Sand Supplementation to Eutrophic Sediments Improves the Growth and Survival of Seagrass Thalassia Hemprichii: Implication for Seagrass Restoration and Management


 Aims Sediment composition is highly crucial for seagrass growth and survival. Eutrophication has been suggested a major cause of seagrass decline globally. We investigated the effects of beach sand supplementation to natural sediments under eutrophic condition on the growth and survival of tropical dominant seagrass Thalassia hemprichii. Methods We cultured seagrass T. hemprichii under the controlled laboratory conditions in three sediment types by combining different ratio of in-situ eutrophic sediment and coarse beach sand. We examined the effect of beach sand mixing to natural eutrophic sediments on the growth of seagrass using photobiology, metabolomics and isotope labeling approaches. Results Seagrass grown in eutrophic sediments mixed with sand exhibited significantly higher photosynthetic activity with high relative maximum electron transport rate and minimum saturating irradiance. Simultaneously, considerably greater belowground amino acid and flavonoid concentrations were observed to counteract anoxic stress in eutrophic sediment without mixing sand. This led to more positive belowground stable sulfur isotope in the eutrophic sediment with lower Eh. Conclusions These results indicated coarse beach sand indirectly enhanced photosynthesis and growth for T. hemprichii by reducing sulfide intrusion with lower concentrations of amino acid and flavonoid. This could possibly explain why T. hemprichii often grow better in the coarse sand substrate. Therefore, it is imperative to consider adding sand soil in the sediments to improve the growth condition for seagrass and restoring the seagrass shoots during transplantation in eutrophicated ecosystem.


Introduction
Seagrass meadows are marine ecosystem engineers and provide important ecological services including nutrient uptake, carbon sequestration, food source and habitat, shoreline and sediment stabilization (Hemminga and Duarte 2000; Larkum et al. 2006). However, global climate change and sustained pressures from coastal developments including dredging and eutrophication (nutrient enrichment) have weakened the capacity of seagrass meadows to support coastal productivity (Jiang et al. 2018; Waycott et al. 2009). Eutrophication stimulates opportunistic macro and micro algae proliferation (Burkholder et al. 2007; de Boer 2007) over the seagrass canopy and thereby reduces the light available to seagrasses below them causing shading effect. Meanwhile, algal proliferation also produced high detritus (Bishop and Kelaher 2013), modifying the origin, grain size and nutrient availability of the sediment (de Boer 2007; Liu et al. 2016). Moreover, anoxic occurred due to eutrophication and induced the sul de intrusion in seagrasses, as assessed by stable sulfur isotope signals, leading to adverse effects as well (Holmer and Hasler-Sheetal 2014). To date, the physiological indicators have largely failed to monitor seagrass health and prevent their decline. This alarming decline highlights a global urgency to implement effective seagrass management strategies to prevent their decline.  Pérez et al. 2007) and the total content of key secondary metabolites to environmental stress (Arnold et al. 2014). However, little is available on the response of the composition of key secondary metabolites by applying targeted metabolomics technique.
