Introduction
The use of chemical fertilizers in agriculture is widespread today (Novara et
al., 2016; Zupanc et al., 2016). This is resulting in environmental damages
to the soil system as chemical fertilizers are part of the modernization of
the agriculture around the world and trigger soil degradation and erosion
(Cerdà et al., 2009; Ochoa-Cueva et al., 2015; Rodrigo Comino et al.,
2016). Grasslands are one of the landscapes that need more attention to the
impact of management (Lü et al., 2016; Pereira et al., 2016) as other
agriculture landscapes are already well studied (Beniston et al., 2016; de
Oliveira et al., 2015; Moreno et al., 2016). The abuse of nitrogen (N) in the
agriculture fields is a well-known topic but little is known about its
impacts on the grassland soil ecosystems (Liu et al., 2016; Zhang et al.,
2016).
Stoichiometric relationships of soil nutrients are reliable indicators of
nutrient availabilities for plants and soil microorganisms (Cleveland and
Liptzin, 2007). Soil carbon : nitrogen : phosphorus (C : N : P)
ratios play essential roles in shaping plant and microbial stoichiometry,
which reveals nutrient allocations, life history strategies, and
physiological adjustment to climate change (Elser et al., 2010). For
instance, enhanced N inputs resulted in higher soil N availability (Wei et
al., 2013) and consequentially a decrease in foliar C : N and an increase
in the foliar N : P ratio (Han et al., 2014). Nitrogen addition was
suggested to cause divergent effects on the plant C : P ratio, which
declined at species level but showed no change at the community level (Han et
al., 2014). Plant species that are stoichiometric
N : P flexibility may have an advantage over those possessing strict
stoichiometric homeostasis under environmental changes (Sardans and
Peñuelas, 2012). Leaves with high nutrient concentrations (both N and P),
which are relatively lower C : N and C : P ratios, tend to be short
lived, with a high specific leaf area (Wright et al., 2004) as well as a high
photosynthetic capacity and dark respiration rates (Reich et al., 2008).
Local nutrient availability from soils is suggested to be one of the most
important contributors to the wide variation of terrestrial plants in
C : N : P ratios (i.e., plants are what they root in; Elser et al.,
2010). Soil exchangeable calcium (Ca) and magnesium (Mg) and available iron
(Fe) and manganese (Mn) are critical metal nutrients for plant growth,
microbial activity, and ecosystem health (Lucas et al., 2011). The ratio of
exchangeable Ca : Mg indicates the relative availability of these two ions
and is reported to influence clay dispersion and surface sealing processes
(Dontsova and Norton, 2002). Maintaining a properly available Fe : Mn ratio
is essential for plant health as a lower ratio might suggest plants suffering
from Fe deficiency and Mn toxicity (Hodges, 2010). However, the
stoichiometric qualification of soil DOC and available nutrients is less
known than plant stoichiometry.
Ongoing increases in anthropogenic N inputs have largely affected soil C and
nutrient cycles in most terrestrial ecosystems (Lü et al., 2013; Neff et
al., 2002). Under N enrichment, higher soil available N (sum of nitrate and
ammonium) has been documented to be positively (Wei et al., 2013), negatively
(Heffernan and Fisher, 2012), or even neutrally (Liu and Greaver, 2010)
related to dissolved organic C (DOC), which is an important linkage for
plant–soil–microbe systems in natural ecosystems. Anthropogenic increases
in reactive N would also shift ecosystem nutrient limitation from N to P by
stimulating P uptake by plants (Menge and Field, 2007). Moreover, soil
acidification caused by N enrichment could help desorption and dissolution of
soil inorganic P, serving as an essential P source for plants, especially in
calcareous soils (Lajtha and Bloomer, 1988; Tunesi et al., 1999). Soil pH is
widely recognized as one of the most influential factors that regulates
nutrient bioavailability (Kemmitt et al., 2006). For example, Piccolo (2001)
suggested that a drop in soil pH elevated the solubility of soil humic
substances. Soil pH is also suggested to influence the soil C : N ratio via
changes in plant litter quality (Schmidt, 1982) and increase soil available P
(AP) by promoting phosphomonoesterase activity (Hogan et al., 2010). Thus, N
enrichment might influence soil available C : N : P stoichiometry through
the alteration of soil pH, and hence aggravate or alleviate plant and
microbial C and nutrient limitations (Elser et al., 2010). Enhanced N inputs
cause leaching of soil exchangeable Ca and Mg and activation of soil
available Fe and Mn as a result of soil acidification, which will lead to
nutrient imbalance in soils (Katou, 2002; Malhi et al., 1998). For example, N
deposition decreases the ratio of exchangeable Ca : Mg as preferential
weathering of soil exchangeable Ca relative to Mg (Lu et al., 2014). Iron
deficiency chlorosis has been found in calcareous soils in grasslands of
America (Rogovska et al., 2007) and Mn toxicity may occur after soil
acidification accompanying leaching of base cations (Lynch and Clair, 2004).
