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OPEN The soil bacterial and fungal

diversity were determined by
the stoichiometric ratios of litter
Received: 24 January 2019
Accepted: 5 September 2019 inputs: evidence from a constructed
wetland
Published online: 25 September 2019

Yunmei Ping1,2,3, Xu Pan 1,2,3

, Wei Li1,2,3, Jinzhi Wang1,2,3 & Lijuan Cui1,2,3

Plant litter is an important component in wetland ecosystems, and the role of plant litter
decomposition is considered to be important for wetland ecosystem functions and services. However,
the consequences of litter inputs have seldom been experimentally tested in real ecosystems such as
constructed wetlands (CWs). The enriched nutrients in CWs might weaken the role of litter inputs on
soil carbon and nitrogen cycling. Here, we conducted a two-month field experiment to examine the
effects of litter inputs on the soils in CWs. Our results showed that litter inputs significantly affected soil
microbial (bacterial and fungi) diversities and properties (soil total nitrogen and nitrogen isotopes), and
litter species with higher stoichiometry ratios, i.e. C/N, C/P and N/P led to higher microbial diversities.
However, litter species had no or weak effects on microbial activities (CO2 and CH4 flux) or on the relative
abundance of microbial communities, indicating that other environmental factors in such a CW might
have stronger effects on those factors than litter inputs. These results highlighted the importance of
submerged plant litter in nutrient-rich wetland ecosystems and provide potential tools for managers to
improve the ecosystem functions and/or services via altering microbial diversities.

Plant litter as the end of primary production entering into detritus food chains plays an important role in wetland
ecosystems, and its decomposition can recycle carbon and multiple nutrients, alter environmental variables, and
affect wetland ecosystem functions and services1–5. Litter decomposition in wetland ecosystems refers to the res-
piration and assimilation of plant litter by microbes and invertebrates6, and it can be divided into three interlinked
processes, i.e. leaching, fragmentation and microbial decay7,8. Moreover, litter decomposition are commonly con-
sidered to be affected by the quality of litter, associated soil microbes and invertebrates and the corresponding
environmental factors7,9–13, but in wetlands such as stream and other freshwater ecosystems, litter inputs and
biotic or abiotic factors are proven to be paramount14. Given the large variation in plant functional traits among
plant species, litter decomposition rate can vary significantly among species due to the various ‘afterlife’ effects of
litter traits (i.e. C/N, lignin, base cations and other decomposition related traits)15–17, and this interspecific vari-
ation in decomposition rates might lead to uncertainties in the effects of wetland plant litter on the soil or water
qualities18,19, and thereby other organism in wetland ecosystems20–22.
Previous studies have proved the significant and diverse effects of plant litter on soil physio-chemical prop-
erties and microbial communities18,23–28, and there were plenty of evidence either from a specific ecosystem type
at local scale or from different biomes at the global scale29,30. It has been proved that soil ecological processes,
including soil C, N cycling, flux of CO2, CH4 are closely related to litter decomposition31,32. There are multiple
pathways that plant litter can affect the soils in wetland ecosystems: (1) litter inputs can have various effects on
soil invertebrates or microbes via different physical and chemical traits; (2) litter inputs can also provide food,
microhabitat or shelter for soil microbes or other soil fauna33,34; (3) litter inputs can have negative effects via

1
Institute of Wetland Research, Chinese Academy of Forestry, Beijing, 100091, China. 2Beijing Key Laboratory
of Wetland Services and Restoration, Beijing, 100091, China. 3Beijing Hanshiqiao National Wetland Ecosystem
Research Station, Beijing, 101399, China. Yunmei Ping and Xu Pan contributed equally. Correspondence and requests
for materials should be addressed to L.C. (email: wetlands108@126.com)

