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PROJECT DESCRIPTION
Introduction
Biological invasions represent great challenges to forest ecologists and managers. Invasions alter
ecosystem structure and function, especially when they change the habitat of other species, alter the
availability or transformation rates of key resources, or compete with or replace native species
(Vitousek 1990). Much of the focus on exotic species invasions has been on aboveground invasions,
which are the most apparent. However, belowground invasions may be equally widespread and may
have as large an impact on ecosystem structure and function as aboveground invasions, although they
are not as well understood or studied (Edwards and Bohlen 1996, Scheu 2001, Wardle 2002).
Invasion of northern forests by exotic earthworms is one such below ground invasion that is
receiving increasing attention (Hendrix and Bohlen 2002, Bohlen et al. 2005). Earthworms are the
best known of the large soil fauna, but many forests in North America lacked earthworm populations
prior to European settlement, probably because of slow northward expansion of native earthworm
populations following the last glacial period (James 1995). Foreign earthworm species, mainly of
European and Asian origin, are currently invading these forests over a wide geographic area (Alban
and Berry 1994, Scheu and Parkinson 1994, Bohlen et al. 2004a). These invasions lead to rapid and
marked changes in the soil environment (Langmaid 1964, Alban and Berry 1994) due mainly to the
role of earthworms in modifying soil structure, redistributing organic matter, increasing nutrient
mineralization and altering the habitat of other organisms living in or on the soil. These invasions
have consequences for the processing of organic matter through the soil food web, the flow of
materials from soil food webs into above ground food webs, and for associated cycling of nutrients
between soil and plant communities (Wardle et al. 2004, Parkinson et al. 2004).
In previous NSF-funded research in two forested landscapes in New York State, we demonstrated
that earthworm invasions had striking but complex effects on forest biogeochemistry, the effects
varying with soil environment and the species of earthworms present (Bohlen et al. 2004a).
Regional-scale understanding of earthworm impacts requires examination of the mechanisms by
which different earthworm assemblages impact pathways of C and nutrient processing across the
landscape. Here we request funds to address the following questions and hypotheses:
1. Earthworms reduce soil carbon storage. Our previous results suggest that earthworm invasion
of forests with no history of cultivation reduces soil C in the topsoil by more than 25% (Bohlen et
al. 2004b). This effect on soil C needs to be examined at regional scales in relation to site
characteristics and earthworm species. In particular, we need to distinguish between transient
and more persistent elevated C losses, and to elucidate the pathways of above- and belowground
C flow to better understand long-term impacts on ecosystem C pools. We propose to combine
regional surveys with more mechanistic studies of C flow to test the hypotheses that:
a. Earthworms are significant relative to other landscape and regional scale factors, such as
soil texture, topography and land use history, in regulating soil C.
b. Earthworm effects on soil C will depend on the balance of two factors: stabilization of C
in aggregates versus stimulation of microbial activity by physical mixing and enzymatic
activation as organic matter passes through earthworm guts.
c. Earthworms both reduce C accumulated in the forest floor as well as alter the movement
of C from different components of current detritus into soil and the soil food web.
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2. Earthworms influence processes regulating availability and retention of N and P. Our
previous results indicated no decline in total soil N with earthworm invasion, despite significant
losses of C and a narrowing of soil C:N ratio (Bohlen et al. 2004b, Groffman et al. 2004a),
suggesting that earthworms stimulate N retention processes. Also, we observed that invaded sites
dominated by Lumbricus terrestris had increased soil P levels, possibly due to transport of
unweathered minerals to the soil surface by this deep burrowing species, whereas sites dominated
by other species had reduced P availability and more immobilization of P in mineral complexes
(Suárez et al. 2004). Now we hope to determine if:
a. Earthworms consistently alter the relationship between soil C:N ratio and N loss; with
lower N losses than would be expected at the low C:N ratio in earthworm colonized sites.
b. Earthworm activity fosters stabilization of N in soil aggregates.
c. Earthworm effects on N retention depend on species, i.e. the Asian earthworm Amynthas
spp. may stimulate nitrification and N leaching more than European lumbricid species.
d. Earthworm effects on soil P pools and availability depend upon species-level effects
related to soil mixing.
3. Earthworm effects on C and N retention correspond to changes in the processing of organic
matter by detrital food webs and the flow of C and N into the aboveground food web.
Earthworm-driven changes in soil structure and organic matter distribution are expected to cause
a shift from the fungal channel towards the bacterial channel of the detrital food web.
Furthermore, earthworms are important prey for predators at the interface of soil and
aboveground food webs, such as terrestrial salamanders (Maerz et al. 2005). However,
earthworms reduce the abundance of many other taxa important in the diets of interface
predators. We hypothesize that:
a. Earthworms shift processing of detritus from fungal towards bacterial channels of the
food web, with increases in protozoans and bacterial-feeding nematodes, decreases in
fungal-feeding Collembola and mites, and a decline in tertiary consumers including
predatory mites, ants, and carabid beetles.
b. Earthworm-driven shifts in soil food webs will be reflected in the diets of woodland
salamanders, which function as key “interface species” between above and belowground
food webs. Changes in salamander diets will alter the amount of litter C and N that flows
from the soil into the aboveground portion of forest food webs.
We propose to test these hypotheses with a suite of approaches. We will survey 100 km of transects
in each of three regions of New York State and make observations of the presence/absence of
earthworms, including the presence of Lumbricus terrestris and Amythas spp. at 10 m intervals.
Geographic information system (GIS) coverages will be used to explore relationships between
earthworm distributions and topography, soil variables and land use history and to select 20 plots in
each region for quantitative sampling of earthworm populations and soil C, N and P pools (to 1 m) in
earthworm-invaded and uninvaded reference sites. At one site, we will use 13C and 15N labeled litter
to explore detailed mechanisms of C and N processing in earthworm colonized and reference sites.
We propose to relate patterns in flows of C and N through different organic matter pools to
hypothesized shifts in soil food web structure as well as the flow of litter C and N into the aboveground food web via woodland salamanders. Simulation models will be used to synthesize data and
to address regional scale questions related to C storage and N retention in northeastern forests. The
research will be coupled with a suite of formal and non-formal education/outreach efforts, based on
the idea that earthworm ecology is a useful tool for getting a broad diversity of people to think about
soils and ecosystems.