Seagrass Thalassia hemprichii, the dominant tropical specie, distributes chie y in sandy sediment or coral substrate (Chiu et al. 2013;Jiang et al. 2017). Over the decade, nutrient inputs into seagrass bed in Xincun Bay, Hainan Island, South China Sea, has increased immensely, leading to high eutrophication (Jiang et al. 2018). Macroalgal and epiphytes have proliferated, covering on seagrasses and contributed large detritus to the surface sediments ). Meanwhile, cage farming and shrimp pond culture here have also produced large quantity of food debris, that has modi ed the sediment particle sizes (Jiang et al. 2018). Sediment particle size has decreased from coarse to ne particles. Hypoxic conditions in the sediments occurs frequently, even the emergence of red tides have been noticed in these areas (Li et al. 2010). Overall, these adverse environmental conditions have resulted in a decline of seagrass beds by decreasing the area about 50 ha ). Interestingly, according to our continuous observation, the occurrence of T. hemprichii in Xincun Bay has declined dramatically, especially in the high intertidal zones. Moreover, we have observed a very low success rate in transplanting and restoring T. hemprichii in this bay (personal observations). The failure might be attributed to desiccation exposure during low tide, sediment composition (mud versus sand) and so on (van Katwijk and Wijgergangs 2004). Nevertheless, far fewer studies have considered the effect of sediment type on seagrass physiology (de Kock et al. 2020), especially the avonoid, the key secondary metabolics. Furthermore, sulfation of avonoids in seagrass might mitigate the sul de intrusion (Grignon-Dubois and Rezzonico 2018), but less evidence is available. Therefore, it is highly imperative to investigate the effect of sediment types on the physiological responses of the tropical dominant seagrass T. hemprichii. We undertook a laboratory manipulative experiment by growing T. hemprichii under three sediment types (by combining different ratio of in-situ eutrophic sediment and coarse beach sand) and assessed its growth performance by evaluating photosynthetic performance, pro ling of avonoid and amino acids together with stable sulfur isotope and elemental compositions. The pulse amplitude modulated uorometry was applied, since it could accurately assess the continuous photosynthetic characteristics of seagrasses in the same leaf in a non-destructive way (Beer and Björk 2000;Ralph et al. 1998), without disturbing sediments as well. We hypothesized that 1) seagrass cultures in sediment with large particle size shows higher photosynthetic e ciency due to oxygenic environment and less sul de intrusion, 2) sediment with small particle size causes hypoxic stress with accumulation of diverse avonoids and amino acids, 3) sediment with small particle size induces higher belowgroundδ 34 S due to the consumption of the sul de intruded into seagrass by enhanced avonoids. The results obtained in this study provide new insights that aid to understand the mechanism controlling seagrass physiological response to sediment types. This information is critical for strengthening the knowledge on improving successful rate for seagrass planting and transplantation in the coastal areas that are eutrophic already or in the process of eutrophication, and to implement effective seagrass management strategies to prevent their decline.

