Changes in Soil Organic Carbon Dynamics in a Native C4 Plant-Dominated Tidal Marsh Following Spartina alterniflora Invasion

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Title: Changes in Soil Organic Carbon Dynamics in the Marsh Dominated by Native C4 Plant Cyperus malaccensis Following Spartina alterniflora Invasion

Author: JIN Bao-Shi, LAI Yuk Fo, Derrick, GAO Deng-Zhou, TONG Chuan, ZENG Cong-Sheng

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Please cite this article as: JIN Bao-Shi, LAI Yuk Fo, Derrick, GAO Deng-Zhou, TONG Chuan, ZENG Cong-Sheng, Changes in Soil Organic Carbon Dynamics in the Marsh Dominated by Native C4 Plant Cyperus malaccensis Following Spartina alterniflora Invasion, Pedosphere (2017), 10.1016/S1002-0160(17)60396-5.

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ACCEPTED MANUSCRIPT PEDOSPHERE Pedosphere ISSN 1002-0160/CN 32-1315/P

doi:10.1016/S1002-0160(17)60396-5

Changes in Soil Organic Carbon Dynamics in the Marsh Dominated by Native C4 Plant Cyperus malaccensis Following Spartina alterniflora Invasion JIN Bao-Shi1,2, LAI Yuk Fo, Derrick3, GAO Deng-Zhou1, TONG Chuan1,4, ZENG Cong-Sheng1,4* 1

School of Geographical Sciences, Fujian Normal University, Fuzhou 350007, China School of Resources and Environment Science, Anqing Normal University, Anqing 246011, China 3 Department of Geography and Resource Management, The Chinese University of Hong Kong, Hong Kong China 4 Key Laboratory of Humid Subtropical Eco-geographical Process of Ministry of Education, Fujian Normal University, Fuzhou 350007, China

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*Corresponding author. E-mail: [email protected].

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ABSTRACT

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Numerous studies have shown that the invasion of C4 plant Spartina alterniflora would increase the soil organic carbon (SOC) contents in the native C3 plant-dominated coastal wetlands of China. However, little is known regarding the effects of S. alterniflora invasion on SOC concentrations and fractions in tidal marshes dominated by native C4 plants. In this study, we conducted a field experiment in a tidal marsh dominated by the native C4 plant Cyperus malaccensis in the Min River estuary. Concentrations of SOC and its various fractions, including dissolved organic carbon (DOC), microbial biomass carbon (MBC) and easily oxidizable carbon (EOC), were measured in the top 50 cm soils at sites dominated by C. malaccensis as well as those invaded by S. alterniflora for 0–4, 4–8, and 8–12 years. Our results showed that both SOC stocks in the top 50 cm soils and mean SOC concentrations in the surface soils (0-10 cm) of the Cyperus marsh increased with the age of S. alterniflora invasion, whereas SOC concentrations at 10–50 cm depth showed a slight decrease four years after Spartina invasion before increasing again. The pattern of changes in labile C fractions (DOC, MBC and EOC) with invasion age was generally similar to that of SOC, while the ratio of labile C to SOC (DOC/SOC, MBC/SOC and EOC/SOC) decreased significantly with the age of Spartina invasion. Our findings suggest that the invasion of exotic C4 plant S. alterniflora into a marsh dominated by native C4 plant C. malaccensis would enhance SOC sequestration owing to the greater amount of biomass and lower proportion of labile SOC fractions present in the Spartina communities. Key Words: Spartina alterniflora; plant invasion; chronosequence; labile organic carbon fractions; redundancy analyses; Min River estuary

INTRODUCTION Invasion of exotic plant species is one of the most important threats to natural ecosystems, reducing biodiversity and affecting both the structure and functions of native ecosystems (Walker and Smith, 2000). Spartina alterniflora, a species native to the brackish marshes of the Atlantic and Gulf Coasts of North 2

