Soil Biology & Biochemistry 88 (2015) 362e371
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Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Soil nematode assemblages as bioindicators of primary succession
along a 120-year-old chronosequence on the Hailuogou Glacier
forefield, SW China
Yanbao Lei a, c, Jun Zhou a, Haifeng Xiao d, Baoli Duan a, Yanhong Wu a,
Helena Korpelainen e, Chunyang Li b, *
a
Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences,
Chengdu 610041, China
b
The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Linan 311300, Zhejiang, China
c
Department of Environmental Science on Biosphere, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 1838509, Japan
d
Key Laboratory of Tropical Forest Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla 666303, China
e
Department of Agricultural Sciences, P.O. Box 27, FI-00014 University of Helsinki, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 December 2014
Received in revised form
14 February 2015
Accepted 10 June 2015
Available online 24 June 2015
Successional dynamics in terrestrial ecosystems is important for interactions between aboveground and
belowground subsystems. In this study, nematode communities in a Hailuogou Glacier Chronosequence
from seven stages were investigated to determine whether changes in soil phosphorus (P) and nematode
assemblages parallel those observed in aboveground communities, and whether the primary succession
in this chronosequence has entered a retrogressive phase after 120 years of succession. The initial 40year succession, including stages 2, 3 and 4, can be viewed as a build-up phase. Especially at stage 3,
vegetation succession from grassland to forest accelerated the accumulation of plant litter and
bioavailable P, paralleled with a sharp increase in nematode abundance. The mature phases covering
stages 5, 6 and 7 displayed most balanced nematode communities, in which abundance, taxon richness,
maturity index and structure index were at highest. However, the last stage 7 appeared to show some
retrogressive characteristics, as suggested by the reduced bioavailability of P and a significant decrease in
nematode densities, along with the disappearance of some rare genera of nematodes from higher trophic
guilds, resulting in decreases in the nematode channel ratio, plant parasite index and enrichment index.
Thus, the Hailuogou Glacier Chronosequence may enter its retrogressive phase during the next decade or
century. A bacterial-based nematode energy channel dominated the chronosequence during the development; by contrast, a fungivore-based channel was activated at the early and late stages, because
fungivores are better adapted to nutrient-poor environments. Our results demonstrated that different
nematode guilds have contrasting responses to chronosequence stages, possibly due to their different
responses to bottom-up and top-down controls. Furthermore, soil nematode communities could be used
as sensitive bioindicators of soil health in glacial-retreat areas.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Bioavailable phosphorus
Hailuogou Glacier Chronosequence
Nematode assemblages
Nematode ecological indices
Plant succession
Retrogression
1. Introduction
Elucidating the driving factors of successional dynamics in
terrestrial ecosystems is an important issue in ecology, as such
knowledge is expected to offer fundamental clues to understanding
some basic questions about nutrient limitation that may develop
* Corresponding author. Tel: þ86 571 63839132; fax: þ86 571 63740809.
E-mail address: licy@zafu.edu.cn (C. Li).
http://dx.doi.org/10.1016/j.soilbio.2015.06.013
0038-0717/© 2015 Elsevier Ltd. All rights reserved.
during ecosystem succession: what is the rate of limitation over
time and how do species and ecosystems respond to nutrient
limitation over successional time (De Deyn et al., 2003)? In some
chronosequences, the simultaneous availability of successional
stages provides necessary conditions to use a “space-for-time
substitution” approach as an alternative for long-term studies on
plant and soil biota succession (Pickett, 1989). Despite some
methodological shortcomings, this approach is often considered
useful for determining long-term successional changes (Walker
et al., 2010). The Hailuogou Glacier Chronosequence, located on the
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
south-eastern fringe of the Tibetan Plateau, provides an excellent
place to study the relationship between vegetation succession and
soil development (Luo et al., 2012; Zhou et al., 2013; Prietzel et al.,
2013a, 2013b). Spatial differences in the lithology of parent rocks,
topography, soil-water conditions and climate can be ignored
because of small gradients in length (2 km), width (50e200 m) and
elevation (127 m from 2855 to 2982 m). Along the approximately
2 km-long belt, a series of sites representing different stages of
vegetation succession from pioneer to climax vegetation communities can be readily recognized (Luo et al., 2012). The relatively
mild and humid climate on this site allows rapid moraine colonization by plants, causes the accumulation of organic material, and
promotes relatively rapid soil development (Alexander and Burt,
1996; Luo et al., 2012). Several studies have investigated how
communities develop during primary succession, including pedogenesis (He and Tang, 2008), soil respiration (Luo et al., 2012), plant
succession simulation (Yang et al., 2014), soil microbial changes
(Zhang et al., 2010), and the availability and transformation of
essential nutrients, particularly phosphorus and sulfur speciation
(Zhou et al., 2013; Prietzel et al., 2013a, b). However, successional
studies have rarely investigated the responses of soil fauna communities to different development stages of ecosystems, and
whether these changes parallel those observed in aboveground
communities.
Interactions between aboveground and belowground subsystems contribute to ecosystem functioning (De Deyn and van der
Putten, 2005). Soil nematode communities are useful biological
indicators of soil health, because they form a dominant group of soil
organisms and live in various types of soil, including chemically
contaminated soil (Yeates, 2003). These communities also represent key links in soil food webs, such as herbivores, bacterivores,
fungivores, omnivores and predators, and their trophic structures
are closely correlated with soil ecosystem processes (Yeates, 2003).
Furthermore, nematodes have short generation times and are
sensitive to environmental changes; thus, they can provide
adequate resolution required to detect changes in soil communities
(Bonger and Ferris, 1999; Williamson et al., 2005). The quality and
quantity of organic matter entering soil food webs possibly alter
nematode communities (Ugarte et al., 2013; Zhao et al., 2014).
Thereby, low ratios of C:N and soluble phenolics:N in soil are often
found in forb communities (Eskelinen et al., 2009). By contrast, high
ratios are generally observed in shrub land and tree communities.