Materials And Methods
About 250 Intact shoots of healthy T. hemprichii in the same patch were collected to avoid patch difference. It's a sand-clay site with a water depth of ~2 m in Xincun Bay (18°24′34″N -18°24′42″N, 109°57′42″E-109°57′58″E) located in southeast of Hainan Island, Southern China. The seagrass density was between 208 and 340 shoots/m 2 , and the biomass ratio of aboveground to belowground tissue was between 0.15 and 0.20. Plants were collected carefully to keep belowground structures intact, and transported immediately to the laboratory in buckets with cover containing seawater. Meanwhile, 2 boxes of in-situ sediment below T. hemprichii and 1 box of coarse beach sand without sieving along the coastline were also collected. Plants were gently washed with in-situ seawater, separated into single shoot, and then cultured in the aquarium with in-situ seawater and sediments for 7 d prior to the start of the experiments. The light intensity at the surface of the seagrass leaves was 150 μmol photons m -2 s -1 , and the temperature was kept at 25 ℃ using air conditioning. Light was applied with 400 W metal-halide lamps, and was set on a 12 hour cycle.

Experimental design
T. hemprichii was placed into 9 glass tanks (270 × 220 × 250 mm) with 20 shoots in each tank, and cultured at three sediment types with in-situ sediment combination with different ratio of coarse beach sand (Fig. 1). The sediment thickness was 8 cm, and the overlying volume of the seawater was 8.91 dm 3 .
The seawater pH, salinity and dissolved inorganic nitrogen were 8.08±0.04, 30.45±0.92, 7.45±0.74 μmol L -1 , respectively. The seawater was aerated without replicating tides, since the seagrasses collected was in the lower intertidal with little air exposure. 1:0 represented the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1 represented the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2 represented the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank. The sediment physiochemical parameters were showed in Table 1. The concentrations of sediment organic carbon, total nitrogen, organic matter, and sulfur under 1:0 treatment were all higher than the other two treatments, whileδ 34 S exhibited a contrast trend. For the particle size, an increased trend was observed for sand composition from 1:0, 1:1 and 1:2, while clay composition showed an inverse trend (Table 1). For the sand composition, a decrease trend was found for the coarse sand composition from 1:0, 1:1 and 1:2, while ne sand indicated a contrast trend. Plants were maintained under these conditions for 21 d. Photosynthetic performance and biochemical analysis A PAM uorometer (Mini-PAM, WALZGmbH) was used to generate effective quantum yield (Φ PSII ) and rapid light curves (RLCs). Photosynthetic performance was measured for the same shoot in each tank in day 6 and day 21. An effective yield measurement was taken using a saturation pulse of 0.8 s, before the actinic light was applied (~quasi-darkness), and at the end of each 10 s irradiance step, resulting in nine Φ PSII measurements (Ralph and Gademann 2005). Without knowledge of the actual amount of light being absorbed, uorescence measurements can be used as an approximation of electron transport rates (Ralph et al. 1998). We used the instrument default AF value (AF=0.84) to calculate RETR, as recommended by Beer et al. (2001). The relative maximum electron transport rate (RETR max ), representing photosynthetic capacity, was derived by tting the RLCs to the double exponential decay function (Platt et al. 1980), using a least-squares non-linear curve-tting algorithm: RETR=Ps(1−e -(αPAR/Ps) )e -(βPAR/Ps) 2 Ps value is a scaling factor which was in turn used to calculate RETR max , α(the initial slope of the light limited relationship) and β (down-regulation).
And the minimum saturating irradiance (E k ) was calculated by dividing the RETR max by the initial slope. All calculations were performed in Statistica 6.0 (Statsoft, Tulsa, Okla., USA). and individually powdered with a grinder to pass through an 80-mesh sieve (the mesh diameter was 0.18 mm) for measuring nutrients and stable sulfur isotope, while the other subsamples were sent for measuring composition of free amino acids and avonoid with dry ice. The concentrations of tissue nitrogen were determined with a CHN analyser (Elementar, Vario EL-III, Germany). Stable isotope sulfur and sulfur content were measured with DELTA V Advantage isotope mass spectrometer and EA-HT Elemental analyzer. Amino acids were measured by Waters Quattro premier XE, while avonoids were measured by Waters ACQUITY UPLC and Triple quadrupole mass spectrometer (AB 4000).

Sediment analysis
The particle sizes of the sediment samples were analyzed using a laser diffractometer (Malvem Mastersizer 2000) capable of analyzing particle sizes between 0.02-2000 μm. Grain sizes (size distribution and statistical expressions) are required to describe sediment attributes. Sand, silt and clay are end members of triangular diagrams that are divided into named textural categories depending on their apparent natural groupings (Folk et al. 1970). Grain sizes were divided into 3 groups: <4 μm (clay), 4-63 μm (silt), and >63 μm (sand) (Folk et al. 1970). The sediment samples were freeze-dried, ground and homogenized with a mortar and pestle. Half of the samples were acidi ed overnight with 1 mol/L HCl at room temperature to remove carbonate. The acidi ed samples were washed with distilled water and dried at 40 ℃ in an oven. All samples were stored in a desiccator prior to analysis. The concentrations of sediment organic carbon and total nitrogen were determined with a CHN analyser (Elementar, Vario EL-III, Germany). Sediment organic matter content was analyzed by sediment calcination in a mu e furnace (550℃ for four hours) (Heiri et al. 2001). The pH of the sediments was measured in distilled water with a 1:2.5 sediment/solution ratio using a portable pH acidometer (PHB-4).
At the end of the experiment, sediment Eh was measured with an oxidation reduction potentiometer (Mettler Toledo, Seven 2 Go).