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America, was deliberately introduced to Luoyuan Bay on the Fujian coast of southeast China in 1979 and subsequently to the Yangtze River estuary in the 1990s for the purpose of accelerating sedimentation and land formation via the so-called “ecological engineering” (Liao et al. 2007). Since then it has gradually expanded its range throughout the coastal regions of China, spreading into both native wetland plant communities and open mudflats. Currently, S. alterniflora can be found from Tianjin (38°56′N, 121°35′E) in the north to Beihai (21°36′N, 109°42′E) in the south, covering a total area of approximately 34,137 ha (Lu and Zhang, 2013). The intertidal zone of the Fujian estuary and coast is the fourth largest area in China in terms of S. alterniflora coverage. While S. alterniflora invasions may exert both positive and negative impacts on coastal wetlands (Wan et al., 2009; Li et al., 2009), the rapid spread of S. alterniflora over the past decade has been considered as one of the main threats to the native plant communities in the estuaries and marshlands of southeast China (Gao et al., 2014). The potential impacts of S. alterniflora invasion on wetland biogeochemical processes have received considerable attention in recent years, especially with respect to net primary production and soil nutrient dynamics (Liao et al., 2007, 2008; Cheng et al., 2008). S. alterniflora is characterized by the C4 photosynthetic pathway, and has a higher rate of net primary productivity than native C3 plants such as Suaeda salsa (Zhang et al., 2010), Scirpus mariqueter and Phragmites australis (Liao et al., 2007), which leads to an increased carbon (C) and nitrogen (N) storage in the plant biomass. In addition, the lower litter quality of S. alterniflora communities would limit the rate of litter decomposition (Liao et al., 2007), particularly that of belowground decomposition, which can lead to a decrease in C release from litter and an increase in the incorporation of litter into the formation of soil organic matter (Berg and McClaugherty, 2003). To date, numerous studies have focused on the effects of S. alterniflora invasion on soil organic carbon (SOC) dynamics in the estuarine and coastal wetlands of China, with reference being made to the bare mudflats or wetlands that are dominated by native C3 plants (Cheng et al., 2006, 2008; Zhou et al., 2008; Zhang et al., 2010; Yang et al., 2013, 2016; Zhang et al. 2014; Zhou et al., 2015). However, to the best of our knowledge, no published research has focused on the influence of S. alterniflora colonization on the SOC dynamics in a marsh dominated by C4 plants. Additional field studies are needed to further our understanding of the impacts of S. alterniflora invasion on SOC in various native ecosystems. Moreover, there is currently a paucity of research on the response of SOC to Spartina invasion over different periods along a chronosequence, as previous studies have often focused on SOC changes only at a single point in time after invasion. SOC is composed of materials with varying chemical complexity with mean turnover times ranging from days to tens or hundreds of years. It is a heterogeneous mix of old and young C in various fractions (Davidson and Janssens, 2006). SOC pools can be chemically divided into labile and recalcitrant fractions by acid hydrolysis (McLauchlan and Hobbie, 2004). In general, recalcitrant C contributes to the majority of SOC and is important for soil C sequestration and soil quality owing to its long-term retention in soils. On the other hand, labile C accounts for only a minor portion of SOC, but is largely responsible for the initial change in soil C pool owing to its high biochemical activity (Rovira and Vallejo, 2002; Belay-Tedla A et al., 2009). Therefore, labile SOC fractions play a crucial role in the overall SOC budget and could reveal the mechanisms that drive SOC changes (Christensen, 2001; Yang et al., 2015). Some SOC fractions, such as dissolved organic carbon (DOC), microbial biomass carbon (MBC) and easily oxidizable carbon (EOC), are more sensitive to short-term perturbations than the overall SOC amount (Carter et al. 1998; Gosling et al. 2013). For example, both DOC and MBC are considered early indicators of C sequestration in soils owing to their rapid turnover rates (Blair et al. 1995; Ghani et al. 2003; Haynes 2005). The EOC is considered a good indicator of soil quality, partly because of its ease of measurement (Weil et al., 2003; Melero et al., 2009). Although analyzing the response of SOC fractions is a more effective approach for predicting SOC dynamics 3

ACCEPTED MANUSCRIPT in response to plant invasion, relatively little attention has thus far been paid to the impacts of S. alterniflora invasion on SOC fractions in wetlands. In view of the above knowledge gaps, the objectives of this study were to: (1) assess the impacts of invasion by the exotic C4 plant S. alterniflora on the soil C and N pools in a marsh dominated by the native C4 plant C. malaccensis; and (2) examine the changes in labile SOC fractions in response to Spartina invasion, and their relationships with SOC dynamics. The tidal marsh of the Min River estuary which was dominated by C. malaccensis but was increasingly colonized by the invasive S. alterniflora was chosen as the study site. We analyzed the plant samples, soil physical-chemical properties, as well as the concentrations of total SOC and some labile C fractions in the top 50 cm soils in a C. malaccensis marsh and areas invaded by S. alterniflora for 0–4, 4–8, and 8–14 years, respectively, along a chronosequence.

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MATERIALS AND METHODS Study area

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The study was conducted in the Shanyutan Nature Reserve, which contains the largest tidal marsh at the mouth of the Min River estuary in southeast China (119°34′12″-119°40′40″E, 26°00′36″-26°03′42″N; Fig.1). This area is characterized by a subtropical monsoon climate with a mean annual temperature of 19.6°C, a mean annual precipitation of 1,350 mm, and a semi-diurnal tidal range of approximately 2.5–6 m (Tong et al., 2010). The average salinity of the tidal water is around 4.2 ppt. The native C. malaccensis communities extend from the intertidal zone to near the bank in the middle-west section of this tidal marsh. Local historical records show that S. alterniflora was first recorded in this marsh in 2002, and has since gradually expanded its coverage from the zone closest to the sea to the intertidal zone (Tong et al., 2011). At present, S. alterniflora has already colonized many parts of the marsh formerly dominated by C. malaccensis and become one of the most widely distributed plants in the marsh.

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Fig. 1

Fig.1 Study area and sampling sites in the tidal marshes of the Min River estuary. CM = C. malaccensis; SA-4 = S. alterniflora (0–4 years); SA-8 = S. alterniflora (4–8 years); SA-12 = S. alterniflora (8–12 years).