Changes in those ratios may markedly affect soil biota communities, leading to a shift between bacterial and fungal dominated
assemblages. There is evidence of more efficient adaptation of
fungal-based channels when compared to bacterial-based channels
in resource-poor conditions (Sun et al., 2013; Carrascosa et al.,
2014). Meanwhile, microbe-feeders are strongly regulated by the
top-down control of predators (both top predators and omnivores,
or predators that feed on more than one trophic level) (De Mesel
et al., 2004). Thus, different trophic groups of nematodes may
show contrasting responses to chronosequence stages, as their
relative responses to bottom-up and top-down controls differ. Soil
organisms are also supposed to play significant roles in nutrient
cycling, which may affect the productivity and competition of plant
assemblages, with potential effects on vegetation trajectories
(Bradford et al., 2002; van der Heijden et al., 2008). However, few
studies have considered a single conceptual framework relating
both above- and below ground linkages to plant succession.
Furthermore, after a long-term absence of catastrophic disturbances, the maximal plant biomass phase is often followed by a
phase of ecosystem decline or a period of ‘ecosystem retrogression’,
during which a long-term reduction in plant biomass and
ecosystem process rates occurs (Vitousek et al., 2010). Such decline
phases have seldom been compared with build-up phases. For this
363
reason, the former remains poorly understood. It is currently
believed that this decline is associated with diminishing plant resources, particularly as for the availability of P, as chronosequences
develop (Richardson et al., 2004). However, previous studies on the
Hailuogou Glacier Chronosequence estimated that biomass accumulation on the oldest sites gradually continues to increase (Luo
et al., 2012). Zhou et al. (2013) also found that annual P and nitrogen (N) requirements for plant growth are smaller than the
bioavailable stocks of P and N, thus demonstrating that the
bioavailable P and N pool of soil presently meets the requirements
for plant-growth at the 120-year-old site. However, rapid carbon
(C) and N accumulation on the Hailuogou Glacier (He and Tang,
2008), as detected also in many other young chronosequences,
and the great increases in soil C:P and N:P ratios in the A horizon at
later stages (Zhou et al., 2013) show that future plants might be
limited by bioavailable P as C and N deposition. Thus, nematode
assemblages, which are vulnerable to environmental changes,
together with nutrient dynamics can be used to predict, whether
the soil ecosystem in the Hailuogou Glacier Chronosequence has
entered retrogressive stages.
In the present study, soil properties and nematode communities
were examined in the well-characterized Hailuogou Glacier Chronosequence after 120 years of succession to test the following hypotheses: (1) based on sensitive nematode assemblages and soil P
availability, the Hailuogou Glacier Chronosequence has entered a
decline phase, and the chronosequence can be divided into distinct
build-up and retrogressive stages; (2) soil nematode communities
follow the changes in soil fertility and vegetation biomass at
different stages of primary succession, thereby highlighting the
importance of resource availability; (3) different trophic groups
display contrasting responses along the chronosequence, because
these groups are affected, to varying degrees, by bottom-up control
relative to top-down control and by abiotic factors. Our research
has implications for integrated studies on biogeochemical impacts
of vegetation changes and belowground communities. Further, our
results will contribute to improved predictions of the direction and
intensity of primary succession, and also to improved management
practices related to nutrient limitation during long-term soil
development.
2. Materials and methods
2.1. Study area
The Gongga Mountain (29 300 to 30 200 N, 101300 to 102150 E,
7556 m a.s.l), located on the south-eastern fringe of the Tibetan
Plateau, is the highest peak in the eastern part of the Tibetan
Plateau and the Hengduan Mountain. The Gongga Mountain lies in
the transition zone of the Tibetan Plateau Frigid Zone and the
Warm-humid Subtropic Monsoon Zone (Fig. 1). The Hailuogou
Glacier, which flows down to the eastern slope of the Gongga
Mountain, is the longest monsoonal temperate glacier in the
Hengduan Mountain Region (Li et al., 2010). The mean annual air
temperature is 3.8 C, with minimum and maximum means
of 4.3 C in January and 11.9 C in July, respectively. The total
annual precipitation is approximately 2000 mm, rainfall mainly
occurring from June to September. The observed recession of the
Hailuogou Glacier began in about A.D. 1823, and has accelerated
markedly since the early 20th century. This study was conducted on
seven sites undergoing long-term primary succession stages from
pioneer communities to climax vegetation communities (Fig. 1).
C Stage 1 spans the first 3 years after the glacial retreat. This
stage is characterized by coarse gravelly sand and bare soil
with some mosses covering 1e5% of surface area.
364
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
Fig. 1. Location of the Hailuogou Glacier Chronosequence and the seven sampling sites (modified from Zhou et al., 2013).
C Stage 2 spans from 3 (not included) to 12 years of the glacial
retreat. This stage is characterized by gray sand with sparse
nitrogen-fixing Astragalus adsurgens and Hippophae rhamnoides Rehder.
C Stage 3 spans from 12 (not included) to 30 years. This stage is
characterized by shrubs dominated by H. rhamnoides, Salix
spp. and Populus purdomii with a high population density of
1340 trees ha1.
C Stage 4 spans from 30 (not included) to 40 years. This stage is
characterized by the dominance of P. purdomii leading to
species competition; consequently, a large proportion of
Salix spp. and H. rhamnoides disappear from the community.
Standing plant biomass was approximately 22.25 kg m2.
C Stage 5 spans from 40 (not included) to 50 years. Betula utilis,
Abies fabri and Picea brachytula constitute a diverse community with a population density of 230 trees ha1 and
biomass of 26.05 kg m2.
C Stage 6 spans from 50 (not included) to 80 years. P. purdomii
is replaced by A. fabri and P. brachytula with a population
density of 280 trees ha1 and biomass of 36.21 kg m2.
C Stage 7 spans from 80 (not included) to 120 years. The climax
community is dominated by P. brachytyla and A. fabri with a
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
population density of 230 trees ha1 and biomass of
39.25 kg m2.
