Spartina alterniflora raised soil sulfide content by regulating sulfur cycle-associated bacteria in the Jiuduansha Wetland of China

The Spartina alterniflora invasion across the southeast coast in China significantly reduced vegetation diversity and generated associated ecological problems. Sulfur (S) is a vital nutrient, while sulfide is phytotoxic and the impact of S. alterniflora invasion on soil S cycle remains unclear. Therefore, this study aims to investigate the impacts of S. alterniflora invasion on the S cycle and associated soil microbial communities. Both field investigation and lab-scale experiments were conducted, analyzing soil sulfide and sulfate contents, soil properties over four seasons in the Jiuduansha Wetland of Shanghai, China, the high-throughput sequencing of soil microbial communities, S cycle-related functional genes and seed germination experiments. The contents of sulfide, soil organic carbon (SOC), and total nitrogen (TN) in the bulk soil of S. alterniflora invaded area were higher than those in the native species S. mariqueter habitat and bare mudflat soils. Spartina alterniflora invasion increased the abundance of the Nitrospiraceae and Desulfarculaceae families and reduced that of Hydrogenophilaceae. The relative abundance of the SO42− reduction functional genes (dsrA + dsrB) in the soil was increased after S. alterniflora invasion, while that of the S oxidation functional genes (yedZ + soxY) in the soil was reduced. Seed germination experiments with different sodium sulfide concentrations (Na2S) revealed that the phytotoxicity of sulfide caused more lethal damage to S. mariqueter than to S. alterniflora. The S. alterniflora invasion significantly increased SOC and TN contents and reduced the abundance of sulfur-oxidizing functional genes, which led to the accumulation of soil sulfide.


Introduction
Plant invasion can dramatically reduce the biodiversity of the native ecosystem by dramatically reducing the abundance and survival rate of native species (Mack et al. 2000). It also generates ecosystem degradation by altering both soil nutrient cycling processes and hydrological parameters (Sanderson et al. alterniflora is an invasive plant in coastal wetlands of China across climate zones, which was introduced to China to reduce soil erosion in the 1990s (Chung et al. 2004;Zhou et al. 2009). As a C4 plant, the S. alterniflora has higher photosynthetic efficiency and water use efficiency than the native species Scirpus mariqueter (Cheng et al. 2006;Liao et al. 2007;Ge et al. 2015). Previous researches revealed that plant invasion could have distinct impacts on soil elemental distribution and transformation (Yu et al. 2015;Feng et al. 2018).
Sulfur (S) plays a vital role in plant physiological processes, while high soil S levels can be phytotoxic (Lamers et al. 2013). The S. alterniflora can raise soil S contents and generate environmental stress on native species during its invasion (Xia et al. 2015). Soil organic matter and total S content increase can promote the reduction of sulfate, leading to phytotoxic sulfide accumulation in soil (Li et al. 2019). Sulfide includes three chemical forms (H 2 S, HS − , S 2− ) depending on soil pH, which are in a complicated dynamic equilibrium (Eq. 1) (Armstrong and Armstrong 2005). When pH is around 7.5 in a coastal wetland, the dominant species of sulfide is bisulfide (HS − ) (Lamers et al. 2013). Soil sulfide content can alter the composition and distribution of vegetation depending on its sulfide tolerance level (Li et al. 2009). High soil sulfide levels can inhibit plant growth by reducing oxidase activity in cell mitochondria (Bagarinao 1992). Previous research indicated that increased organic matter input could result in higher soil sulfide levels by increasing S mineralization and offering substrate for sulfur-reducing bacteria (Scherer 2009). S. alterniflora is a high sulfur-tolerant wetland plant than lots of native plants since it can absorb sulfides and perform sulfide oxidation at the root zone (Carlson and Forrest 1982;Holmer et al. 2002;Martin et al. 2019). The plant S storage in S. alterniflora tissues was found significantly higher than that in native species (Chambers et al. 1998;Zhou et al. 2009).
The S. alterniflora invasion impacts soil S cycleassociated microbial activities. Sulfate can be reduced to sulfide by sulfate-reducing bacteria (SRB) in anoxic environments (Niu et al. 2021). The sulfuroxidizing bacteria (SOB) changes S 2− to a higher (1) H 2 S ↔ HS − +H + ↔ S 2− + 2H + valence state (Xing et al. 2007). Both SRB and SOB play significant roles in the S cycle and the distribution of soil S forms. Our previous research found that S. alterniflora invasion into coastal salt marsh promoted SRB activities and soil S accumulation, which promotes the invasion of the sulfur tolerant plant (Zheng et al. 2017). Zeleke et al. (2013) found that the dsrB (SRB) gene copy number in the soil of S. alterniflora invaded area was 2.9 times higher than that in the soil of P. australis. Cui et al. (2017) found that S. alterniflora invasion significantly increased the abundance and diversity of soil SRB community in China's eastern coast. There is still a lack of systematic research on how S. alterniflora initiate its invasion by affecting microorganisms related to the S cycle.
We aim to investigate the impacts of S. alterniflora invasion on the S cycle and associated soil microbial communities. We hypothesized that (1) the invasion of S. alterniflora could increase SOC and TN; (2) S. alterniflora invasion altered soil S cycling associated microorganisms and resulted in an increase in sulfate reduction and a change in sulfide in the Jiuduansha Wetland; (3) increased soil sulfide level can environmentally stress on native species S. mariqueter, whereas S. alterniflora can survive under high sulfide levels. To test these hypotheses, a field investigation was carried out in the Jiuduansha Wetland, and seed germination experiments were conducted in the laboratory. This study will provide a new perspective for explaining the rapid invasion of S. alterniflora and provide a detailed reference for subsequent ecological protection.