Thus, the available Fe : Mn ratio will be useful in indicating the relative
availability of Fe and Mn as affected by N addition. Under anthropogenic N
enrichment scenarios, clarifying stoichiometric traits of soil available
elements would promote our understanding of soil nutrient imbalance as caused
by environmental changes.
Physical protection by soil aggregation is a main mechanism of soil organic
matter (SOM) stabilization (Amezketa, 1999; Wiesmeier et al.,
2012). Oxygen and water diffusion
rates are generally parallel with soil aggregate sizes as determined by pore
space (Young and Ritz, 2000; Baker et al., 2007). Consequently, available
nutrients like N and P tend to distribute in aggregates of larger size due to
more favorable microclimates for microbial activity herein (Dorodnikov et
al., 2009; Wu et al., 2012). Smaller aggregate-size classes are confirmed as
a preferential site for the physical stabilization of SOM and nutrient
retention (Fonte et al., 2014). Higher clay contents in microaggregates tend
to provide more surface area and binding sites to retain more exchangeable Ca
and Mg (Oorts et al., 2003). Within microaggregates, more microbial-processed
SOM will also chelate or make complex a larger amount of base cations and
micronutrients (Lü et al., 2016). Even though numerous studies have
focused on C and nutrient cycling in the aggregate scale (Fonte et al., 2014;
Six and Paustian, 2014; Wright, 2009), stoichiometric traits of DOC and
available nutrients within aggregates are still unclear.
Schematic diagram of chestnut soil illustrating three soil horizons
(figure modified from the Digital Science and Technology Museum of China,
http://amuseum.cdstm.cn/AMuseum/agricul/6_5_27_ligt.html).
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Semiarid steppes of Inner Mongolia in northern China, playing a vital role in
serving environmental health, regional economy, and the global C cycle, have
undergone degradation during the last 50 years' increased stocking rates and
static grazing management practices (Kang et al., 2007). Non-physically
protected SOM pools, which can be a majority of SOC and nutrients in these
sandy grassland systems, are, however, inherently vulnerable to environmental
changes (Creamer et al., 2011), like elevated N inputs. A 7-year N field
manipulation experiment has already shown significant aboveground changes in
species turnover rates, plant community composition, and community stability
(Xu et al., 2012). Microaggregates (< 250 µm) had the
highest dissolved inorganic N (DIN) concentration as compared to large
(> 2000 µm) and small (250–2000 µm; see Wang
et al., 2015a) macroaggregates, which was not the case for both DOC (see Wang
et al., 2015b) and AP (Wang et al., 2016a). Nitrogen addition significantly
increased DIN (see Wang et al., 2015a) and available Fe and Mn but decreased
exchangeable Ca and Mg across three soil fractions (not for Ca in
microaggregates; Wang et al., 2016b). In this study, we aimed to analyze
stoichiometric traits of DOC, DIN, AP, exchangeable Ca and Mg, and available
Fe and Mn within soil aggregates in this semiarid grassland. We hypothesize
that microaggregates had lower DOC : DIN and higher DIN : AP ratios due
to better N retention capacity in smaller-size aggregates. We also
hypothesized that N addition would decrease DOC : DIN while increasing
DOC : AP and DIN : AP ratios due to higher anthropogenic inputs of
inorganic N and intensive removal of available P from soil by plant uptake.
Due to preferential weathering of Ca relative to Mg during soil
acidification, we expected to detect a significant decrease in the Ca : Mg
ratio under N addition across soil aggregate classes. Due to the fact that
plant growth is commonly limited by Fe deficiency in calcareous grasslands,
enhanced Fe translocation from soil to plants would result in a lower soil
available Fe : Mn ratio under N enrichment.
