Soil Biology & Biochemistry 75 (2014) 1e10

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Soil Biology & Biochemistry

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Effects of experimental nitrogen and/or phosphorus additions on soil

nematode communities in a secondary tropical forest
Jie Zhao a, b,1, Faming Wang a,1, Jian Li a, c,1, Bi Zou a, Xiaoli Wang a, c, Zhian Li a, *,
Shenglei Fu a, **
a
Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences,
Guangzhou 510650, PR China
b
Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125,
PR China
c
University of Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In tropical forest ecosystems, highly weathered soils are often considered as relatively nitrogen-rich but
Received 3 January 2014 phosphorus-poor. Nutrient availability greatly regulates ecosystem processes and functions of tropical
Received in revised form forests. However, little is known about how nitrogen and/or phosphorus additions affect the conditions
18 March 2014
of soil food web which is an important component of belowground ecosystems. In the present study, soil
Accepted 21 March 2014
nematode communities were monitored and served as indicators of soil food web conditions under
Available online 13 April 2014
experimental nitrogen and phosphorus additions in a secondary forest in tropical China. The principal
response curves of soil nematode community structure revealed same tendency of changes under ni-
Keywords:
Nitrogen fertilization
trogen and/or phosphorus additions compared with control, in terms of nematode functional guild
Phosphorus fertilization compositions: apparent successions from communities dominated by He3 and Ba1 to communities
Soil nematodes dominated by Ba2 and Fu2 occurred after nitrogen and/or phosphorus additions. The diversity of soil
Soil food web nematode genera was not sensitive to either nitrogen or phosphorus addition. Phosphorus addition
Tropical forest significantly suppressed total nematode density, density of omnivore-predators, and four nematode
Nitrogen-rich and phosphorus-poor soils faunal indices (i.e. MI25, EI, SI, and SFI), but increased two faunal indices (i.e. CI and BaI). However,
nitrogen addition did not induce remarkable changes of these variables in the present study. Our results
suggest that nitrogen and/or phosphorus additions suppress soil nematodes in tropical secondary forests,
which was inconsistent with our expectation that nitrogen addition was detrimental to and phosphorus
addition was conducive to soil nematodes in this nitrogen-rich but phosphorus-poor soil. Furthermore,
the effects of phosphorus addition are more powerful than the effects of nitrogen addition. Moreover,
phosphorus addition degrades the structure and trophic links of the soil food web, reduces the carbon
utilization of soil nematodes, and leads to a more fungal dominated decomposition pathway. The al-
terations of soil food web conditions might result in altered ecosystem functioning. Our findings could
provide a better understanding of the responses of soil food web to nitrogen and phosphorus additions in
nitrogen-rich but phosphorus-poor soils.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction and 30e50% of terrestrial productivity (Grace et al., 2001). There-

Tropical forests possess great biodiversity (Myers et al., 2000;
Sala et al., 2000), contain up to 40% of global terrestrial biomass
carbon (Dixon and Brown, 1994; Cleveland and Townsend, 2006)
* Corresponding author. Tel.: þ86 20 37252631; fax: þ86 20 37252615.
** Corresponding author. Tel.: þ86 20 37252722; fax: þ86 20 37252831.
E-mail addresses: lizan@scbg.ac.cn (Z. Li), sfu@scbg.ac.cn (S. Fu).
1 anthropogenic activities such as fossil fuel burning and agriculture
These authors contributed equally.
fore, they are considered as one of the most important ecosystems
on earth. Their highly weathered soils are often considered as
relatively nitrogen-rich but phosphorus-poor. Soil phosphorus
level plays a key role on ecosystem functions such as net ecosystem
production (NEP) and litter decomposition in those ecosystems
(Vitousek, 1984; Hobbie and Vitousek, 2000; Wardle et al., 2004;
Cleveland et al., 2006; Kaspari et al., 2008). In addition, global at-
mospheric nitrogen deposition is accelerating through time due to

http://dx.doi.org/10.1016/j.soilbio.2014.03.019
0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
2 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10