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Improvements in Response to Previous Review
This proposal was submitted to the Ecosystem Studies panel in July 2004 (scores: 2 E, 4 VG, 1 F)
and has been extensively revised in response to panel and reviewer suggestions. Reviewers liked the
three-phase approach of regional surveys, experiments using isotopically labeled litter and simulation
modeling, but had concerns about each. Key revisions include:
1. Regional survey. Reviewers were concerned that plots should be located along gradients of
limiting factors and that the survey work be broadened to consider forest types other than sugar
maple. We have completely changed our approach to the regional survey, abandoning the focus
on sugar maple. Our new design allows for evaluation of the effects of earthworms on C, N and P
processing relative to other factors that have been shown to influence both nutrient dynamics and
earthworm dynamics, i.e. soil texture, topography, land use history and vegetation composition.
2. Labeled litter experiments. Reviewers expressed a variety of concerns about the design and
execution of these experiments. In several cases these concerns reflected our inadequate
descriptions of the procedures: 1) each litter box will be sampled only once with a destructive
sampling; 2) existing litter will be removed prior to adding labeled litter; 3) earthworms will be
sampled on every date; 4) soil sampling will be to 1 m depth; and 5) enclosure effects and other
details are better justified, herein. Also, we have added a soil carbon biogeochemist and expanded
our studies of soil C pools. We elected not to include a no-salamander control because
earthworms are the focus of our studies and salamanders are ubiquitous in our region. And we
retain the comparison between uninvaded controls beyond the earthworm invasion front, rather
than using earthworm additions to non-invaded soils because we are less interested in the
transition phase that accompanies earthworm colonization than in longer term effects of invasion.
3. Modeling. We have clarified how the CENTURY model will be modified and how data gathered
in the survey and litter experiments will be used in modeling and extrapolation work. Bill Parton,
developer of the CENTURY model has been added as a consultant.
4. Education and outreach. We have provided more detail about the WWW site that will be
produced and show how our work complements existing outreach work related to earthworms.
Background and Justification
Earthworm invasions are initiated by human introduction associated with fishing, commerce in soil
and plant materials and in earthworms themselves, e.g. vermicomposting (Figure 1, Hendrix and
Bohlen 2002). Persistence and spread of earthworms depends on site factors such as vegetation type,
which influences food quality, and topography, which influences soil moisture and pH (e.g., Suárez
et al. in press a). Several exotic earthworm species are involved in the ongoing invasion of North
American forest landscapes (Hendrix and Bohlen 2002, Bohlen et al. 2005), but little is known about
the patterns of invasion of various species (Hale et al. 2005, Suárez et al., in press a, b).
Earthworms influence ecosystem nutrient cycling processes by modifying soil structure and
redistributing organic matter as a by-product of their feeding and burrowing activities (Figure 1).
These activities vary with different earthworm species; some reside mainly in the upper organic layer
(epigeic species), whereas others mix organic and mineral layers together (endogeic species). Still
other species, such as L. terrestris, the common nightcrawler, form nearly vertical permanent
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burrows up to 1-2 m deep and incorporate litter into the soil and bring mineral soil from different
depths to the surface (anecic species). Earthworm effects on soil organic matter dynamics also
depend on land use history (Bohlen et al. 2004b). In our previous study, we showed that at a site
with no history of cultivation (Arnot Forest) and a thick (3 – 5 cm) forest floor, earthworm invasion
reduced soil C in the top 12 cm of the soil profile by 28%, while at a site with a history of cultivation
and thin forest floors (Tompkins Farm), there was no difference in soil C between invaded and
reference sites (Bohlen et al. 2004b). However, it is likely that the proportional change in soil C
resulting from earthworm invasion varies across a range that depends not only on past land use but
also on earthworm species composition, vegetation and topographic factors. For example, Suárez et
al. (in press c) observed major differences in processing of sugar maple and red oak litter between
earthworm communities dominated by L. terrestris and L. rubellus (a common epigeic species).
Dispersal
Resource Quantity
Resource Quality
Soil Factors
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source population
natural expansion
human act
plant productivity
soil organic matter
ivity
vegetation type
litter C:N ratio
tannins, polyphenolics
moisture, hyrdology
texture, sand/silt/clay
acidity, base cations
streams, rivers
Earthworm
Ivasion
Physical Effects
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Biological Effects
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Geochemical Effects
burrowing and casting
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litter removal
soil aggregates
porosity
hydrology
mixing soil layers
adsorption/desorption
mineral weathering
change in minerology
erosion
change in soil habitat
faster nutrient cycling
less fungally
fewer myco
-dominated
rrhizae
change in rooting zone
altered seedbed
Ecological Consequences
Ecosystem Properties
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C loss (short
Ecological Communities
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- term)
C stabilization (long
term)
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N retention?
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P availability?
Tree nutrition?
plant invasions
loss of native herbs
soil invertebrate
community shifts
microbial community
shifts
Figure 1. Factors that influence earthworm invasions, the three main categories by which invasion influences
ecological systems, and the consequences for ecosystem processes and ecological communities.
Modified from Bohlen et al. (2004a).
Northern forests are thought to be important global C sinks (McKane et al. 1997, Hobbie et al. 2002,
Lal 2004), but our results suggest that earthworm invasions may turn them into temporary C sources
or reduce the magnitude of the C sink. It seems likely that a large proportion of the C loss at our
Arnot Forest site occurred during the initial stages of invasion (Alban and Berry 1994). Now the
need is to understand whether earthworm-invaded sites continue to have a reduced capacity for C
accumulation over the longer-term. Sustained higher soil C loss is suggested by higher microbial
respiration rates that we observed at both the Arnot and Tompkins Farm sites (Fisk et al. 2004,
Groffman et al. 2004a, Li et al. 2002). Clearly there is a need to evaluate the effects of earthworm
invasion on soil C processing and storage and to quantify the importance of invasion relative to other
regional scale regulators of storage such as soil texture, topography and land use history.