Methods
Study site and experimental design
The study site (42∘02′27′′ N, 116∘17′59′′ E,
elevation 1324 m a.s.l.) was located in Duolun County, a semiarid area in
Inner Mongolia, China. The mean annual temperature is 2.1∘ C,
ranging from 17.8∘ C in January to 18.8∘ C in July, and
average precipitation is 379.4 mm yr-1. The soil is chestnut in the
Chinese classification and Calcisorthic Aridisol in the US Soil Taxonomy
classification. The soil texture of the experimental site is sandy loam with
62.75 % sand, 20.30 % silt, and 16.95 % clay (Liu et al., 2009).
The soil contains an Ah horizon of 25–50 cm thickness with dark brown
color, a Bk horizon of 30–50 cm thickness with grayish color, and a C
horizon with dark-grayish brown color (Fig. 1; Heil and Buntley, 1965; Buol
et al., 2011).
In April 2005, seven blocks (107 m × 8 m) were set up in a
split-plot design with water and N being the two treatments. Each block was
divided into two main plots receiving either ambient precipitation or
additional water treatment (180 mm). Each main plot was divided into six
subplots which were randomly treated with four levels of N (0 [CK], 5
[N5], 10 [N10], and 15 [N15] g N m-2 yr-1), P
(10 g N m-2 yr-1), and combined N and P addition. This study
was only concerned about the treatments of four levels of N addition under
ambient precipitation. Nitrogen was added as urea pellets, with half of it
applied in early May and the other half in late June.
The concentrations of soil organic carbon
(SOC, g kg soil aggregate-1), total nitrogen
(g kg soil aggregate-1), dissolved organic carbon (DOC, mg kg soil
aggregate-1), dissolved inorganic nitrogen (DIN,
mg kg soil aggregate-1), available phosphorus (AP, mg kg soil
aggregate-1), exchangeable Ca and Mg (cmol kg soil aggregate-1),
and available Fe and Mn (mg kg soil aggregate-1) within bulk soil and
soil fractions of 0–10 cm soils without field-manipulated treatments (data
from Wang et al., 2015a, b, and 2016a).
Soil aggregates (µm)
Bulk soil
> 2000
250–2000
< 250
SOC
18.9 ± 1.7
18.2 ± 1.0
15.1 ± 2.0
21.6 ± 1.4
TN
1.8 ± 0.2
1.9 ± 0.1
1.5 ± 0.2
2.2 ± 0.1
DOC
69.8 ± 5.6
72.5 ± 6.8
66.6 ± 1.3
71.2 ± 1.5
DIN
21.6 ± 1.5
21.1 ± 0.6
49.5 ± 1.9
57.3 ± 2.0
AP
14.9 ± 2.8
3.7 ± 0.4
5.8 ± 0.7
3.9 ± 0.5
Ca
19.6 ± 0.3
26.6 ± 0.7
19.8 ± 0.4
22.7 ± 2.7
Mg
1.7 ± 0.05
1.8 ± 0.1
1.4 ± 0.05
2.0 ± 0.1
Fe
21.8 ± 0.6
24.0 ± 1.8
20.0 ± 1.4
27.5 ± 2.8
Mn
23.3 ± 2.7
24.5 ± 0.8
20.0 ± 0.8
24.0 ± 1.9
Field sampling and measurements
Soil samples (0–10 cm) were collected from each N plot from four out of
seven blocks in August 2013. For each plot, five random cores were retrieved
after removing the litter layer and stored at 4∘ C under further
analysis. Soil aggregates were isolated according to Dorodnikov et al. (2009)
into three aggregates (> 2000 µm, large macroaggregate class;
250–2000 µm, small macroaggregates; and < 250 µm, microaggregate
class) by a Retsch AS 200 Control (Retsch Technology, Düsseldorf,
Germany). This dry-sieving method was used to maintain microbial activity and
reduce the loss of DOC and available nutrients in soil aggregates. The
separated aggregates were weighed and used for determining soil aggregate
moisture (drying at 105∘ C for 48 h), DOC, DIN, AP, exchangeable Ca
and Mg, and available Fe and Mn.