2.2. Sampling design
At each chronosequence stage, three 5 5 m square plots were
established with a distance of 10 m between plots (except stages 1
and 2 with 2 2 m square plots and a 3-m distance between plots).
Five circular frames with a diameter of 20 cm were then placed on
each plot; one frame in the middle and one at each of the four
corners. Plant litter was collected from each frame and combined.
After collecting the litter in August 2014, soil samples were taken
from 0- to 15-cm depth with a soil corer of 5-cm diameter, and the
samples were combined as one replicate of approximately 1000 g.
Each replicate of soil was passed through a 2-mm sieve and the
roots were separated. Since some plant feeding nematodes live
inside roots, the collected roots were also used to extract nematodes together with the soil samples. The collected soil was divided
into three parts and the material was used for (1) physicochemical
properties analyses, (2) microbial analysis (stored at 4 C), and (3)
nematode community analyses.
2.3. Physicobiochemical analysis of litter and soil
All litter samples were cleaned and oven-dried at 60 C for 72 h
before the final dry weight was recorded. Litter C and N concentrations were determined using a Vario MAX CN element analyzer
(Elementar Analysensysteme GmbH, Hanau, Germany). Soil organic
carbon (SOC) was determined by wet combustion, as described by
Nelson and Sommers (1982), and soil total N was measured by the
semimicro-Kjedahl method (Bremner, 1996). Soil pH was determined with a potentiometer pH meter, and soil bulk density was
quantified using a previously modified method (Maynard and
Curran, 2006). Soil microbial biomass carbon (MBC) was determined using a fumigation extraction method (Brookes et al., 1985).
Concentrations of PO4 3 P in all extracts were determined
applying previously described methods (Murphy and Riley, 1962)
using a UV-VIS spectrophotometer (Shimadzu UV2450) at 710 nm,
as described in detail by Zhou et al. (2013).
2.4. Isolation and identification of nematodes
Nematodes were extracted from 100 g of soil samples obtained
from each plot using a modified cotton-wool filter method
(Townshend, 1963; McSorley and Frederick, 2004). Briefly, each
sample was suspended in 2000 ml aerated water and stirred gently.
Then, the supernatant was passed through a 400 mesh (38-mm
aperture) sieve. Material remaining on the sieve was suspended on
two layers of tissue paper on a 1-mm mesh window screen
mounted on a PVC support frame in an incubation chamber, consisting of a lidded plastic sandwich box of 13.5 13.5 4 cm.
Samples were incubated at 23 C for 96 h, after which the support
frame was removed and extracted nematodes were collected
(McSorley and Frederick, 2004). For light microscopic observations,
the specimens were collected in a small drop of water in an embryo
dish. Formaldehyde (4% with 1% glycerol) was heated to 70 C and
added to fix and kill the nematodes. The fixed nematodes were
processed in anhydrous glycerol following the glycerol-ethanol
method and mounted on microscope slides. At least (when available) 150 nematodes from each sample were counted and identified to the genus level using an inverted compound microscope
ne
l (1998) and Yin et al. (1998). Nematodes were
according to Ha
grouped according to the feeding habit: (1) bacterial-feeding
nematodes (Ba), (2) fungal-feeding nematodes (Fu), (3) plant-
365
feeding nematodes (Pl), and (4) omnivores and predators
(Om&Pr). In addition, nematodes were assigned to five colonizerpersister groups, from microbial feeders with short life cycles and
high fecundity to omnivores and predators with long life cycles and
greater sensitivity to perturbation (Yeates et al., 1993).
Moreover, several ecological indices were calculated to assess
nematode community diversity and structure. These indices
P
included Shannon-Weaver diversity index H0 ¼ pi (ln pi), where
pi is the proportion of individuals in the ith taxon (Yeates and
Bongers, 1999); nematode channel ratio (NCR) ¼ Ba/(Ba þ Fu),
where Ba and Fu are the relative contributions of bacterial-feeding
and fungal-feeding nematodes to the total nematode abundance,
respectively (Yeates, 2003); plant parasite index (PPI), which was
determined in a similar manner for plant-parasitic genera; matuP
rity index (MI) ¼
(vi)*(fi), where (vi) is the cp value of taxon i
according to their r and K characteristics, and (fi) is the frequency of
P
taxon i in a sample; enrichment index (EI) ¼ 100 * ( kene/
P
P
( kene þ kbnb)), where kb is the weight assigned to guilds Ba2
and Fu2, and nb represents the abundance of nematodes in guilds
Ba2 and Fu2, which indicate basal characteristics of the food-web, ke
is the weight assigned to guilds Ba1 and Fu2, and ne is the abundance of nematodes in these guilds (indicating an enriched conP
dition of the food-web); structure index (SI) ¼ 100 * ( ksns/
P
P
( ksns þ
kbnb)), where ks is the weight assigned to guilds
Ba3eBa5, Fu3eFu5, Om4eOm5 and Pr2ePr5, and ns is the abundance
of nematodes in these guilds, representing the structural condition
of a food-web (Ferris et al., 2001).
2.5. Data analysis
All response variables, except those for nematode functional
guilds, were subjected to one-way analysis of variance (ANOVA) to
determine the overall effects of chronosequence stages using SPSS
13.0. Significant differences among means were evaluated by
Tukey's honestly significant difference (HSD) at p < 0.05. The
observed environmental factors were used to construct soil property and nematode diversity matrices for redundancy discriminatory analysis (RDA) in the vegan package (Oksanen et al., 2010) of
the R project (v. 1.17e3, R Development Core Team., 2010).
Detrended correspondence analysis indicated that axis lengths
were less than 3, thus RDA was an appropriate method to analyze
the relationships between nematode communities and environmental factors. Linear regression was performed to analyze relationships between nematode abundance and pH, soil density,
plant litter, SOC, and bioavailable and microbial P levels.