Site description
The study was conducted in the Zhongxiasha area (30°10'N, 122°01'E) of the Jiuduansha Wetland National Nature Reserve in China (Fig. 1). The Jiuduansha Wetland is approximately 423 km 2 and has a subtropical monsoon climate with an average annual temperature of 15-17 °C and annual precipitation of about 1200 mm. Before the introduction of S. alterniflora, S. mariqueter was the local pioneer species in this region. After introducing S. alterniflora, it quickly underwent an ecological invasion and was widely distributed in the Jiuduansha Wetland. It is now a widespread intertidal species (Huang and Zhang 2007). S. alterniflora is mainly distributed in medium-low tidal flats of the Zhongxiasha area, and S. mariqueter is the pioneer plant in the Jiuduansha Wetland. It is distributed in low tidal flats (Yang and Guo 2018).

Sample collection and analyses
As shown in Fig. 1, the dominant habitat types from the beach to the island are mudflats without vegetation, the native species S. mariqueter, and the invasive species S. alterniflora. We chose sampling sites in these three habitat types, which were distributed as transects in the same area. The elevation of the mudflat, S. mariqueter, and S. alterniflora sites were 4.0, 4.2, and 4.9 m. Soil samples were collected in August and November of 2018 and in January and April of 2019 and represented different temperature and humidity features in the four seasons of spring, summer, autumn, and winter. Each sampling site was randomly sampled three times over a distance of no less than 10 m. The stem height and density of the vegetation were measured in situ. Total above-ground plant biomass was measured by drying plant samples until constant weight at 65 °C in an oven.
Soil cores were collected from depths of 0 to 100 cm by using a columnar sampler. The soil samples were kept in ziplock bags and stored at 4 °C. The samples used for further analyses of sulfide and DNA extraction were stored at -20 °C. The soil moisture content was determined by drying at 105 °C in an oven until constant weight. The samples for other physicochemical analyses were air-dried, ground, and passed through a 0.15 mm sieve. Soil pH (1:2.5) was measured in the bulk soil with a pH meter (PHS-25, China), and soil salinity (1:5) was measured with a conductivity meter (Orion 145A + , ThermoFisher Scientific, FL, USA). The soil organic carbon (SOC) content was measured with a potassium dichromate oxidation method . The soil sulfate content was measured using a barium sulfate turbidimetry method (Chen et al. 2018). Soil total phosphorus (TP) and total iron (Fe) levels were determined by ICP-AES (Teledyne Leeman Labs, Hudson, NH, USA) after soil digestion. Soil sulfide contents were determined as described by Niu et al. (2018). The soil sulfide contents were determined using the methylene blue spectrophotometry method (HJ883-2017) (Luo et al. 2018).