fertilization (Lu et al. 2010a; Fang et al. 2011). Chronic elevated
nitrogen depositions impact many aboveground and belowground
ecosystem functions such as NEP, biodiversity or richness, and
global carbon cycling (Vitousek, 1984; Alison et al., 2000;
Nadelhoffer, 2000; Matson et al., 2002; Lu et al., 2010a, 2010b).
Nematodes are the most abundant soil mesofauna, occupy key
positions at most trophic levels in the soil food web, and correlate
with ecosystem processes and functions such as soil nutrient
cycling, litter decomposition, net ecosystem production (NEP), and
ecosystem succession (Van der Putten et al., 1993; De Deyn et al.,
2003; Neher et al., 2012; Zhao et al., 2012; Zhao and Neher,
2013b). They are sensitive to various disturbances (e.g., agricul-
ture management, pollution, land-use change) and it is well
documented that soil nematodes can be useful ecological indicators
(Neher, 2001; Todd et al., 2006; Zhao and Neher, 2013; Zhao et al.,
2013a, 2013b). Soil nematode communities are quite susceptible to
the changes in soil nitrogen and/or phosphorus levels (Todd, 1996;
Sarathchandra et al., 2001; Coyne et al., 2004; Wang et al., 2006;
Liang et al., 2009). Therefore, the additions of nitrogen and phos-
phorus may lead to alteration in soil nematode communities.
Addressing the effects of nitrogen and phosphorus additions on soil
nematodes can improve our insights of how nitrogen and phos-
phorus additions alter soil food web structure and ecosystem
processes (e.g., nutrient mineralization) in tropical forest ecosys-
tems. However, soil nematode communities are usually overlooked
in studies of nitrogen and phosphorus additions, particularly, the
phosphorus addition.
Only few studies have monitored the effects of simulated ni-
trogen deposition on soil nematode communities. The main find-
ings of these studies are that high levels of nitrogen addition
(>80 kg N ha1 yr1) suppressed soil nematode communities but
low levels of nitrogen deposition (<50 kg N ha1 yr1) did not
induce significant changes of soil nematode communities in both
alpine tundra soils in Wyoming, USA (Lokupitiya et al., 2000) and in
grassland soils in Inner Mongolia, China (Ruan et al., 2012; Wei
et al., 2012; Li et al., 2013). Many previous studies monitor the ef-
fects of nitrogen fertilization on soil nematode communities in
agriculture, forest and grassland ecosystems. However, there are
conflicting observations of the same monitored variables even in
similar ecosystems. For examples, mineral N fertilization increased
total nematode abundance of a boreal forest in southern British
Columbia (Forge and Simard, 2001) but decreased that of a boreal
forest in central Sweden (Sohlenius and Wasilewska, 1984),
respectively. Nitrogen fertilization significantly increased the rela-
tive abundance of herbivores and decreased that of omnivores in a
subtropical farm in Florida, US (Wang et al., 2006) but had no sig-
nificant impact on soil nematode communities in a temperate farm
in northeast China (Liang et al., 2009). Moreover, chronic nitrogen
fertilization significantly increased the abundances of herbivorous
and microbivorous nematodes in a tallgrass prairie in Kansas, US
(Todd, 1996) and significantly reduced the abundance of omnivores
in a grass sward in British Columbia (Forge et al., 2005). In addition,
3-yr application of nitrogen fertilizer increased the abundances of
total nematodes and herbivores and decreased the abundances of
fungivores and carnivores in a pasture in New Zealand
(Sarathchandra et al., 2001). However, long-term applications of
different types and levels of nitrogen fertilization did not signifi-
cantly influence the abundances of total nematodes or trophic
groups in a Kentucky bluegrass (Poa pratensis) turf in Ohio, US
nematodes, bacterivores, fungivores and omnivores increased
slightly (statistically insignificant) but nematode maturity index
values did not change after phosphorus fertilization in pasture soils
in New Zealand. Todd (1996) reported that the effects of phos-
phorus fertilization on soil nematodes were limited. Some other
studies focused on how phosphorus fertilization affects specific
nematode species. For example, Coyne et al. (2004) reports that
phosphorus fertilization decreases the abundance of cyst nematode
(Heterodera sacchari) in soils with both upland and lowland rice in
Ivory Coast of Africa. In contrast, Simon and Rovira (1985) and Price
et al. (1995) report that phosphorus fertilization increased abun-
dances of Heterodera avenae and Heterodera glycines.
In the present study, we test the effects of experimental nitrogen
and/or phosphorus additions on soil nematode communities in
soils of secondary forests in tropical China where soils are nitrogen-
rich but phosphorus-poor and face increasing deposition of atmo-
spheric nitrogen depositions (Lu et al., 2010a; Fang et al., 2011).
Therefore, we expected the additional nitrogen application might
aggravate nitrogen-saturation which might be detrimental to soil
nematode communities. Phosphorus application might improve
the available phosphorus levels which was conducive to soil
nematode communities. We hypothesize that: 1) nitrogen addition
may have negative effects on the abundance, diversity, and/or
community complexity of soil nematodes and 2) phosphorus
addition may have positive effects on these variables of soil
nematodes.
2. Materials and methods
2.1. Study site
This study was conducted at the Xiaoliang Tropical Coastal
Ecosystem Research Station (110 540 E, 21270 N), Chinese Academy
of Sciences (CAS), Guangdong Province, China. The climate is
tropical monsoon with a distinct wet (from April to September) and
dry season (from October to March). The mean annual temperature
is 23  C and the annual precipitation is 1400e1700 mm, respec-
tively. The soil is lateritic, formed from highly weathered granite
(Table 1).
Our experiment was conducted in a secondary mixed forest. The
forest started as Eucalyptus exserta plantation in 1959, then 312
species were introduced between 1964 and 1975 (Ding et al., 1992;
Ren et al., 2007). Now, the most common tree species are: Casta-
nopsis fissa, Cinnamomum camphora, Carallia brachiata, Aphanamixis
polystachya, Ternstroemia pseudoverticillata, Acacia auriculiformis,
Cassia siamea, Albizia procera, Albizia odoratissima, Leucaena leuco-
cephala, Aquilaria sinensis and Chukrasia tabularis.
2.2. Experimental design
A nitrogen and phosphorus addition experiment was designed
as a randomized complete block (n ¼ 5) and established in August
Table 1
Means (standard error, n ¼ 5) of soil physical and chemical properties (0e10 cm
depth) in Control (CK), N fertilization (þN), P fertilization (þP), and NP fertilization
(þNP) plots at the study site at the initiation of the experiment in 2009.
Soil properties Treatments