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Litter species
and initial soil TN% TC% TP% C/N C/P N/P C
13 15
N
C 2.37 ± 0.19b 24.36 ± 2.11bc 0.42 ± 0.43a 10.28 ± 0.15b 57.70 ± 0.11b 5.60 ± 0.01c −27.16 ± 0.17d 3.97 ± 0.16d
H 2.51 ± 0.10b 22.76 ± 0.69c 0.49 ± 0.14a 9.08 ± 0.13b 46.20 ± 0.10b 5.00 ± 0.01c −22.39 ± 0.39a 9.41 ± 0.07c
M 3.36 ± 0.07a 35.27 ± 0.91a 0.47 ± 0.21a 10.50 ± 0.04b 75.70 ± 0.22b 7.20 ± 0.02b −25.83 ± 0.11c 10.20 ± 0.07b
P 1.81 ± 0.08c 29.87 ± 0.51ab 0.18 ± 0.15b 16.61 ± 0.94a 164.30 ± 1.47a 9.90 ± 0.05a −24.46 ± 0.19b 19.19 ± 0.10a
IN 0.07 ± 0.00 1.05 ± 0.08 — 14.70 ± 0.39 — — −16.85 ± 0.40 7.08 ± 0.34

Table 1. The initial traits (TN, TC, TP, C/N, C/P, N/P, 13C, 15N) of the four litter species (C: Ceratophyllum
demersum; H: Hydrilla verticillat; M: Myriophyllum verticillatum; P: Potamogeton crispus) and initial soils
(IN). Values are means ± S.E. (n = 3). Values within the same column followed by the same letter indicate no
significant differences at P > 0.05.

releasing leachate from litter which contains detrimental organic carbon and/or other compounds35–37. All above
mechanisms indicated that plant litter traits as litter qualities can strongly influence the chemical and physical
composition of litter inputs, and thereby their decomposability15 and lead to substantial consequences to wetland
ecosystems. However, among various litter traits, litter stoichiometry might form the most important constraints
of carbon: nitrogen ratios on soil microbial communities38,39 and hence act as the key trait to predict the effects of
litter inputs on soil properties and microbial communities in wetlands40.
Moreover, the main methodology to test those effects of plant litter was firstly to sample soils from the field,
and then either directly quantify the soil properties and microbial community composition or activity23,25;
or subsequently set up new controlled experiments in the lab with the litterbag method or soil-litter mixing
method18,26,27,41. For the latter case, knowledge about effects of litter mixing on soil is derived mostly from ‘indoor’
experiments carried out in strictly controlled environments, or via quantifying the litter mass loss and nutrient
release42,43. However, very few investigators have addressed such effects of litter mixing on the wetland soils in
relatively dynamic and unstable environments, such as constructed wetlands (CWs) with irregular waste-water
inputs19. In such kind of constructed wetlands, other environmental factors might weaken the effects of litter
inputs on the soil and thereby other soil organism, such as bacteria and fungi.
Now in this study, we set up a soil-litter mixing experiment (two months) in an ongoing constructed wetland
located in Hanshiqiao wetland, Beijing, China (latitude: 40°07′21.0″, longitude: 116°48′56.7″). Our hypothesis is
that the effects of litter inputs in such a constructed wetland might not be as overwhelming as those proven in
indoor laboratory experiments, in which the related environmental factors are strictly controlled and relatively
stable. We explored the effects of litter traits and incubation time on the soil properties of a constructed wetland
including soil CO2/CH4 fluxes, total carbon (TC), total nitrogen (TN), carbon and nitrogen isotope changes (13C,
15
N), as well as microbial diversities. The purpose of our work was to fully understand the role of plant litter in
constructed wetlands and develop potential tools for the maintenance, improvement and management of con-
structed wetlands using plant litter materials.

Results
Effects of litter inputs on soil properties. There were significant differences between initial litter traits
among four submerged plant species (Table 1). A significant difference was observed for CO2, but no signifi-
cant difference in the other variables between initial soil and the control treatment (Fig. 1, ANOVA 1; Table S1).
Moreover, litter of Potamogeton crispus significantly increased soil bacterial and fungi diversities, and 15N, but lit-
ter of Ceratophyllum demersum significantly increased soil TN, CO2 and CH4, but decreased 15N (Fig. 1, ANOVA
2; P < 0.05). However, litter species had no significant effects on either CO2 or CH4 (Fig. S1, Table S2).