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Collection of field samples

Based on the space-for-time substitution approach, we identified the spatial distribution of S. alterniflora in this marsh in 2006, 2010 and 2014 using aerial images, Google Earth satellite images, and field investigations (Zhang, W. et al., 2011). Given that S. alterniflora began to encroach into the C. malaccensis marsh in 2002 (Tong et al., 2012), we were able to identify three different S. alterniflora communities with an invasion age of 0–4 years (SA-4), 4–8 years (SA-8), and 8–12 years (SA-12), respectively. The C. malaccensis communities (CM) and SA-12 marshes were covered entirely by C. malaccensis and S. alterniflora, respectively. In SA-4, the marsh was dominated equally by the stands of S. alterniflora (50%) and C. malaccensis (50%), while the SA-8 marsh was more dominated by S. alterniflora (> 85%) than C. malaccensis (< 15%). Meanwhile, all the stands that were sampled in the center of SA-4 and SA-8 marshes were purely covered with S. alterniflora. This coastal marsh was rather flat with a < 0.5° slope, and the four sampling sites were parallel to a dam built in 1995 (Liu and Zeng, 2006). Sites in close 4

ACCEPTED MANUSCRIPT proximity to one another with the same elevation were selected in order to minimize environmental heterogeneities, such as salinity, redox potential and tidal inundation frequency. For each plant community, three plots were randomly established for soil sample collection by using a stainless steel cylinder with 10 cm diameter during the ebb tides of November 2014. In each plot, 50 cm soil cores were sectioned at 10-cm intervals, sealed in plastic bags, kept on ice in coolers, and immediately transported to the laboratory. Sixty soil samples (four plant communities × three plots × five soil layers) were included in the analysis. We also randomly established three replicate quadrats (50 × 50 cm2) in each of the four plant communities for destructive sampling to determine aboveground biomass and root biomass in the top 50 cm soils.

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Laboratory analysis

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After carefully removing some identifiable plant residues, coarse root materials and stones with forceps, the soil samples were thoroughly mixed and divided into two sub-samples. One sub-sample was filtered through a 2-mm sieve, sealed in a plastic bag, and preserved in a freezer at 4°C for soil DOC and MBC analyses. The other sub-sample was air-dried, grinded, and filtered through a 2-mm nylon sieve for determining soil pH, electrical conductivity (EC), SOC and EOC, and subsequently through a 0.149-mm sieve for determining total carbon (TC) and total nitrogen (TN). Soil pH was measured using a pH meter (868, Orion Scientific Instruments, USA), and EC was measured as a proxy of soil salinity levels using a EC meter (2265FS, Spectrum Technologies Inc., USA) with a soil/water ratio of 1:5. Soil samples were collected using a 5-mL syringe and then oven-dried at 105°C to determine the soil moisture content and bulk density (BD). Particle size was measured using a particle size analyzer (Mastersizer 2000, Malvern, UK) and classified into clay (< 4 μm), silt (4–63 μm), or sand fractions (63-2000 μm). Biomass was determined by oven-drying harvested materials at 80°C to a constant weight. Concentrations of TC and TN were measured using a CHNS analyzer (Vario EL III, Elementar, Germany). SOC was measured by wet combustion of sediments in H2SO4/K2Cr2O7 (Sorrell et al. 1997; Bai et al. 2005). SOC stock (kg·m-2) in the top 50 cm profile was calculated using the equation (Mishra et al., 2000):

SOCS   SOCi  BDi  H i i 1

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where i represents each sampled layer of the soil profile, SOC is the soil organic carbon concentration (kg·kg-1), BD is the soil bulk density (kg·m-3), H is the thickness (m) of each layer and n is the number of soil layers. DOC was extracted by mixing 10 g of pre-treated fresh soil with 50 mL of deionized water. The mixture was shaken for 30 min in 100-mL polypropylene bottles on a reciprocating shaker at 250 rpm and then centrifuged at 4,000 rpm for 20 min. The supernatant was then vacuum-filtered through a 0.45-um filter membrane into a separate vial for determining C concentrations with a total organic carbon analyzer (TOC-VCPH, Shimadzu, Japan) (Jones and Willett, 2006). MBC was determined by the chloroform fumigation extraction method (Vance et al., 1987). Briefly, two 10 g samples of pre-treated fresh soils were incubated for 24 h in a dark room at 25°C, with one of them being fumigated with chloroform. Both fumigated and non-fumigated soils were extracted with 40 mL 0.5 M K2SO4, shaken at 250 rpm for 30 min, centrifuged at 4,000 rpm for 20 min, and then vacuum-filtered through a 0.45-um filter membrane. The filtrate was diluted with a ratio of filtrate to water of 1:10, and a total organic carbon analyzer (TOC-VCPH, Shimadzu, Japan) was used to determine the C concentrations. Soil MBC was 5

ACCEPTED MANUSCRIPT calculated as Cext/KEC, where Cext was the organic C extracted from the fumigated soil minus that from the non-fumigated soil, and KEC was a constant of 0.38. The dry soil weight of the 10 g of fresh soils used for DOC and MBC determination was estimated by the measured soil moisture content. EOC was determined based on the method of Blair et al. (1995). Air-dried soil samples (1 g) were shaken with 25 mL 333 mM KMnO4 at 120 rpm for 1 h and the suspension was then centrifuged at 4,000 rpm for 5 min. The supernatant was diluted with a ratio of supernatant to water of 1:250, and the absorbance was measured at 565 nm with an UV-visible spectrophotometer (Shimadzu UV-2450, Japan). Blank samples that were identical to KMnO4 but with no soil samples added were included in the analysis. The difference in the concentration of KMnO4 between soil samples and blanks was used to estimate the amount of oxidized C.