3. Results
3.1. Variation in physicochemical properties of soil
All physicochemical properties examined responded significantly to chronosequence stages (Table 1). Soil pH displayed a
gradual decrease as a function of time, from 7.13 (stage 1) to 4.73
(stage 7). Soil density decreased during the early stages. The lowest
soil density was attained at stage 5, while it substantially increased
during the two latter stages. Total N and SOC continuously accumulated from stage 1 to stage 7. Litter quantity increased from 0 to
70 g m2 y1 by stage 3 and it maintained a steady level of
approximately 55 g m2 y1 throughout the following four stages
(Table 1). Litter C/N increased significantly during the three early
stages, stabilized during stages 4 and 5 and increased significantly
during the last two stages. Bioavailable P maintained a relatively
low level during the first two stages and then sharply increased to
its highest value at stage 3. Bioavailable P was stabilized during
stages 4e6 but its amount decreased significantly during the last
366
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
Table 1
Physiochemical characteristics of soil and litter at different stages of the Hailuogou Glacier Chronosequence.
Stand age (years)
pH
Soil density (g cm3)
Total N (g kg1)
SOC (g kg1)
Plant litter (g m2 y1)
Litter C/N
Bio-P (mg kg1)
Mb-P (mg kg1)
Stage 1
Stage 2
Stage 3
Stage 4
Stage 5
Stage 6
Stage 7
3
7.13 ± 0.35a
1.73 ± 0.22a
0.04 ± 0.01e
0.10 ± 0.05d
0 ± 0d
0 ± 0d
4.32 ± 0.65d
4.40 ± 1.22e
12
6.50
1.64
0.19
1.49
20.3
19.5
6.59
44.5
30
6.23 ± 0.12bc
1.57 ± 0.20b
0.25 ± 0.07c
1.59 ± 0.16c
70.4 ± 4.71a
31.1 ± 4.58b
102.6 ± 9.95a
119.7 ± 14.2c
40
5.97 ± 0.15bc
1.32 ± 0.13c
0.28 ± 0.04c
2.30 ± 0.18b
53.2 ± 2.75b
31.9 ± 3.37b
46.15 ± 9.51c
133.1 ± 21.5c
52
5.40 ± 0.20c
1.04 ± 0.15d
0.31 ± 0.08c
2.11 ± 0.11b
56.0 ± 4.45b
35.7 ± 5.98b
69.4 ± 6.21b
227.7 ± 17.8a
80
5.13 ± 0.15cd
1.13 ± 0.17c
0.46 ± 0.08b
2.49 ± 0.18b
56.8 ± 4.16b
62.3.8 ± 9.09a
78.1 ± 7.57b
255.5 ± 25.4a
120
4.73 ± 0.40d
1.17 ± 0.23c
0.54 ± 0.09a
3.50 ± 0.34a
53.0 ± 4.76b
65.3 ± 4.13a
37.7 ± 6.51c
194.3 ± 18.2b
±
±
±
±
±
±
±
±
0.20b
0.14b
0.04d
0.03c
4.32c
1.41c
1.34d
10.2d
SOC: soil organic carbon; Bio-P: bioavailable P; Mb-P: microbial P. Means ± SE (n ¼ 3). Different letters indicate significant differences (p < 0.05) among seven successional
stages according to Tukey's HSD for one-way ANOVA. Bio-P and Mb-P have been published in Zhou et al. (2013).
stage. Microbial P levels increased significantly during the early
stages but fluctuated substantially throughout the later three
stages. The amount of microbial P was lower than the amount of
bioavailable P at other stages except for stage 7 (Table 1).
3.2. Nematode community assemblages
During early successional stages, nematode abundance
increased gradually and significantly to its highest value at stage 5,
almost 11.3 times greater than the value at stage 1, while the
abundance decreased substantially during the last two stages
(Table 2). A total of 42 nematode genera were observed in the
Hailuogou Glacier Chronosequence. Altogether, 10, 22, 26, 25, 39, 39
and 34 genera were detected at stages 1e7, respectively. Among
them, Rhabditis, Cephalobus and Aphelenchoides were the dominant
genera for the first two stages, while Alaimus dominated the last
three stages (>10%). At the middle stages 3 and 4, Cephalobus,
Plectus, Aphelenchoides, Tylenchus, Mononchus and Alaimus were the
subdominant genera (5e10%, Table 2). Several genera were detected only at the intermediate stages, including Malenchus, Eudorylaimus and Aporcelaimellus (stages 3e6); Paraphanolaimus (stages
5 and 6); Dolichodorus (stages 4e6); Epidorylaimus (stages 5 and 6)
(Table 2). Considering the trophic groups of nematodes, we
observed that Ba dominated the nematode communities and
accounted for >40% of the nematodes (Fig. 2). Stage 4 exhibited the
lowest proportions of Ba and Fu, but yielded the highest proportions of Pl and Om&Pr (the latter equal with stage 3). By
contrast, stage 1 displayed a quite different pattern, with the
highest Fu but lowest Pl and Om&Pr proportions (Fig. 2).
3.3. Nematode ecological indices
The lowest values of all of six ecological indices were detected at
stage 1 (Fig. 3). H0 , NCR, PPI and EI showed similar patterns: they
increased at the early stages, and then decreased significantly.
However, they attained the highest levels under different stages, at
stages 5 and 6 for H0 , stages 3 and 4 for NCR, stage 4 for PPI, and at
stages 3, 4 and 5 for EI (Fig. 3). MI and SI increased during the first
three stages and kept high levels during the remaining stages
(Fig. 3).