Statistical analysis
The means and standard errors of all the variables were calculated, and all the data were rst tested to determine whether the assumptions of homogeneity and normality were met. Where these assumptions were not met, the raw data were transformed, and a further statistical analysis was conducted using the dataset that ful lled the assumptions. The effect of sediment type was analyzed by one-way ANOVA using SPSS for Windows version 18. Treatment means were compared and separated by least signi cant difference (LSD) at P<0.05. Multiple comparison test that didn't assume equal variances was Dunnett's T3.

Sediment physiochemical parameters
The Eh in the sediment type of 1:0, 1:1 and 1:2 were -177.0±29.4, -148.7±24.2 and -53.3±17.1, respectively, and the corresponding sediment sulfur content were 0.020±0.001, 0.011±0.001, and 0.006±0.002, respectively (Table 2). Meanwhile, the sediment organic matter also exhibited a decreased trend along the increased sediment particle sizes. Photosynthesis Effects of sediment type on the photosynthetic parameters at two stages were depicted in Fig. 2. No signi cant difference was observed for effective quantum yield at day 6 and day 21 (at the end of the experiment) (Fig. S1). Interestingly, there was also insigni cant difference for ETR max , E k , α (the initial slope of the light limited relationship) and β (down-regulation) among the treatments at day 6. However, markedly difference was found at day 21 for both ETR max and E k , with higher value in the sediment with smaller particle size, whileαand β exhibited a contrast trend (Fig. 2).

Nitrogen and compositions of seagrass amino acids and avonoids
Among amino acids, proline, sarcosine and lysine were the chief three compositions in the aboveground tissue of T. hemprichii, while sarcosine, proline and asparagic acid were the main three compositions in the belowground tissue. Amino acids content in the aboveground tissue was lower than in belowground tissue in sediment type of 1:0, while similar concentration was observed between aboveground and belowground tissue for T. hemprichii in both sediment types of 1:1 and 1:2. Signi cant effects were observed for 11 kinds of the 20 amino acids in the aboveground tissue, while there were 18 kinds for the belowground tissue. Amino acid content in the both aboveground and belowground tissues in 1:0 treatments were signi cantly higher than those in 1:1 and 1:2 treatments. Similarly, sarcosine, proline and alanine in the both aboveground and belowground tissues also showed the same trend. The nitrogen content in the aboveground tissue was signi cantly higher in the larger particle sizes, while the ratio of amino acids to nitrogen in the same tissue showed a contrast trend (Fig. 3, Table 3 and Table 5).
Among avonoids, galuteolin, Luteolin and Isoquercitrin were the prime three compositions in the aboveground tissue of T. hemprichii in the three sediment types. For belowground tissue, catechin isoquercitrin and epicatechin were the major three compositions in the sediment type of 1:0, while catechin, isoquercitrin and luteolin were the leading three compositions in the sediment type of 1:1 and 1:2. Flavonoids concentration in the aboveground tissue was lower than in belowground tissue in sediment type of 1:0 and 1:1, while similar concentration was observed between aboveground and belowground tissue for T. hemprichii in sediment type of 1:2. Flavonoids in the both aboveground and belowground tissues were higher in sediment type of 1:0 than of 1:1 and 1:2 (Table 4 and Table 5).
The relationships of amino acids and avonoids in the aboveground and belowground tissues were both signi cantly positive (Fig. 4). Linear regression tests were performed between the concentration of total avonoids and amino acids and the sediment sand composition. The results showed that the amino acids in the both aboveground and belowground tissues and total avonoids in the belowground tissue were signi cantly negative correlated with sediment sand composition ( Table 6).

Content of δ 34 S
Effects of sediment type on theδ 34 S content in the belowground tissue of T. hemprichii was depicted in Fig. 5. Signi cant difference was observed for theδ 34 S content, with higher value in the belowground tissue in the sediment with smaller particle size. Table 3 Effect of sediment type on the amino acids in the aboveground and belowground tissue of Thalassia hemprichii. 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank. The different lower case and upper case letters indicated signi cant differences for aboveground and belowground tissues among treatments. Table 4 Effect of sediment type on avonoids in the aboveground and belowground tissue seagrass Thalassia hemprichii. 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.