Earthworms have two countrvailing effects onf soil C processing. On the one hand, earthworms limit
microbial activity by protecting decomposable plant material in stable soil aggregates, which leads to
soil C storage, whereas on the other hand earthworms promote organic matter decomposition that
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accelerates C mineralization (Brown et al. 2000). Earthworms promote soil aggregate stability, by
encasing the most easily decomposable, light-fraction of plant material with soil clay (Marinissen
1994, Bossuyt et al. 2005). The result is that C becomes bound into a heavy soil fraction, which has
turnover times of decades to hundreds of years (Post and Kwon 2000, Scullion and Malik 2000). At
the same time, earthworms accelerate C processing by activating, or priming soil microbial activity
(Jenkinson 1966). Enzymes in the earthworm gut (e.g., chitnase, protease, cellulase, glucosidase)
initiate the decomposition process, and the soluble products (i.e., low molecular weight organics)
prime decomposition of organic matter. Moreover, the mixing and burrowing activities of
earthworms shift the surface accumulation of organic matter towards more even distribution through
surface mineral horizons (Görres et al. 1997, Amador et al. 2003, Bohlen et al. 2004b). Bacterial
components of the detrital food web (bacteria, protozoans, bacterial-feeding nematodes) tend to be
more dominant in a mineral soil environment (Holland and Coleman 1987, Maraun et al. 2001, Savin
et al. 2004). Holland and Coleman (1987) hypothesized that increased importance of bacterial
channels of the detrital web directly contributes to higher C loss, via lower growth efficiencies of
bacteria compared to fungi. Furthermore, the bacterial channel is more subject to top-down
influences, with protozoan and nematode populations mediating rapid turnover (Yeates 1981, Wardle
2002), which should also accelerate C loss. We will quantify the importance of these two roles of
earthworms (stabilizing aggregates, activating microbes) by tracing the fate of 13C and 15N labeled
plant litter into different organic matter fractions and microbial groups including 1) soil aggregates ;
2) free versus protected particulate organic matter; 3) light and heavy density fractions of soil organic
matter; 4) different microbial groups using phospholipid fatty acid (PLFA) profiles, total lipids and
ergosterol; and 4) different soil and aboveground food web components.
The effects of earthworm invasion on N cycling and retention appear to be even more complex than
effects on C. Earthworms have been shown to increase mineralization and leaching of N from forest
soils in lab or microcosm studies (Haimi and Huhta 1990, Scheu and Parkinson 1994, Tiunov and
Scheu 2004). However, earthworm invasion did not lead to declines in total soil N or to increases in
N leaching in our plots in New York (Bohlen et al. 2004b). The lack of increase in leaching was
surprising given the lower soil C:N ratio in invaded plots compared to uninvaded plots. Many
studies have found this ratio to be a strong predictor of N loss (Gunderson et al. 1998, Dise et al.
1998, Lovett et al. 2002, Ross et al. 2004). Given the importance of N retention to water quality in
this region (Driscoll et al. 2003), there is a need to determine if earthworm invasion really fosters N
retention in the long term, at the regional scale or might in some instances increase N losses. Studies
using 15N-labelled litter in earthworm colonized
and reference stands are needed to determine just
how earthworms alter the fate of N in forest soils.
Earthworm effects on soil N cycling may vary
Laboratory studies
strongly with species.
comparing L. terrestris, Amynthas hilgendorfi and
Eisenoides lonnbergi (a native) incubated in
common soil showed that inorganic N pools and
nitrification rates were much higher with
Amynthas (Figure 2). Amynthas is invading soils
in the eastern U.S. (Callaham et al. 2003) and may
be affecting N cycling and loss in this region.
In addition to altering pools and fluxes of soil C and N, earthworms may also influence the interface
of soil and aboveground portions of the food web. Though earthworms may increase bacteria,
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protozoans, and nematodes, they may cause marked declines in collembola, mites, and other
arthropods typically abundant in organic horizons (Maraun et al. 2001, McLean and Parkinson
1998a,b, Migge 2001, Migge et al. in review). Much of the primary production of forest ecosystems
is bound in decomposing plant tissues (leaf litter), on the forest floor. If earthworms shift the soil
system from a slower cycling fungal-dominated system, to a faster cycling bacterial-dominated
system, as is commonly held (Wardle 2002), there are likely cascading effects on the abundances of
different soil heterotrophs and the interaction of the soil food web and consumer species at the
interface of the soil and aboveground food web (Lavelle 1997, Scheu 2005). Animals such as
predatory ground beetles and woodland salamanders that prey extensively on soil fauna may be
particularly sensitive to changes in soil food web structure. Because these animals are often the
dominant predators at the interface between the soil and aboveground food web (Burton and Likens
1975a), the flow of nutrients through beetle or salamander populations may affect larger fauna
including birds, snakes, and large mammals (Burton and Likens 1975b, Hairston 1996). Due to their
intimate connection with the soil and presumed importance to forest food webs, salamanders have
been proposed as a sensitive bioindicator of forest health (Welsh and Droege 2001).
It is difficult to predict the effects of earthworm invasions on the flow of nutrients into interface
consumer (e.g. salamander) populations. As prior research shows, earthworm invasions cause C
retention in soils to decline and shift to deeper soil depths. This is likely coupled with a decline in
organic horizon microarthropods (particularly Collembola and Acarina) that are common prey for
salamanders and other forest floor predators (Burton 1976, Maerz et al. 2005). Soil fauna expected
to increase in abundance (e.g., nematodes) with earthworm invasion are seldom found in salamander
diets (Burton 1976, Maerz 2005); therefore, one would predict that earthworm invasions reduce the
flow of C and other nutrients from detritus into aboveground consumer populations. Of course, this
prediction ignores predation directly on earthworms. Earthworms are a major prey for many forest
fauna, including predatory ground beetles, birds, small mammals, snakes, and amphibians. Maerz et
al. (2005) show that invasive earthworms are a large portion of salamander diets in some northern
temperate forests. Therefore, earthworms could increase the rate and total amount of C and N
transferred to aboveground consumers. Which prediction is true will depend on the efficiency with
which earthworms convert litter C and N to tissue, and on the reliability of earthworms as prey.
Maerz et al. (2005) found that earthworm occurrences in salamander diets were highly climate
dependent and more stochastic than other soil fauna. We hypothesize that because earthworms are a
stochastically limited resource for salamanders, reduce the availability of other more stable soil
fauna, and shift the distribution of C and N in soils, that earthworm invasions will reduce the flow of
C and N from decomposing litter into salamander populations.