3.4. Influence of soil properties on nematode assemblages
RDA1 and RDA2 explained 53.67% and 16.25% of the variation
among nematode communities, respectively (Fig. 4). The greatest
difference between nematode communities was observed when
comparing stage 1 and stages 5e7, which was related to differences
in microbial P and litter C/N, according to the length and angle of
axes. The second greatest difference was observed between nematode communities of stage 2 and stages 3e4, which were due to
differences in soil density, pH and litter quantity (Fig. 4). Significant
correlations were observed between nematode abundance and
environmental factors (Fig. 5). Nematode abundance was negatively correlated with soil density throughout the chronosequences
(Fig. 5B). Unimodal patterns were observed for nematode densities
and pH, litter quantity, SOC and bioavailable P: the number of
nematodes increased, as pH increased when nematodes exceeded
600 individuals 100 g1 soil. Likewise, nematode numbers
increased along with the litter content, SOC and bioavailable P,
while decreased when these parameters were higher than the
threshold (Fig. 5A, CeE). Furthermore, nematode abundance
exponentially increased with microbial P during soil development
(Fig. 5F).
4. Discussion
In our study, most soil physiochemical properties and nematode
community structures responded significantly to chronosequence
stages. Based on the studied parameters, the 120-year-old Hailuogou Glacier Chronosequence can be divided into three phases using
RDA (Fig. 4). The initial phase, which lasted approximately 3 years
after the glacier retreat, was characterized by coarse, bare soil (only
5% of the surface was covered by mosses), and highest pH and soil
density, but limited available N and P resources. Furthermore, this
stage displayed the lowest nematode densities and ecological
indices pointing to a most disturbed food web (Table 1; Fig. 3).
Stages 2, 3 and 4 can be viewed as the build-up phase. Especially at
stage 3, vegetation succession from grassland to forest accelerated
plant litter and bioavailable P accumulation, and nematode abundance sharply increased (Tables 1 and 2). As for trophic guilds, the
numbers of bacterial and fungivore nematodes significantly
decreased, and the simultaneous increase in the numbers of plant
feeders, omnivores and predators resulted in higher NCR, MI, EI and
SI (Figs. 2 and 3). The mature phase includes stages 5, 6 and 7, which
displayed balanced, abundant and taxonomically rich nematode
communities, and high MI and SI (Table 2; Fig. 3). Furthermore, the
final stage 7 is associated with a reduced bioavailability of P and a
significant decrease in nematode densities (Tables 1 and 2).
Compared with stages 5 and 6, some rare genera of nematodes
disappeared, especially among the higher trophic guilds of omnivores and predators, followed by significant decreases in NCR, PPI
and EI (Fig. 3). Thus, stage 7 appeared to show some declining
characteristics, although this does not completely support our hypothesis of a retrogressive phase in the Hailuogou Glacier Chronosequence after 120 years of development. Therefore, our results
are not as pronounced as those by Doblas-Miranda et al. (2008),
who found distinct retrogressive stages for the soil communities of
microfauna and macrofauna along the Franz Josef Glacier Chronosequence in New Zealand that spans circa 120,000 years.
Considering the relatively short-term development, the Hailuogou
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
367
Table 2
Nematode abundance (individuals per 100 g dry soil) and proportions of genera (%) among different feeding types at different stages of the Hailuogou Glacier Chronosequence.
Stage 1
Abundance
65.7
Bacterial feeders
Ba1
Diploscapter
0
Diploscapteriodes
3.46
Panagrolaimus
0
Rhabditis
18.49
Rhabditonema
9.57
Ba2
Acrobeloides
1.72
Anaplectus
0
Cephalobus
11.55
Cervidellus