Soil functional bacterial measurements
The total DNA in a representative bulk soil sample from the S. mariqueter, S. alterniflora, and mudflat sites was extracted using a MetaVx™ according to the manufacturer's protocol (GENEWIZ, Inc., South Plainfield, NJ, USA). The quality of the extracted DNA was assayed using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA) to determine DNA sample concentrations, and a series of PCR primers were designed to amplify two highly variable regions (V3 and V4) of 16S rRNA genes using 30-50 ng of DNA as a template. The V3 and V4 regions were amplified using an upstream primer with the sequence 'CCT ACG GRRBGCASCAGKVRVGAAT' and a downstream primer with the sequence 'GGA CTA CNVGGG TWT CTA ATC C'. Additionally, a linker with an index was added to the end of the PCR product of 16S rRNA genes by the Real-time PCR system (Wafergen Biosystems, Fremont, USA) for next-generation sequencing (NGS). Library quality was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and library concentrations were determined with a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA). After the DNA libraries were mixed, 2 × 300 bp pairedend (PE) sequencing was performed according to the Illumina MiSeq (Illumina, San Diego, CA, USA) instrument instruction manual, and sequence information was read by MiSeq Control Software (MCS), which was included with the MiSeq device. For the high-throughput sequencing results, we referred to the classification method of Meyer et al. (2016). We divided the sulfur-related microbial communities in the high-throughput sequencing results into two categories: SRB and SOB, which can respectively reduce sulfate and oxidize sulfide. The quantitative microbial element cycling (QMEC), a high-throughput quantitative-PCR-based chip, was used to obtain the specific functional genes, including sulfur-reducing (dsrA and dsrB) and sulfur-oxidizing (soxY and yedZ) functional genes, for the S cycle (Zheng et al. 2018).

Seed germination under different sulfide levels
A lab-scale seed germination experiment with both S. alterniflora and S. mariqueter seeds was carried out to test their tolerance and survivability of sulfide under different sulfide levels. S. alterniflora and S. mariqueter seeds were collected from sampling sites in the Jiuduansha Wetland in autumn 2018. The seeds were soaked in 5% sodium chloride solution at 4 °C to preserve the vigor of seeds (Vertucci and Roos 1990). The experiment was conducted in the phytotron of the School of Environmental and Chemical Engineering, Shanghai University, in 2019, and the temperature was controlled at 28 ℃ (day) / 20 ℃ (night) (Ricketts et al. 2018), the relative humidity was 80%, and the daily light duration was 12 h.
Twenty seeds of S. alterniflora and S. mariqueter were disinfected with 0.1% sodium hypochlorite solution and rinsed with distilled water. Then the seeds were placed in Petri dishes that were cleaned and disinfected in advance. Three replications were conducted for each treatment. The dishes contained the same amount of washed and disinfected silica sand, and 10 mL of sodium sulfide (Na 2 S) solution from a gradient (0, 10, 20, 50, 100, and 200 mmol/L, pH 7.10 ~ 11.30) was added since 200 mmol/L Na 2 S was the level reported killing S. alterniflora (Shen et al. 2011). After thoroughly mixed silica sand and Na 2 S solution, 5 mL of distilled water was added, while 15 mL of distilled water was added to the control group. The weight of each Petri dish was then recorded. The Petri dishes were placed in a phytotron, and distilled water was added every 24 h to reach the original weight. The plant biomass, root length, shoot lengths were weighed and measured with electronic balance and rulers after 7 days of seed culture.

