PROTOCOL for INTERSITE DECOMPOSITION EXPERIMENTS: I. FINE ROOT, LEAF LITTER, AND WOODEN DOWELS

The need for long-term, intersite experiments was acknowledged at the 1989 Decomposition Workshop held at Wood's Hole, Massachusetts. These studies will play a major role in advancing our understanding of the general principles underlying decomposition and nitrogen cycling processes. This knowledge will be crucial to address the effects that global climate will have upon detrital storage and productivity of ecosystems.

OBJECTIVES AND HYPOTHESES

Leaf and Fine Root Litter

The primary objective of this study is to examine the control that substrate quality and climate have on patterns of long-term decomposition and nitrogen accumulation in above- and below-ground fine litter. These relationships will be explored using a response surface with climatic and substrate quality factors as independent variables. Of particular interest will be to examine the degree these two factors control the formation of stable organic matter and nitrogen after extensive decay.

The decomposition of fine litter is a complicated process controlled by a number of factors including substrate quality, size, decomposer species, edaphic conditions, and climate. We offer the following working hypotheses concerning long-term litter decomposition dynamics fully aware that factors and interactions not considered may be just as important. It is hoped that future experiments will begin to assess the role these other factors play.

EXPERIMENTAL DESIGN

Leaf and Fine Root Litter

The major factors to be considered in this experiment will be site, species of litter, and time. Twenty-eight sites, representing a wide array of moisture and temperature conditions, will be used for litter incubations (Table 1). Nine types of "standard" litters will be sent to each site (Table 2). These include three types of fine roots (graminoid, hardwood, and conifer) and six types of leaf litter (which range in lignin/nitrogen ratio from 5 to 75). Samples will be collected ten times; the time between samples will be one year for all sites except LaSelva and Luquillo which will collect samples every three months. There will be four replicates for each species, site and time.

In addition to the standard litters, each site will be represented by a "wildcard" litter which appears at one site for each sample collection (Table 3). The purpose of the wildcard species is to verify the predictions from the standard species. There will be four replicates for each wildcard species, site and time.

Regression will be the primary form of statistical analysis used to examine results of the long-term decomposition experiment. Dependent variables will include the mass remaining, nitrogen content, and rate constants for total mass, fast, slow, stable, lignin, and cellulose. Climatic independent variables will include mean annual temperature, degree days, total precipitation, and actual evapotranspiration. The primary independent variable used to characterize substrate quality will be the lignin/nitrogen ratio, although other variables such as C/N ratio, and extractive content will be examined as well.

Wooden Dowels

Twenty-eight sites will be used to give a wide range of above- and below-ground environments. Wooden dowels will be used as a common substrate to test the effect of environmental differences upon decomposition.

Analysis of covariance will be the main method used to test if above- and below-ground rates of mass loss are similar. Dependent variables will include total, cellulose, and lignin mass lost, and nitrogen concentration. The covariables used will include climatic descriptors used in fine root and leaf litter analysis.

METHODOLOGY

Litter Collection

Each site was responsible for collecting the litter used in the experiments. For most sites, the leaf litter was collected directly from senesecent plants or as freshly fallen litter. Green leaves were collected from the San Diego, Luquillo, and LaSelva sites. All leaf litter except, Drypetes glauca which was oven dried at 40 C to prevent decay, was air dried prior to shipment to Oregon State University.

Fine roots (<2 mm diameter) were collected by two methods: tropical hardwood (Drypetes glauca) and pine (Pinus elliotii) fine roots were collected by excavating surface roots and washing. Graminoid roots were collected from material exposed along stream banks. Graminoid and pine roots were air dried, whereas the tropical hardwood roots were oven dried at 40 C to prevent decomposition.

In the case of the LaSelva site, the litter was sterilized after the bags were filled to kill all invertebrates, fungi, and virus prior to shipment. Sterilization was conducted at the Battelle National Laboratory by exposing the litter to 20 hours of gamma rays with 60Co as the source. The total exposure was 2 Mrad.

Bag Design

All bags were 20- by 20-cm and filled with 10 g leaves and 5-7 g of fine roots. Each bag was identified with a unique number embossed on an aluminum tag. The bag openings were sealed with six monel staples. The initial air dry weight, calculated oven dry weight, species, site, replicate number for each litterbag were recorded prior to placement in the field. Subsamples of litter material were taken to determine the air dry to oven dry conversion factor and the initial chemistry of the litter. Moisture content of the air dried litter ranged from 2-10% moisture content.

Three types of bags were used in this experiment. For the long-term leaf litter experiment the bags had a top mesh of 1 mm and a bottom of 55 micron mesh. The bags used for fine roots were entirely of 55 micron mesh. The bags used in the mesh size effects study had a top of 7 mm mesh and a bottom of 55 micron mesh.