(Cheng et al., 2008). CK þN þP þNP

Compared with nitrogen, studies focused on the effects of Moisture (%) 25.0  1.3 24.5  1.2 24.1  1.1 25.0  1.4
mineral phosphorus fertilization on soil nematode communities SOC (g/kg) 33.8  0.7 33.8  0.9 32.3  0.7 33.4  0.8
are rare. The effects of phosphorus fertilizer on soil nematode TN (g/kg) 2.60  0.15 2.23  0.21 2.54  0.10 2.57  0.19
community composition and structure are poorly known. TP (g/kg) 0.38  0.04 0.36  0.02 0.40  0.02 0.41  0.03

Sarathchandra et al. (2001) reported that the abundances of total SOC, soil organic carbon; TN, soil total nitrogen; and TP, soil total phosphorus.
 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10
2009. Each block was located in a site more than 50 m apart in the
forest. Within each block, four 10  10 m plots were established and
each plot was surrounded by a 2-m-wide buffer strip in each site.
The treatments nitrogen addition (þN), phosphorus addition (þP),
nitrogen addition with phosphorus addition (þNP), and control
(CK, no addition of mineral nutrients) were assigned randomly to
the four plots within each block. Both nitrogen and phosphorus
were applied at 100 kg ha1 yr1, Briefly, 476.6 g NH4NO3 (equal to
166.6 g N) and/or 808 g Na2HPO4 (equal to 166.6 g P) were dissolved
in 30 L tap water and, then, applied to the corresponding plots near
soil surface using a backpack sprayer in every two months from
2009 to 2011, respectively. Thirty liters of tap water was applied to
control plots in each treatment event. The amount of nitrogen and
phosphorus additions correspond to those studies of experimental
nitrogen (Lu et al., 2010a; Zhao et al., 2013c) and phosphorus (Liu
et al., 2012) additions in neighboring area in Guangdong prov-
ince, PR China, which was lower than the amount of nitrogen and
phosphorus fertilizers that used in tropical rain forest in southwest
Costa Rica (Cleveland and Townsend, 2006).
2.3. Soil sampling and analysis
Soil was sampled in September 2009 before the first mineral
nutrient addition treatment application (background sampling),
September 2010 (after one-year treatment), and September 2011
(after two-year treatment), respectively. Soil cores (5 cm diameter)
were taken at 0e10 cm depth from five randomly selected locations
in each plot in each block. Six cores from the same plot were
combined to form one composite sample. The litter above each
sample area was removed before the cores were collected.
Soil pH was determined in 1:2.5 (w/v) soil:water solutions, and
soil water content (SWC %, g of water per 100 g dry soil) was
measured by oven-drying for 48 h at 105  C. Soil organic C (SOC, g/
kg dried soil) was determined by the loss-on-ignition technique by
ashing in a muffle furnace at 600  C for 6 h, total N (TN, g/kg dried
soil) was measured with an ultraviolet spectrophotometer after
Kjeldahl digestion, total P (TP, g/kg dried soil) was measured with
an ultraviolet spectrophotometer using the procedure of molyb-
denum antimony blue colorimetry, ammonium nitrogen was
determined colorimetrically by the salicylate-nitroprusside
method on a Flow-Injection Autoanalyzer (FIA, Lachat In-
struments, USA), nitrate nitrogen was determined colorimetrically
after cadmium reduction using the same FIA, and available P (mg/
kg dried soil) was extracted with the solution of Bray-1 (0.03 M
NH4Fe0.025 M HCl) and measured colorimetrically (Liu, 1996).
Nematodes were extracted from 50 g of moist soil using the
Baermann funnel method (Barker et al., 1985). After fixation in 4%
formalin solution, nematodes were counted with a DIC microscope
(ECLIPSE 80i, Nikon) (Zhao et al., 2011, 2014a), and the first 100
individuals encountered were identified to trophic group (bacter-
ivores, fungivores, herbivores, omnivores, predators) and func-
tional guild (Bongers, 1990; Yeates et al., 1993a; Bongers and
Bongers, 1998; Okada et al., 2005). The functional guild is defined
by the nematode’s trophic behavior and by its ecological life
strategy as a colonizer or persister (Bongers, 1990; Ferris et al.,
2001). All nematodes were identified to trophic group and func-
tional guild when the sample contained fewer than 100 individuals.
2.4. Data analysis
The diversity of the nematode community was determined by
the ShannoneWiener diversity index (H0 ), the Pielou evenness in-
dex (J), the Margalef richness index (SR), and the Simpson domi-
nance index (l) (Neher and Darby, 2009).
3
X
S
Shannon  Wiener diversity index; H0 ¼  Pi  ln Pi (1)
i¼1
H0
Pielou evenness index; J ¼ (2)
H 0 max
H0 max ¼ ln S (3)
S1
Margalef richness index; SR ¼ (4)
ln N
X
Simpson dominance index; l ¼ Pi2 (5)
in which ‘Pi’ is the proportion of the individuals of “ith” group in the
community, ‘S’ is the total number of nematode genera in the
community, and ‘N’ is the total number of nematodes in the
community.
The nematode data were also used to calculate two maturity
indices (i.e. MI and MI25) (Bongers, 1990). These indices are
calculated by a colonizerepersister (cp) scale that range from a
colonizer (cp value ¼ 1, r-strategy) to a persister (cp value ¼ 5, K-
strategy). Small and large values of these indices represent highly
disturbed and highly stable soil ecosystems, respectively (Bongers,
1990). These indices were calculated as follows:
X
MI ¼ vðiÞcp15  f ðiÞcp15 (6)
X
MI25 ¼ vðiÞcp25  f ðiÞcp25 (7)
Where v(i) is the cp value of free-living taxa i and f(i) is the pro-
portion of that taxa of the total number of free-living nematodes in
a sample; and the superscript ‘cp1e5’ and ‘cp2e5’ indicate the nem-
atodes in cp1e5 and cp2e5 guilds are involved in the calculations,
respectively (Bongers, 1990; Neher, 2001). In addition, the enrich-
ment index (EI) and structure index (SI) values were computed for
each sample, as described by Ferris et al. (2001). The EI value is
calculated from the relative weighted abundance of opportunistic
bacterial- and fungal-feeding nematodes that respond rapidly to
input of food resources; a high EI value indicates an environment
that has been enriched. The SI value is calculated from the relative
weighted abundance of disturbance-sensitive guilds, a high SI value
indicates a complex and stable food web. EI and SI were calculated
as follows:
EI ¼ 100  ðe=ðe þ bÞÞ (8)
SI ¼ 100  ðs=ðs þ bÞÞ (9)
Where ‘b’ is the basal food web component (Ba2, Fu2), ‘e’ is the
enrichment component (Ba1, Fu2) and ‘s’ is the structure compo-
nent (Ba3eBa5, Fu3eFu5, Om3eOm5, Pr2ePr5). Ba, Fu, Om, and Pr
refer to trophic groups (bacterivores, fungivores, omnivores, and
predators, respectively) and the associated subscripts refer to
particular guilds. Moreover, two metabolic footprint indices that,
the enrichment footprint index (EFI) and structure footprint index
(SFI), were computed for each sample, as described by Ferris (2010).
EFI and SFI values indicate the magnitudes of the carbon utilization
(i.e. carbon used for production and respiration) of the enrichment
component (Ba1, Fu2) and structure component (Ba3eBa5, Fu3eFu5,
Om3eOm5, Pr2ePr5) of soil nematode communities, respectively
(Ferris, 2010). The metabolic footprint index for each of the i taxa
were calculated as follows:
4 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10