In addition to altering C and N processing, earthworms appear to have complex effects on soil P
pools that vary with earthworm species (Suárez et al. 2004). In our previous studies, earthworminvaded plots dominated by L. terrestris had significantly more total P in the topsoil than
corresponding reference plots, while plots dominated by L. rubellus, showed significantly less total P
than their reference plots. Invaded plots that had higher amounts of total P had a proportionally
higher amount of unavailable P fixed in Al or Fe hydroxides or primary minerals. The increased
amount of unavailable P forms in invaded plots suggests that the deep burrowing activity of L.
terrestris has mobilized unweathered soil particles from deeper layers of the soil, increasing the
stocks of total P. In contrast, the decrease in total P that we observed in plots dominated by L.
rubellus likely resulted from stimulation of rates of P cycling in the soil by the surface activity of this
species. This stimulation led to increased loss of P in leaching water (Suárez et al. 2004). It remains
to be seen whether these observations can be generalized to other locations.
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Experimental Plan
To address the questions and test the hypotheses outlined in the Introduction, we propose an
integrated suite of three approaches: 1) broad-scale surveys, 2) intensive-plot isotope tracer studies
and 3) simulation modeling. First, we will conduct surveys in three regions of New York State with
contrasting landscapes to examine controls on the distributions of earthworms and their effects on
soil pools of C, N and P. Second, we will produce and add 13C and 15N labeled litter to plots at one
site to explore detailed mechanisms of C and N processing in earthworm colonized and reference
sites. The mechanistic studies will include analysis of soil food web structure by tracing the
movement of 13C and 15N into soil pools and food web components. Third, we will use the
CENTURY soil organic matter model to synthesize results over time, at the regional scale.
Regional Surveys
We propose a series of surveys to; 1) gain a broader understanding of the factors regulating exotic
earthworm distributions in forests of southern New York State, including factors regulating the
distribution of two key species, Lumbrius terrestris and Amynthas spp., and 2) quantify the
magnitude of differences in soil pools of C, N and P in forests with and without exotic earthworm
populations. The surveys will be conducted across environmental, vegetation and land use gradients
in three contrasting regions of southern New York: the Catskills, Finger Lakes and Hudson Valley.
The Catskills are a sparsely-settled, high plateau, the Finger Lakes region is more densely-settled
with a moderate proportion of agricultural and suburban land, while the Hudson Valley has a long
history of human settlement and widespread agricultural activity. There will be two components to
the surveys. We will first sample large numbers of sites along long transects (sample every 10 m
along 100 km of transects) to assess the presence/absence of earthworms (including the specific
presence of L. terrestris and Amynthas spp.). We will then select 60 of these sites (including noninvaded reference sites) for intensive sampling of soil variables.
Sites for regional surveys. Earthworm distributions in forests of southern New York depend
upon a complex suite of factors that differ among regions and between local sites. Our extensive
surveys will be designed to capture the variation in environment, vegetation, land use and landscape
factors that are expected to contribute to patterns of earthworm distribution in the three study regions.
We will establish sampling transects across the typical range of elevation and topography, soil and
forest types and land use history for each region. In the Finger Lakes region we will re-survey
permanent transects in our Arnot Forest study site, both to record changes in earthworm distribution
that have occurred in the six years since the original survey and to sample for the distribution of L.
terrestris and Amynthas. Additional transects in the Finger Lakes region will take advantage of
detailed maps of land use history, vegetation and soils for the lowlands of Tompkins County
compiled by P. Marks and colleagues (Smith et al. 1993, Singleton et al. 2001).
In the Catskills region we propose to establish sampling transects at two sites, one
representative of the western Catskills where small dairy farms occur in the high valleys and the
other in the central Catskills Forest Preserve. The former site coincides with an ongoing program in
where we have been working with farmers to reduce agricultural nutrient loading to New York City
drinking water reservoirs (Hively et al. 2005). At the latter site, Kudish (1979) has documented
detailed land use history and there are extensive ongoing studies of forest management and nutrient
cycling (Lovett et al. 2004, Burns and Murdoch 2005).
In the Hudson Valley, we will establish transects centered on two properties owned by
the Institute of Ecosystem Studies (IES); the Cary Arboretum and Tompkins Farm (site of our
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previous research). These sites have a complex but well-documented history of land use, three
forest community types (Glitzenstein et al. 1990) and a markedly patchy distribution of
earthworms (Burtelow et al. 1998, Verchot et al. 2001). The IES sites are surrounded by large
parcels of private land with similar characteristics. We have a long history of cooperation with these
landowners and we anticipate no problems laying out long transects centered on the IES properties.
Transect sampling. In each region we propose to sample 100 km of transects in both spring
and summer. For every 10 m of transect length we will record earthworm presence or absence. The
presence of L. terrestris and Amynthas will be determined using soil morphological criteria, i.e. L.
terrestris builds obvious permanent burrows and produces distinctive midden piles and casts while
Amynthas are large, highly active and visible at the soil surface during mid-summer and produce
distinctive casts that are readily obvious at the soil surface (Suárez et al., in press a, Bohlen, personal
observation). We will quantify all earthworm species on the sub-set of plots chosen for intensive soil
characterization (see below). Samples for soil pH will be collected at 50m intervals.
For each transect interval the elevation, slope, overstory vegetation composition and GPS
location will be recorded. GIS coverages will be accessed or developed for each sample transect to
allow classification or quantification for each interval of soil map units, topographic index, land use
history and other landscape variables (e.g., distance from roads, settlements, streams, etc.) expected
to influence earthworm distributions.
Soil sampling on intensive plots. Results from the transect surveys will provide a basis for
a stratified random selection of 60 intensive 20 m by 20 m soil sampling plots (20 in each region).
We anticipate that the principal variables for sample stratification will be 1) soil paraenet material 2)
topographic position, 3) forest composition, 4) land use history (previously cultivated or not) and 4)
presence of the selected earthworm species (L. terrestris, Amynthas). Within each stratrum we will
randomly select paired plots with well-established earthworm populations and nearby plots lacking
earthworms. Two different causes of such an arrangement need to be distinguished: 1) situations
where ongoing colonization has created active invasion fronts that separate areas that are similar
except for the presence of earthworms (Hale et al. 2005, Suárez et al., in press b); and 2) situations in
which areas have been accessible to earthworms, but vegetation, soil and other site factors restrict
their invasion or persistence. Our soil sampling program will concentrate primarily on the former to
provide further information on the soil changes that accompany the invasion process. The existence
of an invasion front can be readily established on the basis of the composition and age-structure of
the earthworm community at transitions (Hale et al. 2005, Suárez et al., in press b).