Effects of litter inputs on soil microbial communities. There were significant or marginally signif-
icant relationships between soil microbial diversity and litter stoichiometry ratios, i.e. C/N, C/P and N/P, but
no significant relationships between soil microbial diversity and any other litter trait (Table 2). Moreover, after
two-month incubation, one phylum of soil bacteria, i.e. Planctomycetes, significantly decreased, but another two
phyla of soil bacteria, i.e. Bacteroidetes and Firmicutes, significantly increased (IN vs. CK: Fig. 2, P < 0.01). There
were no significant differences in the soil fungi composition or the other phyla of soil bacteria before and after
incubation (IN vs. CK: Fig. 2, P > 0.05). Overall, different species litter did not drive significant differences in the
relative abundances of soil bacteria or fungi, except for some phyla (Firmicutes and Ciliophora) with lower relative
abundance (<5%) (among four litter species and CK: Fig. 2, P > 0.05).

Discussion
Litter inputs might affect the soil ecological processes, including soil C and N cycling via litter decomposi-
tion31,32,44. We indeed observed significant effects of litter inputs on soil properties (soil TN, 13C and 15N) and
microbial diversities, and those effects to some extent depended on litter species identity (Fig. 1). These results
indicated that in such a real wetland ecosystem mixing litter with soil still played an important role in regulating
wetland soils including isotope signatures and affecting the soil microbial diversities. However, this was different
from our original hypothesis, and in a way highlighted the importance of (even a small amount) litter inputs
might have significant effects on wetland soils. Note that given the initial litter was cut into small pieces before
mixing, the observed effects might be strengthened compared to real litter inputs from submerged plants, which
is similar to previous laboratory studies28,41,45.

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ANOVA 2
ANOVA 2
3 ANOVA 1 1.5
ANOVA 1 ab a
b ab b ab a

Shannon-bacteia
ab b

Shannon-fungi
2 1.0 b
1 ns 0.5
ns
0 0.0

h )
CO2 flux(mg·kg soil-1h-1)

-1 -1
15
a
3
a
10 2 b
b
b b b b b
5 b 1
ns

CH4
0 0

0.20 2.5 a
a 2.0 ab
0.15
b b ab ab

TC(%)
1.5
b
TN(%)

0.10 b 1.0 b
0.05
ns 0.5 ns
0.00 0.0

0 10

b ab b a
-5
ns 8
c
-10 6

N
C
13

15
-15 4
ns
-20
ab a ab ab
2
-25 b 0
IN CK C H M P IN CK C H M P

Figure 1. The soil microbial diversity, soil CO2, CH4 flux and characters before (IN: initial soil without litter)
and after two-month incubation of different litter species (C: Ceratophyllum demersum; H: Hydrilla verticillat;
M: Myriophyllum verticillatum; P: Potamogeton crispus). Values are means ± S.E. (n = 3). CK indicates the
control treatment. The dotted line is used to separate two groups of ANOVA analyses: (1) ANOVA 1 showed
the results between initial soil properties and the control treatment after two-month incubation (without litter
mixing). **Indicated P < 0.01; ns indicated no significant differences between initial soil and CK. (2) ANOVA
2 showed the results among different litter species, including CK. Values by the same letter indicated no
significant differences (P > 0.05).