X   
FI ¼ NðiÞ  0:1  ðWðiÞ=vðiÞÞ þ 0:273  WðiÞ0:75
(10)

Where the N(i) is the number of individuals in each of the i taxon;

W(i) is the body weight of taxon i; and v(i) is the cp value of taxon i.
The i taxa involved in the calculation of EFI and SFI only include the
enrichment component and structure component, respectively. The
values of W(i) are according to Ferris (2010). Additionally, a soil
nematode channel index (CI) (Ferris et al., 2001) was calculated for
each sample to evaluate soil decomposition pathways of soil food
web and a bacterivore index (BaI) (Ferris and Matute, 2003) was
calculated to indicate rates of succession from enrichment-
opportunist (Ba1) to general-opportunist (Ba2) bacterivore nema-
todes. CI and BaI were calculated as follows:

CI ¼ 100  0:8  Fu2 =ð3:2  Ba1 þ 0:8  Fu2 Þ (11)

BaI ¼ 100  0:8  Ba2 =ð3:2  Ba1 þ 0:8  Ba2 Þ (12)

The experiment was set up as a two-factor (22) design. Repeated-

measure COANOVA was employed to determine the effects of two
main factors (N and P) and their interaction (N*P) through the whole
experimental period with the background data (first sampling data)
as co-variables. One-way ANOVA was used to compare effects of the
four treatments (CK, þN, þP and þNP) on soil nematode variables in
each sampling event. Statistical significance was determined at
p < 0.05. ANOVAs were performed using SPSS 16.0 (SPSS Inc., Chi-
cago, IL). The principal response curves (PRC) method was used to
determine the temporal trends of soil nematode community
composition (represented by functional guilds) for each treatment
using CANOCO 4.5 (Ithaca, NY, USA). PRC is based on redundancy
analysis (RDA). The result is a diagram showing the first Principal
Component of the variance explained by treatment on the y-axis
along the sampling periods on the x-axis. The control treatment is
treated as a zero baseline (the horizontal line). The treatment effect
is represented by the deviation of each fluctuating line (i.e. þN, þP
and þNP) from the zero baseline through time. Both environmental
class variables (treatments) and covariables (sampling times) were

coded as nominal 0 or 1 variables (Leps and Smilauer, 2003; Zhao
and Neher, 2013a). Monte Carlo permutation tests were applied to
compute statistical significance (n ¼ 499).