Earthworm populations will be sampled in spring (the peak period of earthworm activity)
using a standard formalin extraction technique (Raw 1959, Bohlen et al. 2004a). Because Amynthas
hatch in spring and reach maximum size in summer, all sample sites will be visited again in summer
to verify presence of this species and to resample plots if necessary. Eight liters of 0.25% formalin
will be applied to four 0.25 m2 areas within each plot and earthworms emerging from the soil will be
collected into vials containing 4% formalin and returned to the lab for identification to species.
Soils will be described using the protocols of Soil Survey Division Staff (1993) with one pit
excavated to a depth of at least 1.0 m or to bedrock. Bulk density will be quantified by depth using
the excavation method. Rock fragment content will be estimated on pit faces using a % area chart,
and depth to a root-restricting layer (fragipan, densipan, bedrock) will be recorded. Soils for
chemical and physical analysis will be collected by genetic horizon. To provide additional areal
coverage and measures of variation, samples of upper soil (0-30 cm depth) will be collected using a
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5-cm corer at five random locations in each plot. Soil texture will be measured by the hydrometer
method (Gee and Bauder 1986). Samples will be analyzed for pH in 0.01M CaCl2 (Robarge and
Fernandez 1987). Organic content will be estimated by loss on ignition (Robarge and Fernandez
1987). Total N will be analyzed with a combustion analyzer. Exchangeable cations (Ca, Mg, Na and
K) P will be determined in 1 mol L-1 NH4Cl extracts (Blume et al. 1990).
Soil P fractions will be isolated using the fractionation scheme described by Hedley et al.
(1982), with modifications described by Tiessen and Moir (1993) and Suárez et al. (2004). In this
procedure, different P fractions are separated by sequentially treating the soil sample with reagents or
procedures of increasing strength in the following order: ion exchange resin, sodium bicarbonate,
sodium hydroxide, hydrochloric acid, and wet digestion with sulfuric acid and hydrogen peroxide. P
concentrations are measured in each of the resulting extracts, and these P fractions are assumed to
have different relative availability to plants (Lajtha et al. 1999, Harmon and Lajtha 1999). P
concentration in extracts will be measured colorimetrically (Murphy and Riley 1962).
Microbial biomass C and N content will be measured using the chloroform fumigationincubation method (Jenkinson and Powlson 1976, Groffman et al. 2004a). We will also measure
inorganic N and CO2 production in unfumigated "control" samples. These incubations will provide
estimates of microbial respiration and potential net N mineralization and nitrification.
Analysis and hypothesis testing from regional survey. The relationships between
earthworm distribution and composition, soil and vegetation factors and land use history will be
analyzed for each region using a combination of statistical approaches that will allow us to answer
questions and test hypotheses about earthworm distributions and effects on C, N and P in a broad
suite of southern New York landscapes. Logistic regression models of earthworm distributions will
be developed for each region, and spatial autocorrelation among transect intervals will be accounted
for with Generalized Estimating Equations that specify an autoregressive error structure within the
transects (Suárez et al., in press a). Analysis of data from the intensive site sampling will focus on
differences between paired colonized and reference plots and how this varies with topography, land
use history, soil, vegetation and earthworm community composition. Standard ANOVA and
multivariate techniques will be used to evaluate variation associated with earthworm colonization
relative to effects caused by other factors that vary among the reference plots. Specific questions that
we will address include:
• Are earthworms a strong controller of C and N storage relative to other factors such as soil
texture, topography and land use history? Earthworm invasions are most likely to be
significant in lower topographic positions that are wet and that also have high levels of soil
C. Does invasion reduce levels of soil C in lowlands to levels similar to uplands?
• Do paired comparisons of worm-invaded and reference plots consistently show loss of C
from the soil profile with little or no loss of N, suggesting a fundamental change in the
relationship between soil C:N ratio and N loss?
• Is Amynthas a common invader in our region, and is this species associated with high rates of
potential net N mineralization and nitrification and soil nitrate concentrations?
• Does the species of invading earthworm (e.g, Amynthas, L. terrestris) matter to C and N
storage or P fractions at the regional scale?
• Is the invasion of forests by L. terrestris limited to areas adjacent to agricultural land?
Similarly, is the invasion by Amynthas restricted to forests adjacent to human settlements or
is it rapidly expanding its range?
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13
C and 15N Labeling Experiment
We will use 13C and 15N labeled litter to explore detailed mechanisms of C and N processing in
earthworm colonized and reference sites. We propose to relate patterns in flows of C and N through
different organic matter pools to hypothesized shifts in soil foodweb structure (specifically increased
mineral-associated organic matter processing corresponding to the bacterial foodweb channel and
higher C loss) as well as flows of litter-borne C and N into the above-ground foodweb (woodland
salamander populations). 13C and 15N concentrations in salamander tissues will be used to estimate
cumulative flows of nutrients from litter into salamander populations, and changes in the ratios of
labeled C and N will be used to infer changes in food chain lengths between leaf litter and
salamander populations (Post 2002), which will be correlated with changes in the composition of the
soil food web. The experiment will contrast earthworm communities dominated by L. terrestris with
non L. terrestris-dominated communities because this species is extremely widespread and its deep
burrowing activities have great potential to influence C and N dynamics in the soil profile.
Producing litter. Sugar maple leaf litter will be labeled with 13C by a two-stage method
designed to separately label primarily structural vs. non-structural organic matter. Saplings will be
labeled by exposing their canopies to 99%-enriched 13CO2 in closed chambers in late-summer, 2006.
