomass:

Poplar leaf production is estimated by collecting poplar leaves as they drop throughout leaf fall (typically late July through mid-November). Leaf litter is collected in two wooden litter trap frames (0.5×0.95 m at KBS, 0.5×0.75 m at Arlington) per replicate plot. Leaves are collected bi-weekly (weekly collections were made at KBS 2008-2014), oven-dried and weighed. Samples are composited by replicate over the season, and subsamples are ground and archived for tissue analysis.

When trees are small and herbaceous growth extensive, traps may not accurately estimate litter fall. When this occurs, litter biomass is measured in late fall by placing a quadrat on the ground at each sampling station and collecting the litter within, and processing samples as above. Note that these samples are cataloged as “ground” samples.

• For leaf biomass, calculate the sum of leaf litter collected over the season for each trap in the replicate, then determine the average cumulative leaf litter biomass per m2 for each replicate (g/m2/y, n=2 litter traps).

Understory biomass:

Understory biomass is estimated by hand-harvesting plants within two 0.5 × 0.95 m quadrats per replicate in late July, using the litter traps described above. Traps are placed a predetermined distance and direction from a selected station flag with the long edge running in both a North-South (NS) and East-West (EW) direction; after sampling, traps are left in place for leaf litter collection. Plant samples were sorted to species until 2018, then unsorted from 2019 onward, oven dried at 60° C, and the weight of each sample (species if sorted) recorded. Samples are then composited by plot and subsamples are ground and archived for analysis.

• For understory biomass, determine the average understory biomass per m2 for each replicate (n=2 quadrats). For samples prior to 2019, sum the biomass of all species in the sampling quadrat. From 2019 onward, samples are not sorted to species so use the “Unsorted” biomass. Surface litter is likely from previous years’ growth and is not included in the current year’s ANPP.

Agronomic practices:

At establishment or between harvests and planting, additional agronomic practices may be needed to manage plots. For example, an oat cover crop was planted during establishment in 2008 and 2009 and plots were left as fallow after the 2018 harvest and then mowed and treated with herbicide before planting. The biomass in these cases is measured and calculated using the methods described above for cover crops. Cover crops are sampled at peak biomass and fallow growth is sampled before mowing and/or herbicide application.

Calculations:

• For annual poplar production, sum the annual woody growth increment (g/m2/y) and leaf biomass (g/m2/y) for each replicate, then calculate the mean annual production and standard error (n=5 replicates).
• For ANPP of the Poplar Treatment, sum the annual woody growth increment, leaf biomass, understory biomass, and biomass affected by agronomic practices (when conducted) for each replicate, then calculate the mean ANPP and standard error (n=5 replicates) for the treatment.