3. Results

Experimental phosphorus addition significantly increased the

soil available phosphorus content (Fig. 1A). There was no significant
interaction effect of phosphorus addition and nitrogen addition on
the content of soil available phosphorus. Experimental nitrogen
addition apparently increased both of the soil ammonium and ni-
trate nitrogen contents during the study (Fig. 1B and C). There was a
significant interaction effect on soil ammonium nitrogen content
(Fig. 1B). Neither phosphorus addition nor nitrogen addition had
significant influence on soil pH (Fig. 1D).
There were altogether 78 nematode genera collected (Table 2).
The most common nematode genera included: Acrobeloides, Pris-
matolaimus, Aphelenchoides, Protorhabditis, Ditylenchus, Dip-
loscapter, Xiphinema, Rhabditonema, Filenchus, Lelenchus, Clarkus,
and Wilsonema. Bacterivores were the most abundant trophic
group followed by fungivores and herbivores (Fig. 2). There were
remarkable negative effects of phosphorus addition on total nem-
atode density (F ¼ 7.602, p ¼ 0.012) (Fig. 2A) and density of
omnivore-predators (F ¼ 4.812, p ¼ 0.040) (Fig. 2E) during the
study. Phosphorus addition tended to decrease the density of fun-
givores (F ¼ 3.288, p ¼ 0.085) (Fig. 2C). Neither nitrogen addition
Fig. 1. Soil available phosphorus (A), ammonium nitrogen (B), nitrate nitrogen (C) and
soil pH (D) under Control (CK), N fertilization (þN), P fertilization (þP), and NP
fertilization (þNP) in each sampling event (2009, 2010 and 2011). Bars indicate stan-
dard errors of means. Non-significant (p > 0.05) main and interaction effects were not
provided.
nor interaction of nitrogen addition and phosphorus addition
affected total nematode density, bacterivore density, fungivore
density, herbivore density, and omnivore-predator density (Fig. 2).
Diversity (mean  S.E.) of soil nematode communities under each
treatment in each sampling event is shown in Table 3. Repeated-
measure COANOVA did not show significant main effect and
interaction effect of nitrogen addition and phosphorus addition on
ShannoneWiener index (H0 ), Pielou evenness index (J), Margalef
richness (SR), and Simpson dominance index (l).
Phosphorus addition suppressed the nematode enrichment in-
dex (EI) (F ¼ 5.711, p ¼ 0.027) (Fig. 3A), the nematode structure
index (SI) (F ¼ 8.413, p ¼ 0.009) (Fig. 3C) and the nematode
structure footprint index (SFI) (F ¼ 8.015, p ¼ 0.010) (Fig. 3D) during
 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10 5

Table 2
Taxonomic and functional group classifications used to characterize the structure of nematode communities in 0e10 cm soil depth in secondary forests in southern China.

Bacterivorous Fungivorous Herbivorous Carnivorous Omnivorous

Genus name cp-value Genus name cp-value Genus name cp-value Genus name cp-value Genus name cp-value

Aspidonema 1 Aphelenchoides 2 Lelenchus 2 Stenonchulus 3 Epidorylaimus 4

Diploscapter 1 Aphelenchus 2 Malenchus 2 Tobrilus 3 Eudorylaimus 4
Diploscapteriodes 1 Aprutides 2 Cacopaurus 3 Tripyla 3 Microdorylaimus 4
Distolabrellus 1 Ditylenchus 2 Criconemella 3 Clarkus 4 Miodorylaimus 4
Mesorhabditis 1 Filenchus 2 Criconemoides 3 Iotonchus 4 Pungentus 4
Panagrolaimus 1 Seinura 2 Helicotylenchus 3 Mononchus 4 Thonus 4
Pelodera 1 Tylenchus 2 Hemicriconemoides 3 Mylonchulus 4 Thornia 4
Protorhabditis 1 Diphtherophora 3 Heterodera 3 Actinolaimoides 5
Rhabditis 1 Dorylaimellus 4 Macroposthonis 3 Aporcelaimellus 5
Rhabditoides 1 Dorylaimoides 4 Paratylenchus 3 Belondira 5
Rhabditonema 1 Tylencholaimus 4 Pratylenchus 3 Belondirella 5
Acrobeloides 2 Rotylenchus 3 Chrysonemoides 5
Cephalobus 2 Longidorus 5 Oxydirus 5
Cervidellus 2 Xiphinema 5 Prodorylaimus 5
Cribronema 2
Drilocephalobus 2
Eucephalobus 2
Fuchsnema 2
Heterocephalobellus 2
Metacrobeles 2
Metacrolobus 2
Osstella 2
Paracrobellus 2
Plectus 2
Tylocephalus 2
Wilsonema 2
Zeldia 2
Aphanolaimus 3
Microlaimus 3
Prismatolaimus 3
Alaimus 4