Dowels

The wooden dowels used in the experiment are made of ramin (Gonystlylus bancannus). This species is a tropical hardwood from southeast Asia. It is not resistant to decay and rated as perishable. The dowels are 13 mm in diameter and 61 cm in length. One half of the dowel is to be embedded vertically into the soil and the other half is to be exposed to aerial conditions. The air dry weight of each dowel was recorded, and a subsample of dowels was measured for diameter, density, air dry moisture content, nitrogen content, and carbon chemistry.

Sample Placement

Samples were placed in the field during fall of 1990 or 1991 by each of the participating sites during the autumn stage of phenology. It will be the responsibility of each site to choose locations that are typical of the major ecosystem represented by their site. If possible, locations should be chosen to be near climatic stations and in areas protected from disturbances that could destroy the litter bags. For example, sites with frequent fire should be avoided, but areas prone to grazing would be suitable. The locations should also be typical of areas that other intersite decomposition experiments might be conducted.

The exact method for placement will vary from site to site, but the following standards should be applied if possible. Deviations from these suggestions may be necessary at some sites; these alterations will be important to note in the study establishment report.

1) Four separate locations should be selected (Figure 2a). These can be in the same stand if access or areas are limited, but it would be preferable if each replicate is placed in a similar but different area to avoid pseudo-replication problems.

2) Each set of bags to be collected will be connected by a cord; these sets of bags should be laid out in parallel lines in a random order (Figure 2b). During placement it is important to note which bags are on which string. This was noted prior to shipment to the sites, but doubling checking will reduce future headaches. This information will also be useful in determining which string is which during future harvests. During harvest it is essential that the correct strings be removed, if the number of wildcard replicates at a site is to be kept at four. Flags are included to mark the strings according to replicate and time of harvest. It may also be advisable to map the layout of the strings to aid in future relocation.

3) Leaf litterbags should be placed so that contact with the underlying litter layer is made. Fine root litterbags will be inserted into the upper mineral soil (humus layer for histosols). A vertical cut with a shovel, the bag inserted the correct depth (0-20 cm), and another cut should be used to press the soil against the bag (Figure 3).

4. Dowels should be installed at the end of the string opposite the fine root bags. A one-half inch steel rebar should be driven into the ground 30 cm to make a hole for the dowel. Do not drive the dowel into the ground without first making a pilot hole, as the dowel likely to break off! Place the dowel so that 30 cm is exposed to the air and 30 cm is embedded in the soil.

Sample Collection Schedule

A list of the litterbags, and prelabeled bags of the samples to be harvested will be sent to each site prior to sample collection. It is crucial that the correct bags be harvested if the number of wildcard replicates are to remain as planned.

All sites, other than LaSelva and Luquillo, will collect the fine litter and dowels on an annual basis for a 10 year period. For tropical sites (LaSelva and Luquillo) sampling will be conducted every three months.

When the dowels are harvested the above and below ground sections should be kept separate. If the entire 60 cm length is not harvested, or some of it is missing then the length missing should be noted.

Sample Processing

Once the litter or dowels are collected they should be oven dried in a paper bags at 55oC until the mass is stable. In the case of fine roots and dowels, a rinse with distilled water to remove adhering soil prior to drying is recommended. Any mosses, lichens, fine roots, or other plant parts that have grown into the bags or dowels should also be removed prior to harvesting. When the samples are dried the weight should be recorded and then the samples should be send on to Oregon State University. A final oven dry weight (55oC for 4 days) will be determined at the Central Processing Laboratory (Andrews LTER), but the site records will be useful as back-up data if the samples are lost in transit.

Ash content of each sample was determined. Samples were be pooled by species, site, and time for carbon and nutrient analysis.

Chemical analyses were performed using two methods. Each pooled sample from each species, site, and time (N=252) will be analyzed for total nitrogen, lignin, and cellulose using near infrared reflectance spectoscopy (Wessman et al. 1988). Internal variability of samples will be estimated by running replicates of high and low lignin species. Twenty five percent of the pooled samples (N=65) will also be sampled for Kjeldahl nitrogen, lignin, cellulose, water extractive, non-polar extractive, and ash content using wet chemical methods. Wet chemical methods will then be used to calibrate the near infrared reflectance spectoscopy methods.

Sample Archiving

Ground, dried material from each species, site, and time will be archived for future reference at Andrews LTER. Over 50 g of this material will be on hand to repeat questionable analyses or for new analyses. Material will be stored in sealed nalgene vials at room temperature. Access to coarse-ground samples will be provided after a short description of the new study has been received.

Data Storage/Access

All information/data concerning these experiments will be stored in the Forest Science Data Bank/Andrews LTER. Along with the data, an abstract describing the experiment, the formats and variable definitions of the data, and the programs used to process the data will be stored.

References

Wessman, C. A., J. D. Aber, D. L. Peterson, and J. M. Melillo. 1988. Foliar analysis using near infrared reflectance spectroscopy. Can. J. For. Res. 18:6-11