Soil pH was measured in a 1:2.5 (w/v) soil-to-water slurry of soil
aggregate samples with a digital pH meter. The concentration of DOC was
extracted by adding 50 mL of 0.5 M potassium sulfate and analyzed by a TOC
analyzer (High TOC, Elementar). Soil DIN concentration was extracted with
50 mL 2 M KCl from 10 g fresh soils (McLeod, 1992) and determined
colorimetrically using an AutoAnalyser III continuous Flow Analyzer (Bran
& Luebbe, Norderstedt, Germany). Soil AP was measured using the Olsen
method (Olsen et al., 1954). Briefly, 2.5 g of the soil fraction was mixed
with 50 mL 0.5 M NaHCO3 (pH 8.5) and 5 g of phosphorus-free
charcoal. The mixture was shaken at 150 rpm for 30 min and filtered. The P
concentration of P in filtration was determined by the molybdenum blue
colorimetric method (Murphy and Riley, 1962). The units of DOC, DIN, and AP
were expressed in mg per kg soil aggregate. Exchangeable Ca and Mg within
soil aggregates were extracted by mixing a 2.5 g soil fraction with 50 mL
1 M ammonium acetate (pH 7.0) and then shaken for 30 min at 150 rpm. After
filtration, the Ca and Mg concentrations in extraction were determined by an
atomic absorption spectrometer (AAS, Shimadzu, Japan). To analyze available
Fe and Mn within soil aggregate fractions, 10 g of soil samples were
extracted by 20 mL 0.005 M DTPA + 0.01 M CaCl2+ 0.1 M TEA
(triethanolamine; pH 7.0). After being shaken at 180 rpm for 2 h, the
extracts were filtered and available Fe and Mn concentrations in the
filtration were determined by the AAS. Soil DOC and available nutrients of
the control plot were listed in Table 1 (data from Wang et al., 2015a, b, and
2016a). The concentrations of exchangeable Ca and Mg were represented as cmol
per kg soil aggregate, and it was a mg per kg soil aggregate for available Fe
and Mn.
Two-way ANOVAs (F values) of the effect of soil aggregate size
(S), nitrogen addition (N), and their interactions on the ratios of
DOC : DIN, DOC : AP, DIN : AP, exchangeable Ca : Mg, and available
Fe : Mn.
DOC : DIN
DOC : AP
DIN : AP
Ca : Mg
Fe : Mn
S
100.86**
0.41
16.32**
1.81
0.25
N
36.91**
3.44*
1.73
1.19
109.23**
N × S
12.64**
1.72
2.43*
2.07
7.41**
* Significance level at P < 0.05. ** Significance level at
P < 0.01.
The ratio of (a) dissolved organic carbon to dissolved inorganic
nitrogen (DOC : DIN), (b) DOC to available phosphorus (DOC : AP), and (c) DIN : AP
as influenced by N addition (0 [CK], 5 [N5], 10 [N10], and 15
[N15] g N m-2 yr-1) in soil aggregates. Data are represented
as mean ± SE. Lowercase letters indicate significant differences
between N treatments within a soil fraction. The capital letters at the top
indicate significant differences between soil aggregate sizes across N
treatments.
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Statistical analysis
The normality of data and homogeneity of variances were determined by the
Kolmogorov–Smirnov test and Levene's test, respectively. Two-way ANOVAs were
executed to determine soil aggregate size (S), N addition, and their
interactive effects (S × N) on the ratios of DOC : DIN,
DOC : AP, DIN : AP, exchangeable Ca : Mg, and available Fe : Mn.
Multiple comparisons with a Duncan design were conducted to determine the
difference between N treatments within each soil fraction. Pearson
correlation analysis was used to determine the relationship between soil pH
and these ratios within each soil aggregate class. The whole correlation
analysis was performed in SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) and
statistical significance was accepted at P < 0.05.
Discussion
The stoichiometric ratios among different soil aggregates fractions
Consistent with our initial hypothesis, microaggregates had lower DOC : DIN
ratios and higher DIN : AP ratios relative to large macroaggregates. It is
generally recognized that more labile SOM concentrates in macroaggregates,
with more recalcitrant SOM in microaggregates (Jastrow et al., 2007).