0
Eucephalobus
0
Paraphanolaimus
0
Plectus
3.41
Tylocephalus
0
Ba3
Microlaimus
0
Prismatolaimus
0
Ba4
Alaimus
0
Fungal feeders
Fu2
Aphelenchoides
23.85
Aphelenchus
11.55
Ditylenchus
0
Fu3
Diphtherophora
0
Fu4
Dorylaimellus
12.04
Tylencholaimus
0
Plant feeders
Pl2
Filenchus
3.41
Malenchus
0
Tylenchus
0
Pl3
Criconmella
0
Dolichodorus
0
Helicotylenchus
0
Hirschmanniella
0
Pratylenchus
0
Paratylenchus
0
Rotylenchus
0
Pl5
Xiphinema
0
Omnivores and Predators
Om&Pr4
Eudorylaimus
0
Epidorylaimus
0
Mononchus
0
Mylonchulus
0
Pungentus
0
Thonus
0
Om&Pr5
Aporcelaimellus
0
Belondria
0
Oxydirus
0
± 14.2e
Stage 2
Stage 3
Stage 4
Stage 5
Stage 6
Stage 7
161.0 ± 39.1d
422.0 ± 84.8c
515.1 ± 66.2c
800.7 ± 91.5a
640.7 ± 31.7b
607.3 ± 51.9b
±
±
±
±
±
0
1.23
0
8.73
8.31
0
2.90
0
9.03
2.94
±
±
±
±
±
0
2.51
0
2.06
2.60
0
2.61
0
4.05
1.45
±
±
±
±
±
0
1.21
0
0.55
1.26
0
1.80
0
3.81
0
±
±
±
±
±
0
0.52
0
1.10
0
1.05
1.03
0.96
1.65
1.08
±
±
±
±
±
0.36
0.24
0.10
0.20
0.25
1.89
1.36
1.67
2.16
1.68
±
±
±
±
±
0.59
0.37
0.88
0.90
0.98
2.40
1.06
1.73
2.43
2.96
±
±
±
±
±
0.86
0.38
1.15
0.46
0.87
±
±
±
±
±
±
±
±
2.98
0
3.17
0
0
0
3.77
0
0.93
0
10.70
6.47
0
0
10.72
0
±
±
±
±
±
±
±
±
1.60
0
3.32
4.87
0
0
2.09
0
3.92
0
8.26
2.99
2.76
0
6.40
0
±
±
±
±
±
±
±
±
1.12
0
0.59
0.81
2.39
0
0.63
0
3.52
0
6.95
4.85
0.58
0
5.92
0
±
±
±
±
±
±
±
±
1.30
0
1.73
0.38
1.01
0
0.75
0
1.60
2.33
7.02
2.78
2.27
3.10
5.96
0
±
±
±
±
±
±
±
±
0.27
0.91
2.60
0.84
0.32
1.47
0.96
0
0
1.78
4.10
2.71
3.06
2.01
2.01
1.76
±
±
±
±
±
±
±
±
0
0.66
3.22
0.99
0.77
0.30
0.65
0.60
0
2.48
4.07
2.11
2.01
0
2.62
1.28
±
±
±
±
±
±
±
±
0
1.17
1.50
1.10
0.32
0
0.40
0.48
±0
±0
7.04 ± 3.13
0±0
5.62 ± 1.01
4.07 ± 0.89
2.55 ± 0.82
4.30 ± 1.61
2.67 ± 0.75
3.10 ± 0.39
4.19 ± 0.75
4.40 ± 1.39
5.10 ± 0.96
5.17 ± 0.81
±0
0.93 ± 1.60
6.83 ± 1.07
6.73 ± 1.03
11.00 ± 2.65
12.10 ± 2.68
10.60 ± 3.05
11.70 ± 0.96
4.75 ± 3.37
0.93 ± 1.60
7.72 ± 0.65
3.15 ± 2.85
1.41 ± 2.45
6.94 ± 1.03
0±0
3.51 ± 0.78
4.73 ± 0.43
2.83 ± 0.82
3.63 ± 1.50
4.21 ± 1.87
7.28 ± 3.12
5.09 ± 2.79
6.66 ± 2.08
5.44 ± 1.70
8.12 ± 1.16
±0
4.77 ± 1.46
0.94 ± 1.63
0±0
4.56 ± 1.16
1.26 ± 0.84
2.15 ± 0.93
± 5.14
±0
2.36 ± 4.09
1.65 ± 2.85
1.82 ± 0.51
0±0
1.84 ± 0.35
0±0
2.37 ± 0.38
1.57 ± 0.25
1.80 ± 0.47
1.05 ± 0.46
2.91 ± 0.15
2.07 ± 0.40
± 3.77
±0
±0
4.92 ± 0.65
0±0
3.38 ± 1.89
4.46 ± 4.00
2.73 ± 2.83
6.25 ± 3.67
7.47 ± 1.09
4.59 ± 1.17
5.27 ± 1.42
4.95 ± 0.56
3.98 ± 0.65
4.51 ± 0.37
5.39 ± 1.95
3.12 ± 1.02
5.76 ± 2.32
6.10 ± 0.26
0±0
5.86 ± 0.47
±
±
±
±
±
±
±
0
0
2.57
0
0
3.05
0
± 5.23
± 3.17
±0
0
0
0
0
0
0
0
±0
±
±
±
±
±
±
0
0
0
0
0
0
±0
±0
±0
±
±
±
±
±
±
±
0
0
2.48
0
0
3.20
0
0±0
0
0
3.13
0.37
0
0
±
±
±
±
±
±
0
0
2.72
0.64
0
0
4.64 ± 1.72
0±0
0±0
0
0
4.24
0
0
3.07
0
±
±
±
±
±
±
±
0
0
0.60
0
0
0.89
0
1.02 ± 0.22
2.56
0
7.25
1.81
0
0
±
±
±
±
±
±
0.32
0
1.46
1.58
0
0
2.56 ± 0.25
0±0
0±0
0
2.82
5.84
0
1.23
4.56
0
±
±
±
±
±
±
±
0
3.48
2.54
0
1.07
4.45
0
1.60 ± 0.43
3.57
0
5.56
0
3.22
0
±
±
±
±
±
±
0.27
0
1.28
0
1.21
0
2.11 ± 0.67
0±0
0±0
0.62
0.83
0.65
0.43
1.56
1.98
1.21
±
±
±
±
±
±
±
0.34
0.45
1.12
0.74
0.92
0.53
0.46
3.94 ± 0.75
1.35
1.08
1.71
0
1.27
0.94
±
±
±
±
±
±
0.10
0.37
0.24
0
0.29
0.26
0.65 ± 0.13
0.79 ± 0.37
0±0
0.69
0.62
1.55
1.59
0
0.99
1.34
±
±
±
±
±
±
±
0.26
0.54
0.50
1.40
0
0.90
0.85
1.84 ± 0.31
0.82
1.32
2.60
1.22
1.55
1.54
±
±
±
±
±
±
0.34
0.12
0.24
0.62
0.58
0.92
0±0
0.67 ± 0.20
0.45 ± 0.13
0.77
0
0.83
1.53
0.68
1.18
1.29
±
±
±
±
±
±
±
0.26
0
0.48
0.96
0.59
0.48
0.47
3.11 ± 0.31
0
0
1.59
0
1.53
0.89
±
±
±
±
±
±
0
0
0.72
0
0.90
0.24
0±0
0.73 ± 0.56
0.56 ± 0.13
Bax, Fux, Plx, Om&Prx (where x ¼ 1e5) represent functional guilds of nematodes: bacterial-feeding nematodes, fungal-feeding nematodes, plant-feeding nematodes, and
omnivores and predators, respectively; and x represents the colonizer-persister (c-p) value according to their r and k characteristics. Means ± SE (n ¼ 3).
Glacier Chronosequence may enter its retrogressive phase during the
next decade or century.