the study. Phosphorus addition did not significantly affect the
nematode enrichment footprint index (EFI); and there were no
apparent effect of nitrogen addition and interaction effect of ni-
trogen addition and phosphorus addition on EI, SI, EFI and SFI
during the study (Fig. 3). Phosphorus addition suppressed MI25
(F ¼ 7.208, p ¼ 0.014), but had no significant effect on MI (Fig. 4A
and B). There were no apparent effect of nitrogen addition and
interaction effect of nitrogen addition and phosphorus addition on
MI and MI25 (Fig. 4A and B). Moreover, phosphorus addition
increased the nematode channel index (CI) (F ¼ 5.203, p ¼ 0.034)
and the bacterivore index (BaI) (F ¼ 5.476, p ¼ 0.030) (Fig. 4C and
D). There were no apparent effect of nitrogen addition and inter-
action effect of nitrogen addition and phosphorus addition on
either CI or BaI (Fig. 4C and D).
Although the background soil nematode community composi-
tion within these plots were somewhat different from each other,
the temporal dynamics of soil nematode community compositions
within þN, þP and þNP plots were similar (Fig. 5). Specifically, the
effect of phosphorus addition was slightly more powerful on soil
nematode community composition than the effect of nitrogen
addition, which was revealed by that the variation of PRC of þP
treatment was larger compared with the variation of PRC of þN
treatment (Fig. 5). In addition, the nematode community compo-
sition showed apparent successions from communities dominated
by He3 and Ba1 to communities dominated by Ba2 and Fu2 after
nitrogen and phosphorus additions (Fig. 5).
4. Discussion
4.1. Effects of nitrogen addition on soil nematode communities
The nematode community, in terms of trophic group composi-
tion, calculated indices of community diversity and structure, was
not sensitive to the nitrogen addition in this study. Previous studies
reported that experimental nitrogen addition, when exceeding
80 kg N ha1 yr1, suppressed soil nematode communities in both
alpine tundra soils in Wyoming, USA (Lokupitiya et al., 2000) and in
grassland soils in Inner Mongolia, China (Wei et al., 2012; Li et al.,
2013), which in general were considered as nitrogen-poor ecosys-
tems. However, little is known about how experimental nitrogen
addition affects soil nematode communities in tropical forests
which are relatively nitrogen-rich. Many previous studies reported
that soil nematode trophic groups responded differently to nitro-
gen fertilizations. Consistent with the present study, there was a
study that reported nitrogen addition had no effects on total
nematode or each trophic group densities in turf soils in Ohio, US
(Cheng et al., 2008). In addition, many previous studies reported
that nitrogen addition did not affect one or more indices
(Sarathchandra et al., 2001; Forge et al., 2005; Wang et al., 2006;
Cheng et al., 2008; Liang et al., 2009).
Although ANOVAs did not detect significant effects of nitrogen
addition on the trophic composition and faunal indices of soil
nematode community structure; nematode functional guild
composition, which revealed by multivariate analysis (PRC), was
significantly altered after two-year nitrogen addition compared
with control treatment. During the study, the importance of Ba1
and He3, as well as the importance of Ba3, Ca4, Fu3, Fu4, reduced
gradually; however, the importance of Ba2 and Fu2, as well as the
importance of Om4, increased gradually. These results indicated
nematode genera in the same trophic groups (e.g., bacterivore and
fungivore) or similar cp-guilds (e.g., cp3e5) responded idiosyn-
cratic to nitrogen addition. Therefore, the idiosyncratic responses of
different genera in a soil nematode trophic group and/or cp-guild
might lead to that the nematode trophic group composition and
community indices were not affected by the nitrogen addition. In
addition, Ba2 and Fu2 are basal components of soil nematode
6 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10

Diversity (mean  S.E.) of soil nematode communities under Control (CK), N fertilization (þN), P fertilization (þP), and NP fertilization (þNP) in each sampling event (2009, 2010 and 2011). H0 , J, SR, and l stand for Shannone

0.15
0.03
0.29
0.03




þNP

1.97
0.78
2.81
0.21
0.07
0.02
0.15
0.01




2.42
0.85
3.69
0.11
þP

0.16
0.04
0.25
0.04




2.23
0.79
3.12
0.18
þN

0.15
0.04
0.19
0.04
Sep, 2011





2.14
0.76
3.11
0.20
CK

0.05
0.01
0.18
0.01




þNP

2.37
0.83
3.50
0.14
0.14
0.02
0.35
0.02




2.37
0.86
3.07
0.13
þP

0.07
0.02
0.11
0.02




2.44
0.82
3.38
0.13
þN
Wiener diversity index, Pielou evenness index, Margalef richness index and Simpson dominance index.

0.10
0.02
0.39
0.01
Sep, 2010





2.59
0.85
3.98
0.11
CK

0.08
0.02
0.12
0.01




þNP

2.53
0.86
3.83
0.10
0.09
0.02
0.19
0.02




2.43
0.83
3.31
0.12
þP

Fig. 2. Densities of total nematodes (A), bacterivores (B), fungivores (C), herbivores
(D), and omnivore-predators (E) under Control (CK), N fertilization (þN), P fertil-
0.07
0.03
0.16
0.01

ization (þP), and NP fertilization (þNP) in each sampling event (2009, 2010 and
2011). Bars indicate standard errors of means. Non-significant (p > 0.05) main and




2.37
0.86
3.02
0.12

interaction effects were not provided.

þN

0.09
0.02
0.13
0.02

communities (Ferris et al., 2001). The increased weights of Ba2 and

Sep, 2009

Fu2 might demonstrate that the succession of the soil food web




2.39
0.86
3.03
0.13

shifted towards a relatively simple system (a basal condition)

CK

under nitrogen addition treatments.