Flavonoids
Aboveground tissue Belowground tissue   Belowground amino acids and avonoids were stimulated to counteract anoxic stress in sediment with smaller particle sizes Amino acids are of great importance for protein biosynthesis, other metabolic pathways and in the signal transduction processes (D'Mello 2015). Proline and sarcosine were the main amino acids in the both aboveground and belowground tissues of T. hemprichii. For P. oceanica, asparagic acid, asparagine and glutamate were the chief amino acids in leaves, while asparagic acid, serine and alanine were the dominant components in the roots. Furthermore, proline were the main amino acids in the both aboveground and belowground tissue of Cymodocea nodosa (Pirc and Wollenweber 1988). The amino acids may change substantially in response to environmental factors. The present study indicated that the total amino acid contents in the aboveground and belowground tissues were both higher in the smaller sediment particle sizes. Especially, the belowground amino acid concentration in the smaller sediment particle sizes exhibited more than two times of that in the larger sediment particle sizes. There were two reasons to explain this phenomenon. One could be explained by that increased ammonium assimilation induced by higher nitrogen content in the sediment with smaller particle sizes takes place at the expense of glutamate and aspartate, and concomitantly with the accumulation of GABA (Good and Muench 1993). Furthermore, leucine and valine, the two branched-chain amino acids, were also enhanced, which could be de novo synthesized from pyruvate (D'Mello 2015). Proline in most of the higher plants under environmental constraints including salinity, drought and anaerobiosis often responses with an increase (D'Mello 2015). The accumulation of proline was due to an increase in proline synthesis from glutamate as well as a decrease in proline oxidation (Huang and Cavalieri 1979; Szabados and Savoure 2010). The increased proline content also like factor of free radicals detoxi cation in ooded corn plants (Yordanova and Popova 2007). Moreover, excess sulfate is also reduced to sul de and incorporated into methionine, the sulfur-containing amino acid (Nikiforova et al. 2006). Signi cantly higher methionine in the belowground tissue was observed in the smaller sediment particle sizes, indicating the biosynthesis of methionine might function as the detoxi cation agent for excess sulfate or sul de. Similarly, Z. marina also detoxi ed gaseous sediment-derived sul de through incorporation and that most of the detoxi cation occurred in belowground tissues, where sul de intrusion was greatest (Hasler-Sheetal and Holmer 2015).
Among phenolic compounds, avonoids have all the potentialities to constitute reliable biomarkers of environmental quality (Cannac et al. 2006). The present study indicated galuteolin and luteolin were the prime avones in the leaf of T. hemprichii, while catechin and isoquercitrin were the main composition in the belowground tissue. For P. oceanica, myricetin and Isorhamnetin were the chief composition of leaf avonols (Cannac et al. 2006). The avonoid of Halophila stipulacea was dominated by apigenin-7-O-β-glucopyranoside co-occurring with other minor avone glucosides containing two malonyl derivatives (Bitam et al. 2010). Seagrasses with larger leaves and/or more pairs of cross veins in the leaves contained sulfated avonoids, while those with smaller leaves and/or fewer cross veins lack these compounds (McMillan 1986). The difference might be associated with the measurement method or speci c difference. Although there were considerable differences among the different methods regarding avonoid content in seagrass, none modi ed the avonoid pro le (Cannac et al. 2007). Low-oxygen stress changed the expression of metabolic genes such as avonoid biosynthesis, and induced the process of avonoid biosynthesis that involve methylation as a modi cation of compounds to accomplish activation or intracellular translocation (Klok et al. 2002). The present study showed that lower avonoids concentration in the belowground tissue was observed in the sediment with larger particle size. Similarly, a decrease in the total phenolic concentrations in Z. marina was also found when grown in high pCO 2 waters (Arnold et al. 2014). This might attributed to a reallocation of carbon to other pathways (Groner et al. 2018). Phenolic compounds are regarded as storage compounds for carbohydrates, which are only produced when the plants cannot convert the carbohydrates into growth (Cannac et al. 2006;Waterman et al. 1984). Ecological plant strategy theory implies that plants investing into biochemical means of stress protection are likely to invest less carbon into constitutive productivity (Grime 2001). A trade-off mechanism between growth and secondary production for protection might occur in the present study, which needed further study. Interestingly, theδ 34  In the present study, a strong positive correlation between avonoids and amino acids indicated that the amino acids were a good indicator of the avonoid accumulation. The available aromatic amino acids are intended for the avonoid pathway and provided by the primary metabolism (Trantas et al. 2015), which was con rmed by that aromatic amino acid includes phenylalanine, tryptophan and tyrosine were higher in the in-situ sediment. Meanwhile, leucine and valine serve as precursors of plant secondary metabolites. Further research will be needed to perform a cross phytochemical/phylogenetic analysis of seagrasses to correlate the phenolic ngerprint and the amino acid sequences of genes encoding the avonoid pathway (Grignon-Dubois and Rezzonico 2012).