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Statistical analysis

RESULTS

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All statistical analyses were performed with SPSS 19.0 (SPSS Inc, Chicago, IL). Data were tested for normality and log-transformed to meet the assumptions for statistical analyses. Differences in mean grain size, pH, salinity, BD and moisture of soils among the plant communities were tested for statistical significance by analysis of variance (ANOVA). One-way ANOVA was also used to test for differences in mean SOC, TN, C:N, DOC, MBC and EOC in the same soil layer among all plant communities, as well as the average DOC/SOC, MBC/SOC and EOC/SOC ratios among all soil layers. Two-way ANOVA was used to test for the effects of invasion age, subplot, and their interaction on mean SOC, TN, C:N, DOC, MBC and EOC. Differences at the P < 0.05 level were considered significant. OriginPro8.0 was used to fit the plot. Redundancy analyses (RDA) using CANOCO 4.5 software (www.canoco5.com) was performed to examine the relationships between SOC fractions and environmental factors. RDA was used for ordination to analyze the change in biotic communities with environmental conditions, with the extra advantage of visualizing the relationships between the ecological community and the environment in terms of individual species (Smilauer and Leps, 2003). Here, SOC and its fractions were treated as biotic factors, and other soil parameters such as pH, BD, and EC were treated as abiotic factors.

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Plant community characteristics and soil physico-chemical properties Our results showed that the average height of C. malaccensis (111 cm) was lower than that of S. alterniflora (168-174 cm). However, the mean plant density of S. alterniflora communities was considerably lower than that of C. malaccensis (896 stem m-2), especially in the SA-4 stand with only 302 stem m-2 (Table I). The aboveground live and dead biomass of S. alterniflora communities increased consistently with the age of invasion, whereas the belowground biomass of S. alterniflora communities four years post-invasion was only about two-thirds of that prior to invasion but increased by nearly two-fold 8 years post-invasion. In addition, the mean C:N ratio of belowground roots and aboveground plant tissues including the leaves and stems, was substantially higher in the S. alterniflora communities as compared to the C. malaccensis communities.

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ACCEPTED MANUSCRIPT TABLE I The characteristics of C. malaccensis and S. alterniflora communities at different ages of invasion. Properties

CM

SA-4

SA-8

SA-12

111±10

168±21

173±15

174±13

896±87

302±45

517±36

713±54

643±143

2143±319

3744±381

5164±428

290±81

616±146

1051±272

1450±243

Belowground biomass (g m )

3192±286

2200±363

4196±418

5786±431

C:N

30.35±1.97

46.13±3.22

44.96±2.74

47.15±2.71

Height (cm) -2

Density (stem m ) -2

Aboveground live biomass (g m ) -2

Aboveground dead biomass (g m ) -2

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CM = C. malaccensis; SA-4 = S. alterniflora (0–4 years); SA-8 = S. alterniflora (4–8 years); SA-12 = S. alterniflora

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(8–12 years).

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The physico-chemical properties of the top 50 cm soils in the C. malaccensis and S. alterniflora communities are shown in Table II. Soil pH was significantly lower in the S. alterniflora communities 8 years post-invasion, whereas soil moisture was significantly lower in the C. malaccensis communities than all the other communities invaded by S. alterniflora (P < 0.05). Soil EC and BD in the S. alterniflora communities beyond 8 years of invasion were significantly higher and lower, respectively, than those without invasion or with a younger invasion age (P < 0.05). The soils across all the communities were dominated by silt particles (69-72%). The percentages of sand and silt particles were relatively stable but showed a slight decrease in response to S. alterniflora invasion, while the percentage of clay particles increased slightly with invasion age, although statistically insignificant (P > 0.05). (Table II).

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TABLE II

Physicochemical properties of the top 50 cm soils (mean ± SE, n = 15) in the C. malaccensis and S. alterniflora Properties pH

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communities at different ages of invasion.

-1

Moisture (g·kg ) -1

EC (mS·cm )

SA-4

SA-8

SA-12

6.44±1.19a

6.28±0.11a

6.32±0.13a

6.09±0.06b

97.93±4.71b

104.23±6.60a

107.75±6.84a

109.21±5.05a

4.22±2.21b

4.15±0.33b

5.62±0.42a

5.77±0.14a

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CM

BD (g·cm )

0.74±0.23a

0.79±0.08a

0.69±0.02b

0.70±0.05b

Sand (%)

3.10±0.64a

3.03±6.51a

3.04±10.24a

3.06±4.67a

Silt (%)

72.09±15.84a

72.15±4.06a

70.12±7.79a

68.96±5.46a

Clay (%)

24.81±20.66a

24.82±1.64a

26.83±2.56a

27.98±1.44a

Different letters within a row indicate significant differences among communities (P = 0.05). EC = electrical conductivity; BD = bulk density; CM = C. malaccensis; SA-4 = S. alterniflora (0–4 years); SA-8 = S. alterniflora (4–8 years); SA-8 = S. alterniflora (8–12 years).