Additionally, SOM in microaggregates experiences more microbial processing
cycles which are more decomposed as compared to macroaggregates (Six and
Paustian, 2014). These resulted in a lower C : N of SOM in microaggregates.
Our previous study suggested that microaggregates retained significantly
higher inorganic N relative to large macroaggregates (Wang et al., 2015a).
However, DOC and AP concentrations showed no difference between large
macroaggregates and microaggregates, as suggested by our previous studies
(Wang et al., 2015b, 2016a). In this context, microaggregates had
significantly lower DOC : DIN ratios (Fig. 2a) but higher DIN : AP ratios
(Fig. 2c) than large macroaggregates. Our results indicated that
microaggregates were preferential sites for retaining DIN relative to DOC and
AP as compared to large macroaggregates (Fig. 2a,c). Microaggregates serve as
an indicator of soil C storage capacity and help elucidate the capacity of
soils to protect SOM and supply N and P nutrients (Du et al., 2015). Higher
DIN : AP ratios in microaggregates would be essential for available N
supply, but it might eventually result in P deficiency under enhanced N
loading rates in the long term.
Effect of N addition on DOC : DIN, DOC : AP, and DIN : AP ratios within soil aggregates
As expected, we observed a significant decrease in the soil DOC : DIN ratio
within all soil aggregate classes affected by N addition (Fig. 2a). This
should mainly be due to a supply of DIN which is directly transformed from
urea fertilizer or indirectly derived from enhanced soil organic N
mineralization and nitrification under urea addition (Chen et al., 2013)
within three soil fractions. Indeed, our previous studies suggested that N
addition increased DIN by up to 128.3, 41.2, and 45.7 % (Wang et al.,
2015a), while DOC only increased by 12.7, 3.7, and 12.7 % within large macroaggregates,
small macroaggregates, and microaggregates, respectively (Wang et al.,
2015b). As suggested by our previous work, N addition decreased soil pH from
7.3 to 5.6 in large macroaggregates, from 7.5 to 5.8 in small
macroaggregates, and from 7.4 to 5.9 in microaggregates (Wang et al., 2015b).
Soil pH was one of the main factors influencing the DOC : DIN ratio, as
suggested by their positive correlations within three soil fractions
(Fig. 4a, b, c). A decline in soil pH was reported to be associated with
accumulation of soil DIN (Aciego Pietri and Brooks, 2008), thus leading to
the decrease in the DOC : DIN ratio (Fig. 3a, b, c).
Soil DOC and DIN are important energy and N sources for microorganisms, and
relative soil C and N availabilities (represented as DOC : DIN) make a
great contribution to controlling microbial activity and nutrient cycling
processes (Dijkstra et al., 2006; Wu et al., 2012). A significant decrease in
DOC : DIN (Fig. 1a) might suggest that higher N inputs induced microbial C
limitation relative to N across soil fractions. However, Wei et al. (2013)
found that N addition increased both DOC and DIN but decreased microbial
biomass C (MBC) concentration, and they suggested that soil microbes were no
longer C-limited due to a non-significant correlation between DOC and
microbial biomass C. In their study, they failed to analyze the relationship
between the DOC : DIN ratio and MBC, which could be used to indicate
relative C and N availabilities in determining microbial growth. In this
study, a coincident decrease in the DOC : DIN ratio (Fig. 2a) and MBC (see
Wang et al., 2015b) indicated that repression of microbial growth under N
addition was due to lower C availability relative to N (Treseder, 2008).
Our previous study suggested that N addition increased soil AP by 60.2 and
84.5 % within large macroaggregates and microaggregates, respectively
(Wang et al., 2016a). Based on this, it was reasonable to detect a
significant decrease in the ratio of DOC : AP within large macroaggregates
and microaggregates (Fig. 2b). Soil acidification could enhance dissolution
of phosphate from mineral-bound P pools, while this was not the case for DOC
(Wang et al., 2016a). Thus, production of DOC might not keep up with
continuous dissolution of AP, resulting in a lower DOC : AP ratio under N
addition (Fig. 2b). Significant positive correlations between soil pH and the
DOC : AP ratio (Fig. 4d, f) also confirmed higher AP dissolution than DOC
supply under soil acidification. Under the scenario of enhanced ecosystem N
inputs, a decrease in the soil aggregate DOC : AP ratio would induce
microbial C limitation relative to P (Treseder, 2008), and continuous release
of AP from rock weathering would shift the ecosystem from N limitation to P
limitation in the long term (Lü et al., 2013).