The abscised foliage from these saplings, containing 13C-labelled non-structural organic matter, will
be collected in October, 2006. Much of the label will be stored in the stems of the saplings
overwinter, and the following spring new foliage will be produced containing the 13C label primarily
in structural organic matter. Subsequently, in fall 2007, this leaf litter will be collected. We have
successfully labeled foliage of sugar maple saplings in this manner; for example, seven days after
labeling 10-yr-old saplings for 2 hours, foliage was labeled at a level over 400 per mil δ 13C (Phillips
and Fahey 2005). For the present application, about 10 hours of labeling will be applied for each
sapling. To conserve the expensive 99%-enriched 13CO2, the label will be applied in a large, closedcirculation Tedlar chamber after scrubbing ambient CO2 from the chamber atmosphere. Although
some respiratory loss of 13C label will be unavoidable, calculations based upon our preliminary
experience indicate that we will be able to produce 6 kg of litter in each year (enough to fill 54 litter
boxes – see below) containing a label of δ 13C exceeding 1000 per mil (1st-year litter -- non
structural) and exceeding 300 per mil (2nd-year litter – structural) for $50,000. These calculations
assume no bias for or against the newly-fixed 13C in respiration and plant storage from the time of
labeling (early September) until leaf abscission, and equal mobilization of stored label to all growing
tissues the following spring (hence, a conservative estimate of label strength).
In addition to enrichment with 13C, saplings will be fertilized with 99% 15NH4+ at a rate of 3
kg N ha-1 y-1, applied in 3 doses (April, June, August) in 2006. As for the 13C, this will produce
highly, but non-uniformly labeled litter in 2006, and more uniformly (including structural tissues)
labeled litter in 2007. Use of a low dose of highly enriched 15N will produce highly labeled, but not
N enriched litter. Structural and non-structural components of litter will be isolated (van Soest 1973)
and analyzed for 13C and 15N before litter is applied to field plots.
Experimental design. Litter will be applied to 18 plots (9 in fall 2006 with the nonstructural label and 9 in fall 2007 with the structural label) to fill out the following experimental
design: 3 worm community treatments (L. terrestris-dominated vs non- L. terrestris-dominated vs.
no-worm control) x 3 replicates x 2 types of labeled litter = 18 plots, with 3 boxes per plot (54 boxes
total) to allow for destructive sampling at three dates.
Plots will be at the Arnot Teaching and Research Forest, a 1,650 ha property located near
Ithaca in south central New York that was the site of much of our previous earthworm research. This
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forest was heavily logged at the end of the 19th century, but never cultivated. Currently the site is
dominated by typical species of northern Allegheny hardwood forests such as sugar maple, American
beech, red maple, white ash and American basswood (Fain et al. 1994, Fahey 1998). We have
extensive data on earthworm communities at this site that will allow us to establish plots to fill out
the experimental design (Bohlen et al. 2004a, Suárez et al. in press – a,b,c).
Litter decomposition methods. We will use 1 m2 “litter boxes” as described by Suárez et
al. (in press – c) to follow decomposition of the double-labeled litter. Litter bags have traditionally
been used to estimate rates of litter decay, but their effectiveness in studies of macrofauna effects is
questionable because patterns of colonization of the litter by soil fauna can be altered by the presence
of the bag (Bocock and Gilbert 1957, Anderson 1973).
Boxes (27) for the first crop of labeled litter (non-structural label) will be established in
November 2006 and boxes (27) for the second crop of litter (structural label) will be established in
November 2007. All original litter from the box area will be removed, and 200 g of labeled litter will
be added to each box. A 25 cm x 75 cm rough-cut maple coverboard will be added to facilitate
recovery of salamanders from boxes. Boxes will be covered with fiberglass screen (1 mm mesh size)
to exclude new litter and to contain added litter. For a brief period just prior to and following litter
introduction, we will collect and permanently mark salamanders from inside the box and from a 0.5
m wide perimeter around the box (marks will allow us to confirm that salamanders collected from
boxes during harvest were the same individuals originally introduced). Some salamanders collected
outside the boxes may be released inside to insure a minimum density of 4 individuals per box
(Bailey et al. 2004), and three salamanders collected outside each box will be taken to measure initial
13
C and 15N levels. Based on recent studies using coverboard (Maerz unpublished data) and fencing
(Bailey et al. 2004) we expect salamander immigration or emigration from litter boxes to be minor
On three sampling occasions (May 2007 or 2008, October 2007 or 2008 and May 2008 or
2009) over the 18 months following litter addition, salamanders and all the litter remaining will be
collected from one randomly selected box of each litter type in each replicate plot. The litter will be
stored in plastic bags and transported to the laboratory, where miscellaneous debris will be removed
(e.g. fine roots, earthworm casts, and plant seedlings) and the litter will be dried to constant weight
(65 ºC) and weighed. Ash content for each sample will be determined by loss-on-ignition (8 hours at
450 ºC) and the percentage of litter remaining will be calculated from the ash-free final weight of
each sample. Earthworms will be sampled as described above from each litter box as it is harvested.
Fate of 13C and 15N. As each litter box is sampled, soils will be separated into forest floor (if
present) and 10 cm increments to a depth of 1 m (or a confining layer). Earthworm casts will be
sampled separately from bulk soil. Samples will be sent to the Stable Isotope Facility at the
University of California at Davis (UC Davis) for analysis of total C and 13C and total N and 15N
contents. Microbial biomass C and N content will be analyzed as described above and gas samples
and extracts will be analyzed for 13C and 15N respectively. Gases will be sent directly to UC Davis
and extracts will be prepared for 15N analysis using the diffusion procedure of Stark and Hart (1996).
A suite of methods will be used to quantify the movement of 13C and 15N: 1) soil aggregates,
2) free and protected particulate organic matter (Bossuyt et al. 2002, 2004); 2) density fractions of
soil organic matter; 3) phospholipid fatty acids (PLFA) (Zelles 1999) and total lipids; and 4)
ergosterol (Malosso et al. 2004). This combination of methods will permit us to characterize the
movement of the labels into different organic matter fractions and microbial groups.
First, wet-seiving of field-moist samples will be used to separate macro (> 250 mm) and
micro (53-250 mm) aggregates for susbsequent analysis of total C and N and isotope analysis.
Density fractionations (in 1.65 g mol-1 polytungstate ) will be used to separate two organic matter
pools, a low-density light fraction (LF) that represents labile, relatively undecayed plant detritus
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(Trumbore 1997) and a heavy fraction (HF) that is more resistant to mineralization (Swanston et al.
2002). Isotope analysis will be conducted on finely-ground portions of each fraction.