4.1. Chrono-function of plant succession and soil properties
Changes in pH, vegetation succession and microbial activity are
considered to be major factors affecting soil succession, including P
speciation and transformation, in the Hailuogou Glacier Chronosequence (Zhou et al., 2013). The pH values of the upper mineral soil
showed a decrease of 2.4 units with progressive succession. In
contrast, Dümig et al. (2011) reported a smaller pH decrease (about
1 unit) on a 140-year chronosequence of the Damma Glacier forefield. The mean annual accumulation rates of organic C and N in the
Hailuogou Glacier Chronosequence reached 28 and 3.5 g m2 y1,
respectively (He and Tang, 2008). These values are significantly
higher than those reported in the Swiss Alpine Chronosequences
over 400 years, with a range of 6.7e9 and 0.33e0.5 g m2 y1 for C
and N, respectively. This difference may indicate that the humid
368
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
100
(A) Ba
100
(B) Fu
80
80
Nematode trophic groups (propotion in %)
60
a
a
a
a
b
ab ab
60
a
40
b
20
0
100
1
2
3
4
5
6
7
(C) Pl
1
2
d
d
3
4
c
c
5
6
b
40
20
7
0
100
(D) Om&Pr
80
80
60
60
a
40
c
20
b
b
40
b
b
d
0
b
a
2
3
a
d
1
2
3
4
5
6
7
1
4
c
b
5
6
c
7
20
0
Fig. 2. Proportions (%) of nematode trophic groups at different stages of the Hailuogou Glacier Chronosequence. Means ± SE (n ¼ 3). Different letters indicate significant differences
(p < 0.05) among seven successional stages according to Tukey's HSD for one-way ANOVA. Ba, Fu, Pl, Om&Pr represent the nematode functional guilds of bacterial-feeding
nematodes, fungal-feeding nematodes, plant-feeding nematodes, and omnivores and predators, respectively.
3.0
2.5
(A) H'
b
b
a
a
b
c
2.0
(B) NCR a
c
1.0
b
b
c
.4
d
.2
.5
Nematode ecological indices
0.0
3.0
2.5
1
2
3
(C) PPI
4
a
5
1.5
6
7
b
c
c
2.0
c
1
2
3
4
5
6
7
(D) MI
b
a
a
a
a
a
0.0
1.5
1.0
e
1
.5
2
b
3
4
5
a
a
a
6
7
1
2
(F) SI
b
d
b
c
3
4
a
a
5
6
a
a
7
0.0
80
a
60
c
40
40
20
20
0
3.0
2.0
d
80 (E) EI
60
0.0
2.5
c
1.0
.5
.8
.6
d
1.5
1.0
a
and mild climate on the site of the Hailuogou Glacier Chronosequence is more favorable to the rapid accumulation of organic
matter.
The relatively low C:P and N:P in soil on 0 to 12 year-old sites
(Zhou et al., 2013) may indicate the co-limitation of C and N for
€ ransson et al., 2011), as indicated by low mimicrobial growth (Go
crobial P (Table 1). Combined with a low amount of bioavailable P,
these limitations hinder the development of biomass-rich vegetation, and only a small number of pioneer N-fixing plants colonize
during the first two stages. At stage 3 (30-year-old site), the vegetation succession from grassland to forest was dominated by Salix
spp., P. purdomii and H. rhamnoides, which possibly accelerated
1
2
3
4
5
6
7
1
2
3
4
5
6
7
0
Fig. 3. Nematode ecological indices at different stages of the Hailuogou Glacier Chronosequence. H0 , Shannon Weaver diversity; NCR, nematode channel ratio; PPI, plant
parasite index; MI, maturity index; EI, enrichment index; SI, structure index.
Means ± SE (n ¼ 3) shown. Different letters indicate significant differences (p < 0.05)
among seven successional stages according to Tukey's HSD for one-way ANOVA.
Fig. 4. Redundancy discriminatory analysis (RDA) of the nematode communities at
different stages of the Hailuogou Glacier Chronosequence. Black inverse triangle: stage
1; white triangle: stage 2; black square: stage 3; white square: stage 4; black diamond:
stage 5; white diamond: stage 6; black triangle: stage 7.
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
1.6
R=0.87
P<0.001
6
R=0.79
P<0.001
5
4
4
(C)
(D)
60
plant litter
1.2
3
R=0.84
P<0.001
40
2
R=0.88
P<0.001
20
1
0
0
(E)
100
bio-availble P
(F)
80
R=0.86
P<0.001
40
20
0
80
60
60
200 400 600 800
nematode abundance
40
20
R=0.73
P=0.001
0
soil organic carbon
pH
7
2.0
soil density
(B)
(A)
Microbial P
8
0
200 400 600 800
nematode abundance
0
1000
Fig. 5. Nematode abundance as a function of pH (A), soil density (B), plant litter (C),
soil organic carbon (D), bioavailable P (E) and microbial P (F) at different stages of the
Hailuogou Glacier Chronosequence as revealed by linear regression. Black inverse triangle: stage 1; white triangle: stage 2; black square: stage 3; white square: stage 4;
black diamond: stage 5; white diamond: stage 6; black triangle: stage 7.
litter and SOC accumulation, leading to a sharp increase in
bioavailable P, microbial P and nematode abundance (Tables 1 and
2). Further vegetation development, especially the presence of
coniferous trees on 30- to 80-year-old sites resulted in a thicker
litter layer, which provided substantial energy supply and favorable
conditions for microbial activity (Fontaine et al., 2011; Zhao et al.,
2014), along with a gradual increase in microbial-P levels. At
these stages, the decreased pH intensified weathering and liberation of mineral P into bioavailable P. However, the large amount of
sequestered bioavailable P in the forest biomass (299.3 kg ha1,
Zhou et al., 2013) reduced bioavailable P concentrations (Table 1).
At stage 7, microbial P exceeded bioavailable P, implying that soil
microorganisms may strongly compete with plants for bioavailable
P (Lajtha and Schlesinger, 1988; Zhou et al., 2013). Thus, the conceptual model developed by Walker and Syers (1976) to explain
long-term (millennial) changes in P dynamics seems valid for
describing short-term changes in P speciation and P availability
within a century of initial soil formation in our chronosequence.
4.2. Chrono-function of nematode communities
The taxonomic richness and diversity of communities (ShannoneWeiner index, H0 ) responded significantly to chronosequence
stages. A total of 42 nematode genera, more than the aboveground
plant richness (data not presented), were observed in the Hailuogou
Glacier Chronosequence. On a local scale, soil biodiversity is believed
to be considerably higher than aboveground diversity (De Deyn and
van der Putten, 2005). This trend is probably caused by the
extremely heterogeneous habitat, with potential for niche partitioning, habitat specialization and species coexistence (Bardgett,
2002).