SOC and TN concentrations The mean SOC concentration in the top 10 cm soils of the S. alterniflora communities increased with the age of invasion from 16.03 g C kg-1 at SA-4 to 22.02 g C kg-1 at SA-12. The mean SOC concentrations at 7

ACCEPTED MANUSCRIPT SA-4, SA-8, and SA-12 increased by 4.65%, 24.23%, and 43.83%, respectively, as compared to the value of 15.31 g C kg-1 in the C. malaccensis communities (Fig.2 A). On the other hand, SOC concentrations at 10–50 cm depth decreased slightly four years after S. alterniflora invasion before increasing further with the age of invasion (Fig.2 A). The SOC stocks in the top 50 cm soil profiles of the communities of C. malaccensis, SA-4, SA-8 and SA-12 were 5.03, 5.43, 6.11, and 8.09 kg·m-2, respectively. Similarly, TN concentrations in the top 20 cm soils increased consistently with invasion age, whereas those in the deeper soil layers decreased initially before increasingly again in the later periods of invasion (Fig.2 B). Meanwhile, the soil C:N ratio in all five soil layers increased following S. alterniflora invasion (Fig.2 C). The results of two-way ANOVA showed that both SOC and C:N ratio were significantly affected by invasion age, while TN was significantly affected by invasion age, subplot, and their interaction (Table III ).

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Fig. 2

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Fig. 2 Variations of (A) SOC and (B) TN concentrations and (C) C:N ratio (mean ± SE, n = 3) at different soil depths in the C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05 in the same layer. SOC = soil organic carbon; TN = total nitrogen. See Fig. 1 for abbreviations of CM, SA-4, SA-8 and SA-12.

TABLE III

Results of two-way ANOVA on the effects of invasion age and subplots on the concentrations of total SOC, various

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SOC fractions, TN, and the ratio of C:N among the C. malaccensis and S. alterniflora communities. df

SOC

TN

C:N

DOC

MBC

EOC

Invasion age

3

46.53**

10.16**

19.29**

4.21*

1.34

12.34**

Subplot

4

1.59

11.41**

1.04

9.15**

5.22**

1.68

12

3.48**

3.97**

1.48

1.26

0.58

2.40*

Invasion

age

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Subplot

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Analysis of variance

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*P < 0.05, ** P < 0.01; SOC = soil organic carbon; TN = total nitrogen; DOC = dissolved organic carbon; MBC = microbial biomass carbon; EOC = easily oxidizable carbon.

SOC fractions

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Changes in the concentrations of the labile SOC fractions (DOC, MBC, EOC) following S. alterniflora invasion were generally similar to those of SOC, except for the concentrations of DOC and MBC at 30–40 cm depth and EOC at 20–30 cm depth of the SA-8 and SA-12 S. alterniflora communities that were consistently lower than those in the C. malaccensis communities (Fig.3 A, B and C). In the vertical soil profiles across the communities, the concentrations of labile SOC fractions were highest in the surface layer (0–10 cm), and the concentrations of DOC and MBC decreased significantly with soil depths (P < 0.05), whereas EOC concentrations had little variations among 10–50 cm depths (Fig.3 A, B and C). Furthermore, DOC was significantly affected by both invasion age and subplot, whereas MBC and EOC were significantly affected by subplot and invasion years, respectively (Table III). In contrast to the labile SOC fractions, the mean ratios of DOC/SOC, MBC/SOC and EOC/SOC in the top 50 cm soils decreased significantly in response to S. alterniflora invasion (Fig.4 A, B and C).

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ACCEPTED MANUSCRIPT Fig. 3 Fig. 3 Variations of SOC fractions, including (A) DOC, (B) MBC, and (C) EOC (mean ± SE, n = 3) at different soil depths in the C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05 in the same layer. SOC = soil organic carbon; DOC = dissolved organic carbon; MBC = microbial biomass carbon; EOC = easily oxidizable carbon. See Fig. 1 for abbreviations of CM, SA-4, SA-8 and SA-12.

Fig. 4

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Relationship between SOC and soil physico-chemical properties

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Fig. 4 Mean ratios of (A) DOC/SOC, (B) MBC/SOC, and (C) EOC/SOC in the top 50 cm soils (mean ± SE, n = 15) of C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05. See Figs. 1-3 for abbreviations of CM, SA-4, SA-8, SA-12, SOC, DOC, MBC and EOC.