We will take advantage of differences in PLFA profiles of bacterial and fungal cell
membranes to describe the relative abundance of these groups and to detect community-wide
compositional differences among samples (Myers et al. 2001). The PLFA technique will be
combined with stable isotope analysis to distinguish groups of organisms responsible for
decomposition of added litter with compound-specific isotope-ratio mass spectroscopy (Boschker
and Middelburg 2002). PLFAs will be extracted using methods described by (Frostegard and Baath
1996), focusing on the approximately 20 compounds unique to soil microorganisms (Zelles 1999).
We will identify fungi specifically by measuring ergosterol, a sterol found in fungi but not in bacteria
using methods described by Welsch and Yavitt (2003). Analysis of the 13C content of ergosterol will
be used to trace C flow through the fungal decomposer community.
To more sensitively test for the movement of labeled litter to lower depths in the soil profile
(especially in areas colonized by L. terrestris), we will install soil gas sampling probes (Burton and
Beauchamp 1994), at 10, 30, 50, 70 and 100 cm depth. Samples will be collected and analyzed for
CO2 and 13C on two dates in spring and fall – the times of maximum earthworm activity.
Food web analysis. We will use a combination of direct “snapshots” of soil fauna and
salamander diets and measurement of 13C and 15N in salamander tissues to evaluate the effects of
earthworm invasions on (1) the structure of soil food webs at the interface with the aboveground food
web, and (2) the rate of C and N flow from litter into salamander populations.
Characterization of the soil food web. Before destructive sampling of litter boxes, we will
collect 4 randomly located 5 cm diameter cores of litter and soil to a depth of 3 cm. Two of the cores
will be used for heat extraction of mites, collembola, and other microarthropods (Macfayden 1961).
Nematodes and Enchytraeidae will be sampled from the other two cores using wet extraction (Scheu
et al. 2003). Macrofauna will be extracted from a 20 cm diameter by 3 cm depth core with heat on
Berlese funnels. All fauna will be preserved in 70% EtOH (non-arthropod macrofauna, nematodes,
and enchytraeids will be fixed in 4% buffered formalin before preservation). Fauna will be identified
to the highest taxonomic resolution possible and assigned to one of three trophic guilds (sensu Scheu
et al. 2003, Maraun et al. 2001): microbe-detritivores, herbivores, and predators.
Measurement of salamander diets and stable isotopes. We will kill salamanders immediately
in MS-222, and place the carcasses on ice for transport to the lab. At the lab, we will remove the
stomach to eliminate the potential influence of fresh prey on isotope levels, and freeze the remaining
carcass for isotope analysis. Frozen tissues will be sent to UC Davis for δ13C and δ15N analysis.
Each prey in salamander stomachs will be identified to the highest taxonomic resolution possible and
its volume measured as an index of its biomass (Jaeger 1980, Maerz et al. 2005).
Statistical analysis. We will use three factor MANOVAs to test whether land use, worm
community or samping period has an effect on the abundances or biovolumes of different soil fauna
categories in (1) soil and litter samples and (2) salamander diets. We will use standardized canonical
variables to compare the responses of different faunal groups to different factors (Scheiner 1993).
We will use canonical analyses to correlate changes in salamander diets with changes in soil fauna
among treatments. We will use a four factor MANOVA with earthworm community, land use, litter
type, and sample period as fixed factors to test the hypothesis that earthworm invasions affect litterborne C and N flow into salamander populations (indicated by different δC13 and δN15 in salamander
tissues). Modeling the movement of stable isotopes through all parts of the food web is beyond the
logistical scope of this project. Instead, we will use patterns of stable isotope levels in soil fractions,
soil fauna change, and stable isotope levels in salamanders to infer how earthworm impacts on soil
food webs are affecting the flow of C and N in forest ecosystems.
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Simulation Modeling of C and N Data
Analysis of changes in soil C and N pools requires a long-term perspective due to the large size and
slow turnover of these pools. Simulation models have long been used to provide a long-term
perspective to soil C and N cycle studies, allowing for evaluation of the long-term effects of
dynamics observed in short-term experiments. We will build on previous work using the CENTURY
model (Parton et al. 1988) to synthesize data from our regional surveys and 13C and 15N labeling
experiments on the movement of C and N from litter to soil organic matter pools and to evaluate the
long-term impacts of earthworms on soil C and N pools in northeastern forest soils.
In our previous project, we designed a new sub-routine for the CENTURY model to simulate
the effects of earthworm activity on soil C dynamics in collaboration with the Natural Resources
Ecology Laboratory of Colorado State University. This new sub-routine was intended to represent
three basic processes related to earthworm activity: 1) the incorporation of surface litter C into
mineral soil (mixing), 2) the increase of organic matter decomposition as a result of casting and
burrowing activity, and 3) the protection of organic matter in stable aggregates. For each type of C
(structural and metabolic) the sub-routine includes parameters that determine the fractions of C that
are consumed and respired by earthworms and the fate of the C that they release. The model flows
that were altered included: direct incorporation of surface litter into the mineral soil layer, enhanced
decomposition of slow, structural and metabolic pools and stabilization of organic matter in the slow
soil organic matter pool due to earthworms. All of these changes have been incorporated into the
DAYCENT version of the CENTURY model (Parton et al 2001) which uses a daily time step to
simulate trace gas fluxes, nutrient cycling, plant production and soil organic matter dynamics. The
most recently developed version of DAYCENT includes the separation of slow organic matter
derived from surface litter decay and root litter decay and thus will be better able to better simulate
the soil O horizons in forest soils and the potential mixing by earthworms of surface soil organic
matter pools into the mineral soil layer.
The model was successfully used to simulate the C content of the first 20 cm of the Arnot
forest and the new subroutine simulated a 30% reduction in soil C - similar to what we measured at
the site. However, the model was mechanistically weak, as there is little information about how
metabolic and structural C are modified once they are processed by earthworms and deposited in cast
materials throughout the soil profile. An additional problem is that CENTURY simulates SOM
dynamics for the first 20 cm of the soil profile, while earthworms have effects in much deeper layers.