Bacterial-feeding nematodes dominated throughout succession (Fig. 2A), which was consistent with higher NCR and EI
(Fig. 3B, E) and suggested abundant resources and fast nutrient
369
turnover, often associated with high ecosystem productivity. This
trend would partially explain the rapid accumulation of organic
matter and the succession from herb to forest communities. At
intermediate stages, percentages of fungal-feeding nematodes
significantly decreased, as the densities of omnivores and predators increased (Fig. 2). This trend is consistent with previous
studies that imply strong top-down control of microbe-feeding
€l€
nematodes by predation (Mikola and Seta
a, 1998; Xiao et al.,
2014). The decline in several communities of plant-feeding and
predatory nematodes during the later stages of the chronosequence could be attributed to a lower quality of litter from
dominant tree species, as indicated by higher litter C:N (Table 1).
The decline in plant feeding nematodes may also relate to coniferous plants producing more defensive secondary metabolites in
the roots to deter herbivores and pathogens under poor nutrient
conditions (van der Putten, 2003). The unimodal pattern observed
between the nematode densities and the litter quantity, SOC and
the bioavailable P levels, with a threshold of 600 individuals
100 g1 soil, suggested the importance of nutrient resources in
shaping nematode communities (Fig. 5). An alternative interpretation could be that at the early successional stages 1e3 vegetation succession from mosses to nitrogen-fixing grasses and shrubs
accelerated the accumulation of plant litter and nutrient resources, as there was a positive relationship between nematode
abundance and plant litter quality and SOC. By contrast, at the
later stages 4e7 of the forest establishment, there was no clear
relationship between nematode abundance and environmental
factors (Fig. 5). These results confirmed our second hypothesis,
since the soil nematode communities reflected changes in soil
fertility and vegetation succession at different stages and resource
availability was shown to be an important determinant of soil
organism communities.
The decline in NCR during the final chronosequence stage
implied the greater importance of the fungal-based channel during retrogression (Fig. 3B). The fungal-based channel is more
efficient than the bacterial-based one in terms of nutrient retention (Wardle, 2002). These results agreed with the common
observation of succession from bacterivory to fungivory in nematode faunas (DuPont et al., 2009), with fungal-based decomposition channels predominating during late succession (van der
Heijden et al., 2008). Higher values of PPI during the build-up
period, especially at stage 4, suggested the presence of a high
rate of mineralization and faster cycling rate via plant feeders by
grazing, which may contribute to high biomass accumulation and
rapid succession. By contrast, lower PPI indicated slower rates of
mineralization via decomposition channels in the detritus food
web of initial and final stages (Fig. 2C). Nevertheless, the most
balanced nematode communities appeared during intermediate
stages, especially at the mature stage with the highest taxa richness and abundance, and a higher abundance of omnivores and
predators in higher trophic guilds. High SI (above 50 except for
stage 1) indicated the presence of a structured soil food web with
diverse trophic linkages and “k-selection” feedback control during
the ecosystem development. The abundant and complex soil food
webs composed of diverse interacting elements at the intermediate stages of succession may provide biological buffering,
thereby preventing individual organisms (i.e. nematode pests)
from becoming dominant (Stirling and Eden, 2008). Different
nematode trophic groups and ecological indices peaked at
different stages along the chronosequence, in accordance with our
third hypothesis. In all, plant succession, as well as chemical
signaling among plant and soil organisms, becomes very complex
when both direct and indirect interactions are considered, and
these developments are closely tied to the soil-fauna community
structures.
370
Y. Lei et al. / Soil Biology & Biochemistry 88 (2015) 362e371
5. Conclusions
Our results showed that the 120-year-old Hailuogou Glacier
Chronosequence can be divided into initial (stage 1), build-up
(stages 2e4) and mature (stages 5e7) phases based on the studied soil physiochemical parameters and nematode assemblages. At
build-up stages, vegetation succession from grassland to forest
accelerates the accumulation of plant litter and bioavailable P.
Nematode populations shifted toward more diverse and balanced
communities, especially at the climax stage after 52 years of succession. However, the last stage 7 appears to show some retrogressive characteristics, suggested by the reduced bioavailability of
P and a significant decrease in nematode densities, along with the
disappearance of some rare nematode genera from higher trophic
guilds. Considering the relatively short-term development, the
Hailuogou Glacier Chronosequence may enter its retrogressive phase
during the next decade or century. Moreover, nutrient limitation
during succession is considered as a major stimulus for nematode
communities during long-term changes in the ecosystem. Although
the bacterial-based energy channel dominated several stages of the
chronosequence, the fungivore-based channel activated at early
and final stages may adapt better to low nutrient availability. We
showed that different nematode guilds have contrasting responses
to chronosequence stages, probably because they differ in their
relative responses to bottom-up and top-down controls. In addition, soil nematode communities are sensitive bioindicators of soil
health in glacier retreat areas. Further research should be conducted to determine the most efficient approach to integrate plant
succession, nutrient availability, and soil bacterial and invertebrate
community dynamics into models of ecosystem development and
succession. These models would be helpful for prediction and
management of nutrient limitation during long-term soil
development.
Acknowledgments
The authors are grateful to the Gongga Mountain Alpine
Ecosystem Observation Station, Chinese Academy of Sciences for
logistic support, Dr. Lei YU and Mr. Quan LAN for assistance in
collecting samples, and Professor Douglas A. Schaefer for insightful
suggestions and comments to improve our manuscript. This work
was supported by the National Science Foundation of China (No.
31370607), Chinese Academy of Sciences (Nos. SDSQB-2012-01,
SDS0-135-1207) and Japan Society for the Promotion of Science,
Postdoctoral Fellowship for Foreign Researchers (No. P13080).
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