Initial litter traits

Shannon C/N C/P N/P TN% TC% TP% 13
C 15
N
diversity
index R 2
P R 2
P R 2
P R 2
P R2
P R 2
P R2
P R2 P
bacteria 0.90 0.01 0.64 0.07 0.91 0.01 0.13 0.55 0.28 0.21 0.00 0.93 0.16 0.27 0.31 0.19
fungi 0.67 0.06 0.60 0.08 0.51 0.11 0.08 0.65 0.03 0.37 0.03 0.78 0.14 0.29 0.59 0.08

Table 2. Relationship between initial litter traits (C/N, C/P, N/P, TN, TC, TP, 13C and 15N) and the change value
of soil microbial diversity (Shannon diversity index) before and after two-month incubation (the value = after −
before). Values where P < 0.05 are in bold and P < 0.1 are in italic.

The different responses of wetland soils to litter mixing might result from the different reactions of
micro-organisms to different litter traits and/or to different chemical fractions released during litter decomposi-
tion processes46. In our study, we observed the highest TN and the lowest 15N in the soil mixed with C. demersum
litter after two-month incubation (Fig. 1), and this might be due to the lowest 13C and 15N in the initial litter of
C. demersum (Table 1). Isotope signatures can represent the ratios of heavier element (13C and 15N) to lighter
element (12C and 14N), to some extent determining the decomposition rates of plant species litter47–49. Moreover,
we also observed the highest microbial diversities and the highest 15N in the soil mixed with P. crispus litter after
two-month incubation, and this might also result from the stoichiometric or isotope ratios of initial P. crispus
litter (Table 1). The imbalance among plant litter, microbial biomass and soil stoichiometry might explain this
phenomenon18,50, but regrettably we did not have the data for microbial stoichiometry. Instead, we indeed found
litter inputs might offset the imbalance between soil and mixed litter, and there was a significant positive corre-
lation between litter stoichiometric ratios and microbial diversities (Table 2), indicating that litter traits related
to stoichiometry ratios were still important predictors for the effects of litter inputs on soil C and N cycling50.

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others
100 Planctomycetes
Firmicutes
80 Bacteroidetes
Actinobacteria

pecent of bacteria(%)
Chloroflexi
60 Acidobacteria
Proteobacteria
40

20

100
others
Ciliophora
80 Basidiomycota
Ascomycota
pecent of fungi(%)

60

40

20

IN CK C H M P

Figure 2. The relative abundance of bacteria and fungi in soil of different litter species before and after two-
month incubation (IN: initial soil without litter mixing; CK: control treatment; C: Ceratophyllum demersum; H:
Hydrilla verticillat; M: Myriophyllum verticillatum; P: Potamogeton crispus).

However, we did not find significant effects of litter inputs on the relative abundances of microbes except for sev-
eral minor groups (Fig. 2), and the relative abundance of microbes might largely be determined by the long-term
waste water inputs rather than the short-term litter inputs.
Litter inputs might also affect the CO2 and CH4 flux via the interactions between microbes and litter
sources26,44,51, and litter traits such as C/N ratios were expected to drive those differences52,53. However, our results
showed no significant differences in CO2 and CH4 flux among plant species (Table S2), but only the C. demersum
litter led to the highest CO2 and CH4 flux after two months (Fig. 1). There might be due to the weaker effects of
litter mixing than other factors from the constructed wetlands (Fig. 3), such as temperature and the C/N ratios of
the waste water inputs. The continuous inputs of wasted water with extra carbon and nutrients in our study site
might be more overwhelming than the effects of such a small amount of litter inputs. Note that the quantity of
plant litter and the time of incubation might also matter27, and we suggested that future studies should incorpo-
rate both litter quality and quantity effects and put them in a relatively longer incubation period.

Conclusion
In conclusion, our results provided empirical evidence for the effects of submerged plant litter on the soil prop-
erties and microbial diversities in a constructed wetland. These findings might have multiple implications for
the design, maintenance and management of constructed wetlands: (1) when designing a constructed wetland,
it is better not only take the species identity into consideration, but also for the stoichiometry of different growth
forms19; (2) for constructed wetlands, plant litter of submerged plants should not have been always considered as
wastes and being directly refloated from the CWs. Instead, it is possible to shift the ‘unfavored’ submerged plant lit-
ter to ‘useful’ tools to improve the ecosystem functions and services of constructed wetlands; (3) plant litter might
be a feasible and economic materials for improving the microbial diversities of CWs, and it is worth to comprehen-
sively study the role of wetland plants in constructed wetlands, especially for the role of plant litter, and this might
provide valuable suggestions for managers about the maintenance and management of constructed wetlands.