Data analysis
The stem height, stem density, and above-ground biomass of S. alterniflora and S. mariqueter were calculated by averaging morphological data of four seasons in the field investigation. The soil water content, pH, and salinity in five soil layers (0-20 cm, 20-40 cm, 40-60 cm, 60-80 cm, and 80-100 cm) in the range of 0-100 cm were calculated by averaging the values of four seasons. The soil sulfide and sulfate concentrations of different seasons were calculated by averaging the data in five depths, and significant differences between four seasons and three sites were analyzed using analysis of variance. The annual average sulfide, sulfate, SOC, TN, TP, and total iron concentrations in soil were calculated by averaging all data in five depths and four seasons. Statistically significant differences between vegetation types were analyzed using one-way analysis of variance and Tukey's range test at 5% significance level (Dat et al. 2000). All reported differences were statistically significant at a P < 0.05 level. The above statistical analyses of the data were performed using SPSS 18.0 (SPSS for Windows, SPSS, Inc.), and diagrams were created with OriginPro 8.5. The relationships between sulfide and soil parameters (water content, SOC, TN, TP, Fe, sulfide, sulfate) were analyzed using principal component analysis (PCA) using Origin 8.5 software and Pearson correlation analysis using SPSS 18.0.
A principal coordinates analysis (PCoA) was also applied, to explore the difference of bacterial communities among the soils collected from different sites.

Vegetation and soil properties
As shown in Table 1, the plant stem height and above-ground biomass of S. alterniflora were 8 and 3 times higher than those of S. mariqueter, respectively. In contrast, the stem density of S. alterniflora was significantly lower than that of S. mariqueter. Both soil moisture content and salinity in S. alterniflora invaded area were higher than those in S. mariqueter and mudflat soils. In contrast, the S. alterniflora invaded area's soil pH was lower than that in the mudflat soil.
The sulfide concentrations of soils from the mudflat were generally the lowest, with an average value of 22.10 ± 19.87 mg/kg. The sulfide concentrations in S. mariqueter soil were higher than those in the mudflat soil, from 8.35 ± 1.22 mg/kg to 286.88 ± 79.22 mg/ kg with an average value of 79.65 ± 65.53 mg/kg. The highest sulfide concentration was observed in S. alterniflora soil (210.97 ± 133.41 mg/kg), which was 9.56 and 2.65 times higher than that in the mudflat and S. mariqueter soils, respectively ( Fig. 2A). The Table 1 Vegetation and soil properties of three sampling sites (Mudflat (MF); S. mariqueter (SM); S. alterniflora (SA)). The data are expressed as means ± SDs, which are the annual averages  S. alterniflora (SA)). The boxes display the upper and lower quartiles of the data, and whiskers represent the standard deviation above and below the mean of the data. Different letters indicate significant differences between three sampling sites at a p < 0.05 level  (Table 2). Among soils covered by S. alterniflora and S. mariqueter and in the nearby bare mudflat, the sulfate contents were generally higher in spring and summer (P < 0.05) ( Table 2). The specific sulfide and sulfate concentrations of five depths and three sites in four seasons were shown in Fig. S1 and Fig. S2. The SOC content in S. alterniflora soil was 6.77 ± 1.94 mg/g, which was much higher than that in S. mariqueter soil (3.68 ± 1.43 mg/g) and mudflat soil (3.08 ± 1.49 mg/g) (Fig. 2C). The TN content for S. alterniflora soil was 286.65 ± 64.56 mg/ kg, which was higher than that for S. mariqueter (155.57 ± 36.72 mg/kg) and mudflat soils (137.64 ± 31.42 mg/kg) (Fig. 2D). The specific SOC and TN concentrations of five depths and three sites in four seasons were shown in Fig. S3 and Fig. S4. There was no significant difference in the averaged TP (Fig. 2E) and Fe content (Fig. 2F). In summer, the TP content for S. alterniflora soil was lower than that for the mudflat and S. mariqueter soils (Fig. S5). In all depths of soil in summer and 0-40 cm of soil in autumn, the total iron contents of S. alterniflora soil were higher than those of the native S. mariqueter soil. In winter, the total iron content of S. mariqueter soil was higher on the contrary (Fig. S6).