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The results of RDA showed that several soil physico-chemical properties, including C:N ratio, TN, EC, and moisture, were positively correlated with both SOC and the labile fractions, while both soil pH and BD were negatively correlated with SOC as well as the labile fractions. Moreover, the results of RDA that SOC and its various fractions were significantly and positively correlated (Fig. 5). Fig. 5

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Fig. 5 Redundancy analysis of SOC fractions and soil physico-chemical properties in the C. malaccensis and S. alterniflora communities with different invasion ages (n = 60). The total percentage of variability explained by all the canonical axes was 86.9%. SOC = soil organic carbon; TN = total nitrogen; DOC = dissolved organic carbon; MBC = microbial biomass carbon; EOC = easily oxidizable carbon; EC = electrical conductivity; BD = bulk density. DISCUSSION

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Effects of S. alterniflora invasion on SOC

Plant invasions have previously been found to alter C and N cycles in various ecosystems (Ehrenfeld, 2003). Cheng et al. (2006) found that TC and SOC were significantly higher in the invasive S. alterniflora communities as compared to those in the native Scirpus mariqueter communities, although the soil inorganic carbon contents were not significantly different. They further concluded that S. alterniflora invasions would increase C storage in soils by sequestering organic C. Numerous studies have shown that the accumulation of organic matter in S. alterniflora soils is the result of a higher level of primary production that in turn increases C inputs to soils via surface litter and roots (Cheng et al., 2006; Zhang et al., 2010; Yang et al., 2013). Our results indicated that in SA-4 communities, the aboveground and belowground biomasses were higher and lower than those in the C. malaccensis communities, respectively (Table I). Therefore, the higher SOC concentrations in the top 10 cm soils of S. alterniflora communities, although statistically insignificant (P > 0.05) (Fig.2 A), could be a result of the higher inputs of aboveground litter as compared to the C. 9

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malaccensis soils, which was similarly reported in other studies (Zhang et al., 2010; Yang et al., 2013; Zhang et al., 2014). Meanwhile, the mean SOC concentration at 10-50 cm depths in the soils of S. alterniflora four years after invasion was slightly lower than that of C. malaccensis (Fig.2 A), because of the lower amount of belowground biomass and thus litter inputs. As a result, SOC concentrations at 0–10 cm and 10–50 cm depths exhibited different responses to S. alterniflora invasion during this initial period. We also found that the belowground biomass in the S. alterniflora communities 8 years post-invasion was nearly two-fold higher than that in the C. malaccensis communities (Table I), which would account for the higher SOC concentrations at 10–50 cm depths in the former communities (Fig.2 A). Moreover, the amounts of aboveground live biomass in the SA-8 and SA-12 plots were 3,744 and 5,164 g·m -2, respectively, which were considerably higher than those found in the native C. malaccensis communities (643.32 g·m-2) (Table I). Therefore, the higher SOC concentrations in the 10–50 cm layers of S. alterniflora communities after over 4 years of invasion were likely a result of the greater abundance of both surface litter and roots (P < 0.05) (Fig.2 A). Meanwhile, litter decomposition is an important pathway that controls nutrient cycling in wetlands (Jordan et al., 1989). Tong et al. (2011) demonstrated that S. alterniflora had a lower litter decomposition rate than C.malaccensis because of its higher C:N ratio, which was consistent with our results of a higher aboveground dead biomass in S. alterniflora communities (Table I). A higher ratio of recalcitrant C to easily decomposable organic C in the soils of S. alterniflora could favor greater C accumulation as compared to that of C. malaccensis. The continual increase in SOC concentrations in the top 50 cm soils with the age of S. alterniflora invasion suggested that soil C storage had not yet reached a maximum 12 years post-invasion, which was similar to the pattern reported in the Wanggang coastal wetland of Jiangsu province, China with an invasion age of 14 years (Zhang et al., 2010). Liao et al. (2006) also observed in the surface soils of a grassland that SOC concentration rose by approximately five-fold without showing any signs of stabilization after more than 120 years of invasion by woody plants. In the vertical soil profile, the mean SOC concentrations in the top 20 cm soils increased markedly 8 years post-invasion as compared to those in the deeper soil layers (Fig.2 A), which was likely due to the majority of S. alterniflora roots being grown in the upper 20 cm soil layers (Liao et al., 2007; Darby et al., 2008). Moreover, the greater input of aboveground plant litter in S. alterniflora communities would contribute to an increase in organic matter accumulation in surface soils. Given that C and N cycles often interact closely in ecosystems, we also quantified soil TN contents in the four plant communities and found that their variations closely resembled the patterns of SOC (Fig.2 B). The higher levels of TN in soils were largely the result of an increase in biomass inputs (Davidson and Janssens, 2006; Fissore et al., 2009). When coastal wetlands dominated by C. malaccensis were invaded by S. alterniflora, soil TN would likely increase owing to the occurrence of epiphytic N fixation in S. alterniflora stems and sheath litter (Currin and Pearl, 1998). The N fixed during litter decomposition could be further incorporated into soil organic matter and increase the soil N stocks (Knops et al., 2002). The observed increase in soil TN in the S. alterniflora communities as compared to the C. malaccensis ones could also be the result of a lower rate of N mineralization in soils, as supported by the observation of a lower N loss from litter and higher soil N content at sites invaded by S. alterniflora in the same study area (Wang et al., 2015). Hence, the effects of S. alterniflora invasion on soil TN could be attributed to the higher plant-derived N and the lower N mineralization rate. The molar ratio of soil C:N ranged from 10.43 to 11.62, which suggested that organic matter in the Min River marsh might be derived primarily from macrophytes (Fig.2 C), and was in agreement with the previous results presented by Zhang et al. (2015). Moreover, the soil C:N ratio was consistently higher in the communities dominated by S. alterniflora than C. malaccensis, because of the much higher C:N ratio in the biomass of the exotic Spartina (Table I). 10

