# Assessment of Nutrient Limitation in Floodplain Forests with Two Different Techniques

Authors: Matthew A. Neatrour; Robert H. Jones; Stephen W. Golladay
Journal: Research Letters in Ecology
 (2008)

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## Abstract

We assessed nitrogen and phosphorus limitation in a floodplain forest in southern
Georgia in USA using two commonly used methods: nitrogen to phosphorus (N:P) ratios in
litterfall and fertilized ingrowth cores. We measured nitrogen (N) and phosphorus (P) concentrations in
litterfall to determine N:P mass ratios. We also installed ingrowth cores within each site containing native
soil amended with nitrogen (N), phosphorus (P), or nitrogen and phosphorus (N + P) fertilizers or
without added fertilizer (C). Litter N:P ratios ranged from 16 to 22, suggesting P limitation. However,
fertilized ingrowth cores indicated N limitation because fine-root length density was greater in cores
fertilized with N or N + P than in those fertilized with P or without added fertilizer. We feel that these two
methods of assessing nutrient limitation should be corroborated with fertilization trials prior to use on a
wider basis.


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## Body

## 1. Introduction

Many ecologists
have applied Liebig’s law of the minimum to forest ecosystems and have sought
to determine what nutrient limits net primary production (NPP). Globally, NPP generally
is considered to be limited by nitrogen (N) [1], but many forests may be
switching from nitrogen to phosphorus limitation because humans have increased atmospheric
nitrogen inputs to forests through fossil fuel combustion, nitrogen fertilizer
production, and other human activities [2]. Assessing nutrient limitation,
however, has been historically problematic because it typically has required laborious
and time-consuming fertilization trials [3]. Within the last decade, ecologists
have increasingly used simpler methods to determine nutrient limitation in
forests, such as foliar N : P ratios [4–11] or root growth into fertilized
ingrowth cores [12–18], but no study to date has explicitly compared these two
techniques.The root ingrowth
core method was originally developed to measure root production and involves excavating
a soil core that is replaced with root-free growth medium (e.g., native soil,
vermiculite, peat), sometimes enclosed in a mesh bag [18]. The core is
extracted after a given period of time and roots growing into the core are
removed. The
ingrowth core technique was modified by Cuevas and Medina
[13] to measure
nutrient limitation by comparing root growth into cores fertilized with
different nutrients. Their modification makes use of the well-known plastic
response of plants to proliferate roots into nutrient-rich patches [19] and
assumes that roots respond more strongly to limiting nutrients. Raich et al. [17]
corroborated this technique in forests with known nutrient limitation, and it
has recently come into widespread use as a result of their work [12, 14–16, 18].N : P ratios in plant
tissue have been used as diagnostic indicators of N or P limitation, and
thresholds have been established for some ecosystems [3, 9, 20]. The use of N : P
ratios to determine N or P limitation is based on the assumptions that either N
or P is limiting and that N or P concentration in plant tissue reflects nutrient
deficiencies in soils. For wetland plant communities in Europe,
Koerselman and Meuleman [3] suggested that communities with an N : P ratios >16
were P-limited while those with an N : P ratios <14 were N-limited. Lockaby and
Conner [20] extended this work to forested wetlands of the southeastern United States
by examining N : P ratios in litterfall and proposed that N : P ratios <12 were
N-limited and those >15 were P-limited.Here, we used the fertilized
ingrowth core and litter N : P ratio techniques to assess nutrient limitation in
a floodplain forest along a blackwater stream in southwestern, Ga, USA. Blackwater
streams typically have lower inorganic materials and suspended sediments in
their waters and therefore floodplain forests associated with these systems generally
are thought to be nutrient poor [21]. In other blackwater systems in the southeastern
United States,
P is generally considered to be limiting [8, 22]. Thus, we expected both
techniques would indicate P limitation.