Relationship between sulfide, sulfate, and environmental factors
In the mudflat soil, the sulfide content was substantially correlated with total iron (r = 0.497, p < 0.05) and TP (r = 0.571, p < 0.01), and sulfate was significantly correlated with TN (r = 0.668, p < 0.01). Meanwhile, the total iron was positively correlated with TP in the mudflat soil (r = 0.761, p < 0.01) (Fig. 3a). In the S. mariqueter soil, there was a negative relationship between sulfate and total iron (r = -0.595, p < 0.01) (Fig. 3b). In the S. alterniflora soil, the sulfide content exhibited a strong correlation with SOC (r = 0.616, p < 0.01) and total iron (r = 0.472, p < 0.05), and sulfate was significantly correlated with TP (r = 0.547, p < 0.05). Besides, SOC was positively correlated with total iron and TP, with coefficients of 0.546 (p < 0.05) and 0.468 (p < 0.05), respectively. The total iron in the S. alterniflora soil exhibited a significantly positive correlation with TP (r = 0.578, p < 0.01) (Fig. 3c).

Effects of plant invasion on S cycle-related soil functional bacteria
The PCoA mapping analysis results based on the Bray-Curtis distance matrix (Fig. 4) showed that the bacterial communities were different among the soils collected from different sites. The PC1 and PC2 axes together accounted for 52.46% of the variation. The relative abundance of SRB measured at the family level was lower for S. alterniflora soil than in the other soils (Fig. 5A). The relative abundance of SRB in S. alterniflora soil was 7.32%, which was lower than that in S. mariqueter soil (14.9%) and higher than that in mudflat soil (7.28%). The main groups of SRB in S. alterniflora soil were Desulfobulbaceae, Desulfuromonadales, and Desulfobacteraceae, which accounted for 68.2% of the SRB S. alterniflora community, and with the invasion of S. alterniflora, Nitrospiraceae and Desulfarculaceae increased (Fig. 5A). The relative SOB abundances were lower for S. alterniflora soil (3.03%) than for S. mariqueter (3.75%) soil and for mudflat (4.49%) soil (Fig. 5B). The S. alterniflora communities mainly consisted of Rhodobacteraceae and Hyphomicrobiaceae, while Hydrogenophilaceae was the main SOB component in the mudflat and S. mariqueter soils. The QMEC test results showed that the variation trend of the relative abundance of sulfur-reducing functional genes was similar to that of the sulfide content, and the main sulfur-reducing functional gene was dsrA. The highest sulfur-reducing functional gene abundance, which was 1.49%, appeared in the soil of S. alterniflora, while in the mudflat and S. mariqueter soils, the abundances were 1.27% and 1.17%, respectively (Fig. 5C). The primary sulfur-oxidizing functional gene was yedZ. The lowest abundance of sulfur-oxidizing functional genes was observed in the soil of S. alterniflora, which was 0.19%, while in the mudflat and S. mariqueter soils, the abundances were 0.25% and 0.32%, respectively (Fig. 5D).

Effects of sulfide on the growth of S. alterniflora and S. mariqueter
After seven days of the seed germination experiment, the root length, shoot length, and biomass of S. alterniflora and S. mariqueter showed variability along the gradients of Na 2 S incubations (Fig. 6). The root tissues of S. mariqueter ceased to grow when the Na 2 S concentration reached 50 mmol/L. Although low concentrations of Na 2 S had no significant effect on S. alterniflora root length, S. alterniflora root length showed a decreasing trend with the increase of Na 2 S concentration. The root tissue of S. alterniflora was still alive at the concentration of Na 2 S was as high as 200 mmol/L (Fig. 6A). When the concentration of Na 2 S reached 50 mmol/L, a significant decrease in the shoot lengths of S. mariqueter occurred. However, with the increase of the concentration of Na 2 S, there was no significant effect on the shoot lengths of S. alterniflora increased (Fig. 6B). Na 2 S concentrations over 100 mmol/L significantly reduced S. mariqueter plant biomass, while the biomass of S. alterniflora remains the same at high levels of Na 2 S concentration (Fig. 6C).