Nitrogen addition usually can facilitate abundance and com-
Indices

munity complexity (as indicated SI) of soil nematodes directly or

Table 3

indirectly by bottom-up control via plant growth or microbial

SR
H0

l
J

activity (Yeates et al., 1993b; Vestergård, 2004; Liang et al., 2009).

 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10 7

Fig. 3. Enrichment index (A), enrichment footprint index (B), structure index (C), and structure footprint index (D) under Control (CK), N fertilization (þN), P fertilization (þP), and
NP fertilization (þNP) in each sampling event (2009, 2010 and 2011). Bars indicate standard errors of means. Non-significant (p > 0.05) main and interaction effects were not
provided.

Fig. 4. The nematode Maturity indices (A and B), Channel index (C) and Bacterivorous index (D) under Control (CK), N fertilization (þN), P fertilization (þP), and NP fertilization
(þNP) in each sampling event (2009, 2010 and 2011). MI and MI25 are the maturity indices of all free-living nematodes and of cp2e5 free-living nematodes, respectively. Bars
indicate standard errors of means. Non-significant (p > 0.05) main and interaction effects were not provided.

Additionally, nitrogen addition commonly reduces soil pH, and
causes ammonium and aluminum toxicity or introduces sufficient
salt to harm soil biota (Russell et al., 2006; Liang et al., 2009; Wei
et al., 2012). The trade-off between positive and negative effects
determined the responses of soil nematodes to nitrogen application
(Zhao et al., 2014b). In the current study, experimental nitrogen
addition significantly enhanced the contents of soil ammonium and
nitrate nitrogen, which potentially had negative impacts on soil
nematodes. In addition, studies in neighboring area of the present
study have demonstrated that the diversity or cover of the under-
story vegetation significantly reduced and the aluminum mobility
changed in nitrogen addition plots than that in control plots in both
secondary and old-growth tropical forests which are nitrogen-
saturated (Lu et al., 2010a, 2010b). Therefore, the negative
changes of both plant diversity or cover and soil properties after
nitrogen addition might result in shifts in soil nematode
communities.
4.2. Effects of phosphorus addition on soil nematode communities
Studies focused on the effects of mineral P addition on soil
nematode communities are rare. The effects of phosphorus
8 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10
Fig. 5. Principal response curves (PRC) with weights of density of each soil nematode
functional guild under Control (CK), N fertilization (þN), P fertilization (þP), and NP
fertilization (þNP) in each sampling event (2009, 2010 and 2011). The horizontal axis
represents the control treatment. Soil nematode functional guild designation is the
composite of trophic group and cp value: Ba, bacterivore; Fu, fungivore; Ca, carnivore;
Om, omnivore; He, herbivore.
addition on soil nematode community composition and structure
are not clear. In the present study, phosphorus addition did not
significantly affect MI. Similarly, Sarathchandra et al. (2001) found
that the MI was not significantly affected by phosphorus addition in
pasture soils in New Zealand. However, phosphorus addition
significantly reduced MI25 in the current study. The main differ-
ence between those two maturity indices is that cp1 (or Ba1) free-
living nematodes are involved in the calculation of MI rather than
MI25. Therefore, the alterations of Ba1 nematode abundance might
result in the idiosyncratic responses of MI and MI25 to phosphorus
addition during the study. In addition, phosphorus addition
significantly reduced omnivore-predator density (K-selected) and
SI in our study, which indicated the soil food web had fewer trophic
links after phosphorus addition. Fewer links or connectances might
lead to decline in functional resilience of food webs to disturbance
(Wardle et al., 1995; Ferris et al., 2001). In the present study, the
relative weight of fungal mediated decomposition pathway was
significantly increased after phosphorus addition, which indicated
by the significant increase in nematode channel index (CI) (Fig. 4C)
and the abundance of Fu2 guild (Fig. 5) after phosphorus applica-
tions. This finding was consistent with the results of a previous
study that evaluated soil decomposition pathways using soil mi-
crobial communities in secondary subtropical mix-forests in
southern China near to the present sites (Liu et al., 2012). Addi-
tionally, phosphorus addition induced remarkable reduction of the
carbon utilization of structure component of soil nematodes, which
is indicated by the significant increase in nematode structure
footprint index (SFI) (Fig. 3D) after phosphorus applications.
Therefore, the alterations of soil food web complexity, soil
decomposition pathways, and carbon utilization of soil organisms
after phosphorus applications may result in the changes of
ecosystem functioning and processes.
In contrast to our hypothesis, we found negative effects of
phosphorus addition on soil nematode community. The most likely
reason is that phosphorus fertilization improves the soil available
phosphorus condition and results in the decrease of resources input
from plants to soil organisms in the short-term. Plants efficiently
and economically allocate resource to nutrient acquisition
(Treseder and Vitousek, 2001). When ecosystem is phosphorus-
poor, plants allocate substantive resources (include extracellular
phosphatase) to soil organisms that decompose litter and release
phosphorus to soil which can be used by plants. In conditions of
phosphorus addition, plants can directly obtain enough available
phosphorus from soil; thus, plants will reduce the resources input
to soil organisms. In fact, experimental phosphorus addition
significantly increased the soil available phosphorus content during