## 2. Methods

Three26×26 m plots
were established within the floodplain forest of the Chickasawhatchee Wildlife
Management area of Baker and Calhoun counties, Ga, USA. The overstory was dominated at
all sites by Liquidambar styraciflua L, Acer rubrum L, and various Quercus species (Quercus lyrata Walt., Quercus laurifolia Michx., Quercus nigra L). The understory consisted primarily of Smilax spp., Toxicodendron radicans (L.) Kuntze and Sabal minor (Jacq.)
Pers. Soils were Typic Alabaqualfs (Megget series) or Typic Fluvaquents (Muckalee
series) [23, 24]. Mean annual temperature during the study year was 19∘C and
annual precipitation was 141 cm, which was 11% above normal (National Climatic
Data Center, Asheville, North Carolina).Forty cores of
mineral soil (2-cm diameter× 20-cm deep) were collected with a soil probe from
each plot. Soil was sieved through a 2-mm mesh screen to remove coarse
fragments, combined by plot, and air dried. Soil pH was measured using an
Accumet pH meter, and total C and N was determined using a Perkin Elmer Series
II CHNS/O analyzer (Perkin Elmer Inc., Boston,
Mass, USA).
Soils were analyzed for extractable phosphate on a Lachat Quickchem AE
autoanalyzer following double-acid extraction [25].Five 0.25-m2 litter traps were placed randomly in each plot in September 2003, and litterfall
was collected in November 2003 to determine litter N and P concentrations. Litter
was dried to a constant mass (60∘C), weighed, ground using a Wiley mill (Thomas
Scientific, Swedesboro, NJ, USA), and pulverized with a ball Spex 8000D ball
grinder (SPEX CertiPrep Group, Metuchen, NJ, USA). Litter N concentration was
determined using the dry combustion method on a Perkin Elmer Series II CHNS/O
analyzer. Litter P was measured with the dry ash method [26] followed by
analysis on a Lachat Quickchem AE autoanalyzer (Lachat Instruments, Milwaukee, Wis, USA). Litter N : P ratios were calculated as
mass ratios typically used for forest and wetland vegetation.For the ingrowth
core study, each plot was divided into2×2 m grid cells consisting of 196 grid
intersections and 44 of the intersections were designated randomly for
installation of ingrowth cores. In early July 2003, a 10.16-cm diameter × 20-cm
deep core was extracted at each selected location. The core was filled with
soil collected from a nearby site that was sieved through a 2-mm screen to
remove roots. Soil was packed to approximately the same bulk density as the soil
that was removed. The core was either left unfertilized or had phosphorus, nitrogen,
or phosphorus and nitrogen fertilizers mixed into the top 5 cm. Treatments were
replicated 11 times per plot. Nitrogen was added as 0.75 g of POLYON coated
urea (43-0-0 NPK, Harrell’s Inc., Lakeland, Fla, USA), and phosphorus was added
as 0.40 g triple superphosphate (0-46-0 NPK, Southern States Cooperative,
Christiansburg, Va, USA) to raise soil nutrient availability by 400 kg N/ha and
100 kg P/ha, respectively. Cores were harvested after 4 months in late November 2003 by extracting a 7.62-cm diameter
× 20-cm deep core from the middle of each ingrowth core. Seven cores were not
collected due to damage by animals. Cores were washed over a 1-mm mesh screen
to remove adhering soil particles. Roots were separated from soil organic
matter in the lab and subsampled for root length analyses. Each subsample was
scanned and analyzed using WinRHIZO software (Regent Instruments, QC, Canada)
to determine specific root length (SRL, cm of root per g of root). Root samples
were dried to a constant mass at 60∘C and weighed. Root length density (km root
per m3 of soil) was calculated by multiplying SRL by root mass. We ran
a one-way ANOVA to compare root length density among fertilization treatments with
plots as blocking factors using the GLM procedure of SAS version 8 (Statistical
Analysis System, Cary, NC, USA). We also used to 95% confidence limits to
determine whether N : P were different from threshold ratios for 12 and 15, for N
and P limitation, respectively.