S. alterniflora might increase soil microbial biomass and activity by increasing soil SOC and TN
Our findings suggest that the S. alterniflora invasion leads to increased soil SOC (Fig. 2C) and TN (Fig. 2D), which might provide abundant substrate for more active microbial activity. The increase in plant biomass can promote the input of organic matter, carbon, and nitrogen, which can further change the ratio of carbon and nitrogen, forming a different environment for soil microorganisms (Hu et al. 2016). Plant invasion changes soil properties mainly through the litter and root exudates , and soil microbial structures were selected by the composition of plant litter. S. alterniflora has a more vital ability to enhance above-ground net primary productivity and preserve soil organic carbon than S. mariqueter due to the larger biomass of S. alterniflora, as aboveground plant biomass is the main input route for soil organic carbon (Carrera et al. 2009). In addition, the increase in soil SOC and TN contents arise due to the higher input of S. alterniflora tissue decomposition. Relatively higher nitrogen content was also found in S. alterniflora tissues, which may contribute to the raised soil TN (Zheng et al. 2017). Fig. 6 A Root lengths, B shoot lengths, and C biomass of seed germination of S. alterniflora (SA) and S. mariqueter (SM) under different concentrations of Na 2 S. Lowercase letters and uppercase letters respectively indicate significant differences (P < 0.05) of S. alterniflora and S. mariqueter between means of different stands S. alterniflora altered soil S cycle-related microbial activities and resulted in soil sulfide accumulation We reported that the increase in the abundance of S reduction-relative functional genes (dsrA and dsrB) and the decrease in the abundance of S oxidation-relative functional genes (soxY and yedZ) were consistent with the increase in phytotoxic soil sulfide after S. alterniflorafor the first time. Sulfide is reductive phytotoxicity in the rapid redox reactions with sulfate under tidal conditions in coastal wetlands (Lamers et al. 2013). According to our investigation, the invasion of S. alterniflora changes the community structure and living environment of soil microorganisms into a more suitable condition for sulfide production and accumulation. The differences between the horizontal and vertical projections of PCoA in the two sets of comparisons of S. alterniflora and S. mariqueter implied that these two plants possessed different mechanisms for changing the microbial community structure in soil (Fig. 4).
The composition and structure of S-associated bacterial communities in the soil were altered after S. alterniflora invasion (Fig. 5A). Desulfobulbaceae, Desulfuromonadales, and Desulfobacteraceaeare strictly anaerobic SRB that can transform sulfates into sulfides in a flooded environment and play a central role in methane formation and dissimilatory sulfate reduction (Kuever 2014). The lower relative abundance of Hydrogenophilaceae in the soil of S. alterniflora (Fig. 5B), which is a type of SOB, might lead to the oxidation of reductive soil sulfide being inhibited and further contributed to the accumulation of soil sulfide ( Fig. 2A). DsrA and dsrB are functional genes involved in the S cycle; they catalyze the reduction of sulfite (SO 3 2− ) to sulfide (S 2− ) in the S cycle (Muyzer and Stams 2008). We found that the invasion of S. alterniflora promoted the microbial sulfur reduction process by increasing the relative abundance of the dsrB functional gene in the soil and changing their community structure (Fig. 5C). A trend was found in the relative abundance of dsrA, while it was not statistically significant, thereby led to the increase of soil sulfide content ( Fig. 2A). The invasion of S. alterniflora resulted in a decrease in the relative abundance of the SOB in the soil (Fig. 5D), which reduced the oxidation of sulfur compounds and led to the accumulation of sulfide compounds ( Fig. 2A). The SOB is composed of autotrophs and facultative autotrophs (Mattes et al. 2013), which can consume H 2 S and produce oxidized S compounds such as sulfides, elemental sulfur, sulfites, and thiosulfates (Meyer et al. 2016). Therefore, the increasing SOC after S. alterniflora invasion cannot accelerate the growth of SOB. Sox family can ultimately reduce sulfide compounds to sulfate (Friedrich et al. 2001). The sox multienzyme system is considered to be the basic and original molecular mechanism of S oxidation (Ghosh and Dam 2009); yedZ is considered to have a sulfite oxidation function (Brokx et al. 2005;Zheng et al. 2018).