Effect of N addition on exchangeable Ca : Mg and available Fe : Mn ratios
Within large and small macroaggregates, a significant decrease in the
exchangeable Ca : Mg ratio supported our hypothesis. Soil acidification, as
developed under increased SO2 and NOx emissions, results in
weathering and release of base cations (Lucas et al., 2011). Selective
weathering of certain base cations
(Ca > Na > Mg > K) would result in a
change in base cation budgets and an imbalance of metal ions in soils (Lu et
al., 2014). Preferential loss of exchangeable Ca relative to Mg was the main
reason for the significant decrease in the Ca : Mg ratio in large and small
macroaggregates (Fig. 3a). Correlation analyses also suggested a coincident
decline in the Ca : Mg ratio and soil pH, indicating that more exchangeable
Ca was leached than Mg during soil acidification in macroaggregates. However,
the unaffected Ca : Mg ratio in microaggregates contradicted our hypothesis
that the Ca : Mg ratio would decrease across three soil fractions. In this
sandy grassland, microaggregates containing more minerals and less sand would
be a more favorable site for base cation binding and adsorption, which was
less affected by N addition and sequential soil acidification compared to
macroaggregates (Gunina and Kuzyakov, 2014). Moreover, more decomposed SOM as
indicated by δ13C (Wang et al., 2015a) would provide an additional
strong binding surface for exchangeable Ca and Mg in microaggregates. Thus, a
significant relationship of the Ca : Mg ratio with soil pH was not detected
in microaggregates (Fig. 5c).
The soil Ca : Mg ratio affects plant Cu uptake and lower Ca : Mg might
increase or decrease plant Cu uptake and toxicity depending on plant species
and soil types (Lombini et al., 2003). Under enhanced N inputs, a significant
decrease in the Ca : Mg ratio would further influence nutrient balance,
i.e., Cu uptake of plants in this calcareous grassland. Researchers proposed
that relative availabilities of exchangeable Ca and Mg were more important
than their absolute amounts (Bear et al., 1951; Albrecht, 1975; Kopittke and
Menzies, 2007), and optimal Ca : Mg ratio ranges for plant growth were
identified for various plant species since the study from Loew (1892). In
addition, different soil aggregate classes were characterized by their
various nutrient supply capacities (Mikha and Rice, 2004). Soil structure
could modify the effect of cation ratios on plant growth (Zhang and Norton,
2002). These shed light on the essential role of studying the soil
exchangeable Ca : Mg ratio of different soil aggregate fractions in
evaluating soil fertility and plant growth.
For all soil fractions, a significant increase in the available Fe : Mn
ratio contradicted our initial hypothesis. A decrease in the Fe : Mn ratio
might be caused by the antagonistic relationship between Fe and Mn during
plant uptake (Somers and Shive, 1942). Soil acidification promoted weathering
and desorption of micronutrients from soil minerals, which increased soil
available Fe and Mn concentrations (Malhi et al., 1998). Even though Fe
availability generally limits productivity in calcareous grasslands, enhanced
plant Mn uptake might retard plant Fe acquisition, leading to higher
available Fe retained in soil (Tanaka and Navasero, 1966; Tian et al., 2016)
under soil acidification. Correspondingly, negative relationships of soil pH
with Fe : Mn ratios (Fig. 5d, e, f) suggested accumulation of available Fe
relative to Mn under soil acidification as affected by enhanced N inputs.
Consistent with our findings, Tian et al. (2015, 2016) found soil
acidification enhanced Mn uptake and reduced Fe absorption by forbs which
result in lower photosynthetic rates and growth and loss of forb species.
Plant Fe deficiency was reported to induce chlorosis and decrease
photosynthesis in calcareous grassland (Rogovska et al., 2007). An increase
in the soil available Fe : Mn ratio might pose a threat to grassland
productivity such as potential aggravation of Fe deficiency and the
occurrence of Mn toxicity to plant species (Tian et al., 2016). Soil nutrient
imbalance would not only influence both the quantity (yield) and quality of
plants, but also cause an impact on the health of animals grazing on these
plants (Mikha and Rice, 2004).