the study. Therefore, the decrease of soil nematode density might
be due to the resource/food limitation after phosphorus addition.
Phosphorus fertilization reduced the water soluble organic carbon
in a low land tropical rain forest in Costa Rica (Cleveland and
Townsend, 2006) and reduced the phosphatase activity and
mycorrhizal colonization in Hawaii rain forests (Treseder and
Vitousek, 2001), which might support this view. Phosphorus
addition also inhibited the mycorrhizal symbiosis in a tallgrass
prairie in Kansas (Bentivenga and Hetrick, 1992) and suppressed
the mycorrhizal formation of two poplar clones in South Germany
(Baum and Makeschin, 2000). Consistent with these results, we
found phosphorus addition tended to reduce the density of fun-
givorous nematodes in this study. Another likely reason is that the
additional mineral phosphorus input to soil leads to salt toxicity
that harms soil nematodes. In the present study, the amount of
applied phosphorus is 100 kg ha1 yr1, which might be sufficient
to introduce salt toxicity to soil nematodes. And as noted above, the
content of soil available phosphorus was indeed increased after
phosphorus application. In addition, phosphorus addition in proper
levels can improve plant growth, however, excess phosphorus
addition has no further positive effects on plant growth. For
example, Sarathchandra et al. (2001) reported that dry matter
production significantly increased from 0 to 50 kg ha1 yr1 P rate,
but no further increases were observed at 100 kg ha1 yr1 P rate.
Therefore, the negative effect of phosphorus addition might be
more powerful than its positive effect on the abundance and
community complexity of soil nematodes, which would have
resulted in a net negative effect (Zhao et al., 2014b).
4.3. Interaction effects of nitrogen and phosphorus addition on soil
nematode communities
Nitrogen and phosphorus are the most common elements in
limiting primary productivity in most terrestrial ecosystems. In
agricultural practices and experimental studies, nitrogen and
phosphorus fertilizers (chemical fertilizer usually with other
nutrient elements) are usually used together. In the present study,
although no interaction effect of nitrogen and phosphorus additions
on the monitored variables of nematode community was observed,
the þNP treatment tend to have the most powerful effects on all
tested variables among the four treatments. In addition, PRC showed
a tendency for the effect of þN and þP combined to be greater than
the effect of þP or þN alone. Therefore, the effect of þNP treatment
on soil nematode community might be a superposition effect of þN
treatment and þP treatment. Unfortunately, we did not find any
studies that quantitatively compared the effects between NP addi-
tion and N or P addition on soil nematodes in field. Previous studies
reported that nematode abundances were higher under N applica-
tion than under NP application in the rhizospheres of barley plants
in pot experiments (Vestergård et al., 2004; Bjørnlund et al., 2006),
which was inconsistent with our finding. However, the responses of
soil nematode abundance to nutrient applications were mediated by
bottomeup control through resource (or food) quality and/or
quantity changes in those pot experiments (Bjørnlund et al., 2006).
Furthermore, previous field studies focused on the effects of NP
addition on soil nematode communities have revealed conflicting
results. For examples, NP addition had no effects on nematode
 J. Zhao et al. / Soil Biology & Biochemistry 75 (2014) 1e10
trophic groups in grassland (Todd, 1996; Forge et al., 2005); how-
ever, NP addition significantly reduced the herbivorous nematode
density in the soybean phase of a soybeanewheatecorn rotation in
northeast China (Pan et al., 2010). In addition, chemical fertilizers (N,
P and K) significantly increased total nematode density and bac-
terivorous nematode density of 2-year study in a Japanese soybean
field (Okada and Harada, 2007), but decreased total nematode
density in a 20e25 year old stand of Scots pine located in central
Sweden (Sohlenius and Wasilewska, 1984). Moreover, chemical
fertilizers (N, P and S) did not influenced the total nematode density,
bacterivore density, fungivore density and omnivore density, but
significantly reduced predator density in mature forests of British
Columbia (Forge and Simard, 2001).
5. Conclusion
In summary, our results suggest that experimental nitrogen and/
or phosphorus additions significantly affect soil food web and soil
decomposition pathways in tropical secondary forests. Particularly,
the effects of nitrogen and/or phosphorus additions on soil nema-
tode communities were negative and the effects of phosphorus
addition are more powerful than the effects of nitrogen addition in
relatively nitrogen-rich but phosphorus-poor ecosystems. Nitrogen
and/or phosphorus additions degrade the structure of and trophic
links within the soil food web, reduced the carbon utilization of soil
nematodes, and made the fungal mediated decomposition pathway
more important, which may affect other forest ecosystem processes
and functions (e.g., carbon sequestration, nutrient mineralization,
ecosystem succession, and NEP). In addition, our findings could
provide a better understanding of the responses of soil food web to
nitrogen and phosphorus additions in nitrogen-rich but
phosphorus-poor soils.
Acknowledgments
We thank Deborah A. Neher for her help in preparing the
manuscript. This study was conducted at Xiaoliang Tropical Coastal
Ecosystem Research Station, Chinese Academy of Sciences (XLRS).
This work was funded by National Basic Research Program of China
(2011CB403200), NSFC-Guangdong Joint Project (U1131001), and
National Natural Science Foundation of China programs given to Jie
Zhao (31300448) and Faming Wang (31300419), respectively.
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