In 2017, our team found an increase in the copy numbers of the dsrB functional gene in SRB after S. alterniflora invasion in Dongtan Saltmarsh located in Chongming Island (Zheng et al. 2017), but no significant variation in soxB was observed. It might be because the sampling sites of S. alterniflora were invaded for over 20 years with stable soil physicochemical properties and microbial structure. Previous studies have found the dsrAB coded by the dsr genes in certain strains of SOB, which may lead to the insignificant variation in sox observed with the offset by increasing dsrAB (Loy et al. 2009). In the researches of Cui and Zeleke, a higher abundance of dsrB genes was observed in the soil of S. alterniflora than the native species Suaeda salsa (S. salsa) and P. australis in the Dongtan Wetland in the Yangtze River estuary and Yancheng Nature Reserve, which consisted with our findings (Zeleke et al. 2013;Cui et al. 2017).
S. alterniflora invade successfully with its high tolerance to high sulfide concentration Higher tolerance to sulfide was revealed in S. alterniflora since the decrease in S. alterniflora biomass was much smaller than that of S. mariqueter biomass (Fig. 6A), and the negative effect on the growth of S. alterniflora roots and shoots was significantly weaker than that on the growth of S. mariqueter roots and shoots (Fig. 6B, C), according to our lab experiment. The phytotoxicity of sulfide mainly influences the growth of roots suppressing water and nutrients absorption from the soil, which was consistent with published results that the toxicity of sulfide to plants mainly affects roots by preventing the roots from transporting energy needed for plant growth (Raven and Scrimgeour 1997).
Our findings confirmed that S. alterniflora is a high-sulfur-tolerant plant with a variety of mechanisms to resist sulfide toxicity, such as secretion of oxygen from roots, oxidation of sulfides in tissues (Carlson and Forrest 1982;Martin et al. 2019) and formation of insoluble iron sulfide metals (Marbà et al. 2007). More developed root tissues in S. alterniflora than in the native species P. australis and S. mariqueter also lead to more resistant vitality of S. alterniflora under high sulfide conditions (Stribling 1997;Seliskar et al. 2004). Iron can form FeS and FeS 2 with S to reduce sulfide toxicity (Van Der Welle et al. 2006;Marbà et al. 2007;Sjøgaard et al. 2017). The differences in oxygen transport patterns and root radial oxygen loss (ROL) might lead to differences in Fe plaque formation in the roots to protect plants from adverse and toxic environments (Zhang et al. 2021). Raymond et al. (1999) also reported that root tips from S. alterniflora exhibited a substantial capacity to catalyze sulfide oxidation, especially in a high sulfide environment. Thus, the formation of iron plaque in the roots of S. alterniflora might be one of the mechanisms by which S. alterniflora resists the toxicity of sulfides, and it might result in Fe enriched on the surface of roots which was unable to explore in our result of bulk soil. Simkin et al. (2013) proved that sulfide in pore water helps explain the reduction in plant species diversity and the reduction in coverage of many plant species. However, we did not observe significant variation in soil Fe content. Howes and Teal. (1994) reported oxygen loss from S. alterniflora, and Maricle and Lee. (2002) reported that aerenchyma formation occurred in S. alterniflora. On the contrary, no discovery of aerenchyma in S. mariqueter was published till now. Compared with the S. mariqueter and mudflat soils, S. alterniflora soil showed the shortest anaerobic reduction environment and shortest flooding time caused by its location in the most inland area. Under such flooding conditions, the highest sulfide content was still found in the soils of S. alterniflora. Thus, native species lacking sulfide tolerance capacities could be disadvantaged in the competition with S. alterniflora due to the high soil sulfide content derived from soil S-cycle microbial changes after S. alterniflora invasion was more lethal to the growth of local species.

Conclusions
We found that S. alterniflora invasion significantly altered the physicochemical properties of the soil and sulfur-related bacterial community. First, the contents of SOC and TN in soil increased with the invasion of S. alterniflora, which could provide more substrates for sulfur-reducing bacteria to cause sulfide accumulation in the soil. Second, the abundance of sulfur-oxidizing functional genes decreased after the invasion of S. alterniflora and thus reduced the consumption of sulfide, possibly through root exudates and litter pathways. Third, the phytotoxicity of the increased soil sulfide levels caused much more lethal damage to S. mariqueter than to S. alterniflora, so that S. alterniflora could survive in a high-sulfide environment while S. mariqueter could not. Our findings provide a new perspective on how S. alterniflora is able to rapidly replace the native species S. mariqueter in the Jiuduansha Wetland.