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Dynamics of SOC fractions

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Labile SOC fractions, including DOC, MBC and EOC, have been widely used as indicators of soil quality because of their sensitivities to the variations in environmental conditions and consistent responses to SOC changes (Zhang, M. et al., 2011; Guo et al., 2015). In this study, we observed a significant correlation between SOC and each of the three main labile SOC fractions (Fig. 5). The DOC and MBC mainly come from the leaching of plant residues, SOC hydrolyzation, root secretion, and microbial metabolites (Muller et al., 2009; Wickland et al., 2007). In the stands invaded by S. alterniflora, the higher C input and soil moisture could contribute to an increase in both DOC and MBC. The concentration of EOC was considerably higher than that of DOC and MBC, and its variation was largely associated with an increase in total SOC. Since both DOC and MBC were major energy sources for soil microbes and were affected by microbial activities (Marschner and Bredow, 2002), it was reasonable to find that these two fractions were significantly correlated with each other (Fig. 5). In addition, we observed consistently higher levels of the three labile SOC fractions in the top 10 cm soils across all plant communities, which could be attributed to the high availability of plant litter at the surface layer and the general reduction in biological activity and root biomass with increasing soil depth (Banger et al., 2010; Jackson et al., 1996; Hobbie et al., 2004). The ratio of labile SOC fractions to total SOC is indicative of the rate of recycling and turnover of the soil carbon pool (Jiang and Xu, 2006; Melero et al., 2009; Yang et al., 2016). Singh et al (1989) suggested that the ratios of MBC to SOC constituted the microbial quotient (MQ), with higher MQ values reflecting a trend towards SOC accumulation (Cotrufo et al., 2002). Yet, several other studies have reported a negative relationship between MQ and SOC (Jiang, H. et al., 2006; Zhang et al., 2011; Yang et al., 2013). We found lower MQ values in the soils of the S. alterniflora communities as compared to that of C. malaccensis, and with increasing age of invasion (Fig.4 A). Together with the results of SOC dynamics (Fig.2 A), our findings suggest that the lower MQ values and hence lower microbial carbon use efficiency would play a role in enhancing soil C accumulation in response to Spartina invasion by reducing litter decomposition (Godoy et al., 2010). Both the ratios of DOC and EOC to SOC were lower in the S. alterniflora soils than the C. malaccensis ones, and decreased with invasion age which were similar to the pattern of MQ changes (Fig.4 A, B and C). These were in accordance with the findings of Yang et al. (2013) and Wang (2010) that lower ratios of DOC/SOC and EOC/SOC indicated a decrease in soil C availability and C oxidation efficiency, respectively, which increased SOC stability and supported greater C accumulation in soils. The proportion of labile C (including DOC, MBC and EOC) to total SOC was lower following S. alterniflora invasion, owing to the litter of S. alterniflora containing inherently more recalcitrant substances (e.g., lignin) with a lower quality that reduced the rate of litter decomposition (Yang et al., 2009; Tong et al., 2011). The higher soil moisture and salinity as well as a lower pH in the S. alterniflora soils than the C. malaccensis soils (Table II) would limit the supply of available nutrients and hinder microbial activities belowground (Yang et al., 2013; Fig.5). Moreover, the high C:N ratio of the organic matter of S. alterniflora communities would reduce microbial activities when they entered the nitrogen-limited Min River estuarine wetlands (Wang et al., 2014). Thus, it could be concluded that S. alterniflora invasions would lower the ratios of labile C to SOC by decreasing the concentrations of soil available C, suppressing microbial C use, and reducing the C oxidation efficiency per unit of SOC.

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Numerous studies have reported the SOC levels in S. alterniflora marshes, which are widely distributed in the estuarine and coastal zones of China (Table IV). Our results showed that the SOC concentration of S. alterniflora marshes in the Min River estuary was an order of magnitude higher than that in most other coastal wetlands, including those in Jiangsu Province, the Yangtze River estuary, and Hangzhou Bay, which could be related to the fact that the wetlands in our study site was already dominated by another C4 plant C. malaccensis before the arrival of Spartina. The SOC concentrations in the top 50 cm soils of C. malaccensis communities ranged between 15.31 and 17.16 g kg-1, which were even higher than those of the S. alterniflora communities in other coastal wetlands. As such, the background level of SOC in the marshes of our study was already high prior to Spartina invasion. In addition, the biomass of S. alterniflora was higher in our study area as compared to other regions because of the lower latitudinal location and higher mean temperature. For example, the aboveground biomass of S. alterniflora in the Xinyanggang coastal wetlands 3 years post-invasion was only 762.11 g m-2 (Wang et al, 2013), while that in the Min River estuary was three-fold higher with a mean of 2,383.83 g m-2 (Tong et al, 2011). Furthermore, the SOC concentrations in wetland soils could be affected by sampling time and flooding frequency (Zhou et al., 2008; Bai et al, 2012; Yang et al, 2013). Our study was conducted in November, which was the time with the seasonal maximum in both the biomass of S. alterniflora and SOC (Tong et al, 2011; Yang et al, 2013). Also, our sampling sites were located in the tidal beach with higher elevation for SOC accumulation, which experienced less frequent tidal flooding and less C oss by tidal water (Kulawardhana et al, 2012). All the above factors together contributed to higher SOC concentrations in the S. alterniflora communities of the Min River estuary than other coastal wetlands.