## 3. Results and Discussion

The fertilized ingrowth core method indicated N limitation because root
length density in cores fertilized with N or N + P was nearly double that of root
length density in P-fertilized cores or in cores with no added fertilizer (P<.01, Figure 1). In addition, root length density did not differ between
P-fertilized cores and cores without fertilizer (P<.99). According to 95% confidence intervals, litter N : P ratios (mean = 19.1) were
greater than 12 but not than 15, the thresholds for N and P limitation, respectively,
in floodplain forests of the southeastern United States
[20]. This suggested P limitation or colimitation by N and P in this forest. Low
N (0.8%) and P (0.04%) compared to other floodplain forests [20] may support colimitation (Table
1).
However, high nutrient resorption proficiency of P, defined by Killingbeck [27]
as the degree to which plants can reduce the level of a given nutrient in
senescing leaves, lends support for P limitation in this forest. Proficiency was
<0.05% for P and >0.7% for N, which suggested nearly complete resorption
of P and intermediate resorption of N [27]. The discrepancy between the two
techniques is somewhat puzzling. In the following paragraphs, we provide
possible reasons for these contradictory results.Table 1
Soil and
litter chemistry attributes for the three floodplain forest plots in the Chickasawatchee
Wildlife Management Area. Values are means with ranges in parentheses.
AttributeAmountSoilsExtractable PO4-P
(μg/g)1.0(0.7–1.3)Total N (%)0.07(0.06–0.08)Total C (%)1.6(1.3–1.9)pH5.0(4.2–5.4)LitterP
concentration
(%)0.04(0.03–0.05)N concentration
(%)0.80(0.67–0.98)C concentration
(%)49.8(49.2–50.8)N  :P ratios19.1(16.1–21.7)C : N ratios63.7(50.0–76.0)Figure 1
Fine root response to fertilized
ingrowth cores (+1 SE). C = control, P = phosphorus fertilization with
triplesuperphosphate (100 kg P/ha), N = nitrogen fertilization with coated urea
(400 kg N/ha), N + P = nitrogen and phosphorus fertilization with
triplesuperphosphate, and coated urea (100 kg P/ha + 400 kg N/ha). Different
letters indicate significant differences among treatments(P<.05).We feel there are three likely explanations why roots responded only to N
fertilization in potentially P-limited conditions. First, the use of fertilized
ingrowth cores to measure nutrient limitation is centered on the assumption
that roots respond more strongly to limiting nutrients than to those that are
“non” limiting. However, this assumption may not always be valid for all
forests.  Hodge et al. [28] suggested that proliferation may be an adaptive response
by plants to obtain nutrients when in competition with other plants rather than
a response based on immediate nutritional needs. If this is the case, plants
may not respond more strongly to limiting nutrients because they are constrained
by their evolutionary history. For example, plants species adapted to soils
that are historically N-deficient only may have the capacity to proliferate
roots into N-rich patches even when P is limiting. Second, the ingrowth core
method operates on the implicit assumption that all plants within a community
can proliferate roots into nutrient-rich patches. However, not all plants can
proliferate roots [29], and nutritional status of individual species may differ
from that of the whole plant community [30]. Therefore, proliferation may be a
nutritional response by individual species that may or may not reflect nutrient
limitation of the whole community. Third, P may have become rapidly unavailable
to plants due to adsorption into Fe and Al complexes or leaching from the cores.
The type of fertilizer used in our study, triplesuperphosphate (TSP), is water
soluble and fast releasing even though the level of P we used was relatively
high (100 kg/ha). Although this may help to explain the lack of response to P,
it does not offer any insight as to why roots responded strongly to N
fertilization.There are also several reasons why litter N : P ratios may be poor
indicators of P limitation in N-limited conditions. First, the use of N : P ratios
to indicate nutrient limitation requires that either N or P limits NPP. N : P
ratios cannot detect limitation by another resource (i.e., other nutrient,
light, water) or colimitation by multiple resources. The fertilized ingrowth
core as used in our study also could not determine nutrient limitation by other
resources besides N and P but has the flexibility to at least test limitation
or colimitation by other nutrients. Second, assessing nutrient limitation
through N : P ratios also necessitates that these ratios are indicators of
nutrient deficiencies in soils. However, N : P ratios of individual species do
not always vary across a gradient of known nutrient limitation [31], but Aerts and
Chapin [30] warned that N : P ratios of individual species may differ from
community N : P ratios. Third, thresholds for N or P limitation often are hard to
establish. Although there is a general consensus that low N : P ratios indicate N
limitation, there is no agreement as to whether intermediate to high N : P ratios
indicate P limitation [32]. Some researchers have found N limitation or
colimitation by N and P when N : P ratios are high [9, 33]. Finally, all of the
above problems may have been compounded by the use of litter N : P ratios instead
of N : P ratios in live foliage because of differences in retranslocation
efficiencies between N and P. The use of foliar N : P ratios in conjunction with
litter N : P ratios may have provided more insight into what nutrient was
limiting.In conclusion, we found that these two techniques gave contradictory
results as to what nutrient was limiting (i.e., N or P) and therefore cannot be
used to reliably indicate nutrient limitation for all forest ecosystems. We
feel both techniques should be corroborated with fertilization trials prior to
use on a wider basis.



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*Source: 260754-2008-05-15.xml*