Materials and Methods

Study site. Our study site was located in the Hanshiqiao wetland, Beijing, China. There was a constructed
wetland, which was used to purify the waste water from the pleasure boat area, restaurant and the public toilet in
the Hanshiqiao wetland Park (Fig. 3). The CW consists of seven treatment sections, and we conducted a soil-litter
mixing experiment in one section of a constructed wetland (CW), i.e. the section III as the study site (marked as
✩ in Fig. 3). The area of the section III is 582.30 m2. The water average depth is about 2.5 m. The dominant plant
species in section III are Iris wilsonii, Zizania latifolia, Typha orientalis and Sagittaria sagittifolia, and there were
irregular waste water inputs flowing into our study site through early April to late November every year.

Experimental design. Plant litter of four submerged species (eg., Ceratophyllum demersum, Myriophyllum
verticillatum, Hydrilla verticillata, Potamogeton crispus) were collected from Hanshiqiao wetland park in July 2017
(but not from the constructed wetland). All plant litter (Fig. 3, B1) was air-dried at room temperature for at least
one month. The litter was subsequently cut into pieces (<5 mm) in order to increase the decomposition rate of
litter and to maximize the effect of litter inputs on the soil in the CW. Initial soil samples (Fig. 3, A1) (upper 10 cm
layer) were collected from five random locations in the CW by shovel, and then were thoroughly mixed. Any
visible roots or contaminants were removed before the experiment. We prepared 15 plastic buckets (Fig. 3, the
bottom diameter is around 22 cm and the height of the bucket is around 27.5 cm), and each bucket has only one
litter species (litter species treatments) or no litter species (the control treatment). Within each plastic bucket, we

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Figure 3. Conceptual framework and experimental arrangements for our study. Four litter species are: C:
Ceratophyllum demersum; H: Hydrilla verticillat; M: Myriophyllum verticillatum; P: Potamogeton crispus; CK:
control treatment. “ → ” in the constructed wetland is water flow direction.“⭐” is the study site.

put 400 g wetland soils and 4.0 g air-dried litter, and thoroughly mixed them in order to keep the dry-weight ratio
of soil vs. litter consistently across treatments18,27. Finally, we randomly placed all the buckets at the bottom of our
study site (submerged by water in the CW) and all the buckets were incubated in the same environment only with
different litter input treatments. The distances between each bucket was around 30 cm. The whole experiment last
from August 31, 2017 to November 2, 2017, which is also the main senesced period in the study region.