Our labeling experiment is explicitly designed to provide the data necessary to make
CENTURY capable of depicting earthworm effects on soil C and N dynamics. Our first batch of
litter will be strongly labeled in the “metabolic” C pool and the second batch of litter will be strongly
labeled in the “structural” C pool allowing us to evaluate the effects of earthworms on these two key
pools. Further, we will make measurements of C dynamics at depth, allowing us to apply the model
to the entire extent of the soil profile affected by earthworm activity. The result will be a version of
CENTURY that will allow us to evaluate earthworm invasion effects on soil C and N dynamics at
large temporal and spatial scales using data, GIS coverages and regression relationships from our
regional survey as well as regional scale soils databases for the northeast (Shirazai et al. 2003). This
work will be done by one or more of the Cornell and/or Appalachian State graduate students in
collaboration with Dr. William J. Parton, the developer of the CENTURY model, who will be a
consultant to this project (please see letter in “supplementary documents” section).
Task Management and Responsibilities
Groffman and Fahey will work with one Cornell graduate student to oversee the regional sampling
effort, with Bohlen overseeing sampling and characterization of earthworm populations. Fisk
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(working with a graduate student from Appalachian State) will carry out the soil P analysis and
Groffman will be responsible for the microbial biomass and activity sampling in the regional survey.
Yavitt will work with an additional Cornell graduate student and Fahey to carry out the isotope
experiment and trace the movement of 13C and 15N into different microbial groups, food web
components and organic matter pools. Maerz and Fisk will work with graduate students from
Appalachian State and the University of Georgia on the food web and salamander research.
Modeling will be done by one or more of the Cornell and/or Appalachian State graduate students in
collaboration with Dr. William J. Parton as described above.
Education and Outreach
Our previously funded research motivated IES scientists and educators to develop successful,
inquiry-based educational programs and materials focusing on earthworms (e.g., Berkowitz and
Bohlen 1996, Harvey et al. 2004), building on people’s natural fascination with these animals. The
idea that earthworms are “good” is legion in popular literature and in the gardening and K-12
ecology education worlds. We propose to study what people know and don’t know about earthworm
ecology, and then to develop a web-based educational resource. The goal is to take a diversity of
users from a general interest in earthworms and a willingness to think about their effects on
ecosystems, to a more nuanced and informed view based on our research results as well as related
research. The resource will have applications in formal and informal educational settings, such as in
our IES school programs and in a new Ecosystem Teaching Tools feature of the IES website. We
have begun consulting with other groups developing education/outreach materials on earthworms
(e.g. Minnesota WormWatch) and will work to produce complementary materials.
The work will start with a combination of web-based surveys and focus group and individual
interviews with: 1) elementary, middle and high school students and teachers in the Baltimore
Ecosystem Study (BES) Investigating Urban Ecosystems (IUE) Program (Berkowitz is the BES
education team leader, and one IUE unit focuses on soil ecology and earthworm investigations); 2)
youth and educators in the IES Ecology Field Programs, 3) urban youth and educators in a local
Green Teen program (with Cornell Cooperative Extension) and 4) adults in the IES Continuing
Education Program (representing general public and gardening enthusiasts). These will reveal what
the different groups know, or think they know, about earthworms (ecology and impacts), where their
ideas come from, and what barriers or positive pathways exist to developing a richer and more
accurate view of earthworms in northeast ecosystems. This research will contribute to our
understanding of how people develop ideas about organisms and ecosystems, a dimension of
ecological literacy (Berkowitz et al. 2005).
The results of the surveys will be used to craft an engaging discovery center as part of the Changing
Ecosystems portion of the new Ecosystem Teaching Tools feature of the IES website. Content will
be layered in a way that reaches three main audiences: youth, teachers and the gardening-interested
general public. For each audience, there will be a compelling “story” with animations putting the
research results into interesting and accessible terms. We will use interactive problems, short video
clips and simulations, graphics and links to a more in-depth treatment of our research. For gardeners,
we plan to develop a dynamic Q&A, where practical questions about worms in garden soils and
compost piles, as well as in woodlots and forests, will be addressed by project scientists and
educators. For the school audience, the website will include a set of student-friendly datasets from
our and other research. Linked to the datasets will be investigations that students can do on their own
– both new protocols and links to the excellent protocols available elsewhere. Emphasis will be on
concepts that form central foci in national and state science standards (food webs, biotic/abiotic
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interactions, nutrient cycling, soil processes, disturbance and ecosystem change) and on those that are
receiving much attention in the popular press (ecosystem services, invasive species). The teacher
portion of the website will include a table showing connections to National Science Standards and
Benchmarks. Finally, we will include assessment tools, some web-based, that will give teachers and
our project team feedback about student learning.
Pilot versions of components of the site will be tested with small groups from the same target
audiences as in the initial interview phase. The final website will become an integral part of the
programs listed above, and will be made available through the Urban Ecology Collaborative
education working group (Berkowitz is an active member), the LTER Education Network (Berkowitz
sits on the LTER Education Steering Committee), and other web-based science resources for teachers
(e.g., SciNet Links of NSTA, the Biological Education Network, etc.). A unit focused on
undergraduate students, suitable for the Ecological Society of America’s Teaching Issues and
Experiments in Ecology (TIEE) on-line publication will be developed by the project’s education
team in conjunction with the research PIs who teach undergraduate courses.
We will involve undergraduates in the proposed ecological research through traditional Research
Experiences for Undergraduate supplements (if available from NSF) and through an intern program
at one of our sites, the Arnot Forest. For ~$4500, undergraduate interns live on site and participate in
a research program that includes work on a project, interaction with other interns, and capstone
projects such as a paper and oral presentation the following fall.
Training of graduate students will also be an important component of the proposed research. As part
of this training, we will use ideas about incorporating quantitative modeling approaches into the
“toolkit” of empirically-oriented ecologists developed in an NSF-funded “Ecological Circuitry
Collaboratory (ECC) ” that was partially funded by a supplement to our previous earthworm research
project (Ewing et al. 2004, 2005, www.ecostudies.org/cc). We hope to apply lessons learned from
the ECC to the proposed research and have all graduate students include both empirical and mode
components to their research. Opportunities for modeling range from the CENTURY-based soi
and N analyses described above to the movement of stable isotopes through food chains, as has been
done in aquatic systems (Hall et al. 1998, Pace et al. 2004), to analysis of multiple ecosystem
services at large spatial and temporal scales (Groffman et al. 2004b).
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