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TABLE IV

A comparison of SOC concentrations (g kg-1) in different estuarine and coastal S. alterniflora marshes in China.

Location

Sampling time

Soil depth (cm)

Invasio n years

Yancheng Natural Reserve

33°22′N, 20°42′E

Every month

0-20

10

Wanggang coastal wetland

33°17′N, 20°45′E

April, July, October, December

0-20

4

Wanggang estuarine wetland

33°09′N, 20°47′E

October

0-10

8,12,14

Yancheng Natural Reserve

32°36′34°28′N, 119°51′E121°5′E

April, June, October, December

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Study area

Invaded community Suaeda salsa

mudflat

SOC b)

Reference

13.55

Yuan et al. (2015)

3.354.70

Suaeda

3.67-

salsa

4.96

Zhou et al. (2008) Zhang et al. (2010)

mudflat, Suaeda

0-30

10

salsa,

6.1-10.1

Yang et al. (2013)

4.0-5.7

Cheng et al. (2006)

Phragmites australis

Yangtze River estuarine wetland

31°03′31°17′N, 121°46′122°45′E

October

0-100

7

Scirpus mariqueter

12

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Hangzhou Bay wetland

30°18′N, 121°5′E

Phragmites

May

0-30

ND

australis, Scirpus

6.466.78

Zhang et al. (2014)

mariqueters

Min River estuarine wetland

26°00′26°03′N, 119°3419°41′E

Novembe r

0-50

0-12

Cyperus

14.34-

malaccensis

23.50

This study

ND = No Data.

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CONCLUSIONS

Foundation items

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We found that invasions by the exotic C4 plant S. alterniflora significantly increased SOC, TN, C:N ratio, DOC, MBC and EOC as compared to the native C4 C. malaccensis marshlands, although the response of SOC in the surface (0–10 cm) and deeper layers (10–50 cm) showed different patterns along the chronosequence. The lower ratios of DOC/SOC, MBC/SOC, and EOC/SOC in the soils of S. alterniflora than that of C. malaccensis indicated a lower soil C availability, a lower microbial C use efficiency, a lower C oxidation efficiency, as well as an accelerated SOC sequestration in this estuarine marsh subsequent to Spartina invasion. Our findings suggest that coastal wetlands dominated by C4 plants that are subjected to S. alterniflora invasion have a high potential to sequester additional C and N in soils through the increase in litter inputs and decrease in the proportion of labile C to total SOC.

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National Science Foundation of China (Grant No 41671088), the Program for Innovative Research Team at Fujian Normal University (IRTL1205), CUHK Direct Grant (4052119) and Research Grants Council of the Hong Kong Special Administrative Region, China (CUHK458913).

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Fig.1. Study area and sampling sites in the tidal marshes of the Min River estuary. CM = C. malaccensis; SA-4 = S. alterniflora (0–4 years); SA-8 = S. alterniflora (4–8 years); SA-12 = S. alterniflora (8–12 years).

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3 4 5 6 7 8 9 10 11 12 13 14 15

Fig. 2 Variations of (A) SOC and (B) TN concentrations and (C) C:N ratio (mean ± SE, n = 3) at different soil depths in the C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05 in the same layer. SOC = soil organic carbon; TN = total nitrogen. See Fig. 1 for abbreviations of CM, SA-4, SA-8 and SA-12.

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Fig. 3 Variations of SOC fractions, including (A) DOC, (B) MBC, and (C) EOC (mean ± SE, n = 3) at different soil depths in the C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05 in the same layer. SOC = soil organic carbon; DOC = dissolved organic carbon; MBC = microbial biomass carbon; EOC = easily oxidizable carbon. See Fig. 1 for abbreviations of CM, SA-4, SA-8 and SA-12.

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Fig. 4 Mean ratios of (A) DOC/SOC, (B) MBC/SOC, and (C) EOC/SOC in the top 50 cm soils (mean ± SE, n = 15) of C. malaccensis and S. alterniflora communities with different invasion ages. Different lower case letters indicate statistical significance among communities at P = 0.05. See Figs. 1-3 for abbreviations of CM, SA-4, SA-8, SA-12, SOC, DOC, MBC and EOC.

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Fig. 5 Redundancy analysis of SOC fractions and soil physico-chemical properties in the C. malaccensis and S. alterniflora communities with different invasion ages (n = 60). The total percentage of variability explained by all the canonical axes was 86.9%. SOC = soil organic carbon; TN = total nitrogen; DOC = dissolved organic carbon; MBC = microbial biomass carbon; EOC = easily oxidizable carbon; EC = electrical conductivity; BD = bulk density.

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