Sampling and measurements. Before incubation, three soil samples (Fig. 3, A2) and five litter samples for
each species (Fig. 3, B2) were selected for the initial soil properties and initial litter trait measurements. The initial
soil and litter measurements (listed in Table 1) included total carbon (TC), total nitrogen (TN), soil total phos-
phorus (TP), and stable isotopes (13C and 15N). Total C and Total N content of were assessed using the VarioMAX
CN element analyzer (Macro Elemental Analyzer System GmbH, Hanau, Germany). The TP concentration was
analyzed by inductively coupled plasma emission spectroscopy (Perkin Elmer Optima 3000 ICP Spectrometer,
Waltham, MA, USA) and the isotopes of C and N were subsequently analyzed using an isotope ratio mass spec-
trometer (Isoprime100; Isoprime Ltd, UK).
Initial soil microbe community, i.e. bacteria and fungi, was a measured using the second generation next
throughput sequencing technology (MiSeq high throughput sequencing, 16Sr DNA sequences). Microbial
DNA was extracted from soil samples (or from litter-soil mixtures, see below) using the E.Z.N.A. soil DNA
Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s protocols. The bacteria 16S and fungi
®
18S ribosomal RNA gene were amplified by PCR using primers 515F 5′-GTGCCAGCMGCCGCGG-3′, 907R
5′-CCGTCAATTCMTTTRAGTTT-3′and SSU0817F 5′-TTAGCATGGAATAATRRAATAGGA-3′ and 1196R
5′-TCTGGACCTGGTGAGTTTCC-3′ respectively, where barcode is an eight-base sequence unique to each
sample. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL
of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA.
Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit
(Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions and quantified using
™
QuantiFluor -ST (Promega, U.S.). We calculated the Shannon diversity indices to represent the diversity of soil
bacteria and fungi.
In addition, the CO2 and CH4 fluxes of soil was also measured. Concentrations of CO2 and CH4 were meas-
ured using a gas chromatograph (Agilent 7890 A, Santa Clara, CA). At first, we collected soils samples (at the
beginning of experiment) or mixtures (2 weeks, 4 weeks, 6 weeks, 8 weeks) (about 10 g dry weight) from the CW
by shovel or buckets by self-zip plastic bag. And they were putted in the glass bottle (100 ml). Before gas sampling,
we sealed the glass bottle with airtight butyl rubber stoppers. After 24 h of incubation, the headspace gas of the
glass bottle were sampled using airtight syringes. All gas samples were measured within 24 h after sampling.

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During incubation, litter-soil mixtures were sampled every two weeks and then brought to the lab for the
measurement of CO2 and CH4 fluxes. For the last sampling, the properties and microbial communities of
litter-soil mixtures were again measured using the methods mentioned above. The whole experiment last two
months from August 31, 2017 to November 2, 2017. After incubation, all the plastic buckets were removed from
the CW to avoid the continuous disturbance for the CW.

Statistical analysis. All data were checked for assumptions of normality and homogeneity of variance before
analysis. We firstly compared the differences of initial litter traits, such as TN, TC, TP, C/N, C/P, N/P, 13C, 15N,
among four submerged plant species. Secondly, we conducted one way ANOVAs to examine the differences of soil
microbes (relative abundance and Shannon diversity index), TC, TN, 13C, 15N, CO2 and CH4 between the initial
soil (before incubation) and the control treatment after two-month incubation in the constructed wetland (Fig. 1,
ANOVA 1) and examine the effects of plant species on soil microbes (relative abundance and diversity), TC, TN,
13
C, 15N, CO2 and CH4 after two-month litter mixing (Fig. 1, ANOVA 2). Thirdly, we analyzed the relationship
between initial litter traits (TN, TC, TP, C/N, C/P, N/P, 13C, 15N) and the change value of soil characters (micro-
bial diversity, TC, TN, 13C, 15N, CO2 and CH4) before and after two-month incubation using regression analysis
respectively. In the end, the effects of plant species on microbial respiration (CO2, CH4) during two-month litter
mixing were analyzed using repeated measure ANOVA. Differences between means were tested with Fisher LSD
tests; effects were considered significant at P < 0.05. All the ANOVA analyses were conducted in SPSS Statistics
(SPSS, Chicago, IL, USA), and regression analyses were conducted in R software 3.5.2 (R core Team)54.

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Acknowledgements
This work was funded by the National Key R&D Program of China (2017YFC0506200) and Fundamental
Research Funds for the Central Non-Profit Research Institution of CAF (CAFYBB2017SY045). We thank the
teachers and students of Hangzhou Normal University for the assistance of analyzing litter qualities. We also
thank Zhangjie Cai and Zhiguo Dou for helping with the experiment.

Author Contributions
L.J.C. and X.P. designed the experiment; Y.M.P. and X.P. executed the experiment; Y.M.P., X.P. and W.L.
contributed to analyzing the data and making the figures. Y.M.P., X.P., W.L., J.Z.W. and L.J.C. contributed to
writing and editing the manuscript.

Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-50161-9.
Competing Interests: The authors declare no competing interests.
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