Ecological Signi cance
Sediment type is undoubtedly a key factor in uencing seagrass distribution, growth, abundance and the success of seagrass transplantation (de Boer 2007; Livingston et al. 1998). In recent years, the modi cation of sediment structure and composition by removing polluted sediment and adding exogenous matrix has often been applied in better protecting submerged plants and the ecological restoration project of rivers and lakes Lin Liu et al. 2016;Smart 1986). However, modi cation of sediment type was less considered and applied in the ecological restoration of wetland, especially in the seagrass beds suffering from eutrophication. Seagrasses in the sediment with smaller particle sizes exhibited lower ETR max and E k , indicating a decrease in light tolerance (Fig. 6). Organic matter input from shrimp pond culture along the coastline in Xincun bay resulted in smaller sediment particle size. This induced that T. hemprichii in the high intertidal suffered more from high light stress during air exposure, causing faster decline in the high intertidal than in the lower intertidal. The present study proved that adding coarse beach sand would reduce sediment total nitrogen, organic matter and sulfur content, and enhance oxygen permeability in the hypoxic/anoxic sediment, leading to less synthesis of amino acid and avonoid. This would bene t seagrass photosynthesis and allocate more carbon to growth. Furthermore, stimulated photosynthesis also led to less toxic substance accumulation by increasing oxygenated condition in the rhizosphere ( (Livingston et al. 1998). It is also very important to change the sediment type to improve the growth condition for seagrass and to enhance the successful rate in planting and transplanting seagrass shoots in the eutrophicated ecosystem. Furthermore, T. hemprichii is gradually being replaced by Enhalus acoroides due to decreasing sediment particle size. Meanwhile, sediment type also affected interspeci c competition between the salt marsh plants (Li et al. 2014).
Thus, further study on the effect of changing sediment on interspeci c competition and community succession for seagrasses are also needed and to be enhanced.
Together, our results indicate that coarse beach sand addition could indirectly enhance the photosynthesis of Thalassia hemprichii by improving sediment condition with lower total nitrogen, organic matter and sul de intrusion. Meanwhile, considerably greater belowground amino acid and avonoid were observed to counteract anoxic stress in the sediment with smaller particle size, leading to more positive belowground δ 34 S. Consequently, there is possible that sediment could be modi ed in the eutrophicated bay to improve the growth condition for tropical dominant seagrass T. hemprichii. However, more detailed analysis and eld experiment are required to determine this. Furthermore, further studies are needed to examine the metabolic pathway of key primary and secondary metabolics of seagrass, and the trade-off mechanism between growth and defense, under sediment modi cation.
Abbreviations RETR max , relative maximum electron transport rate; E k , minimum saturating irradiance; α, the initial slope of the light limited relationship; β, down-regulation; 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.

Declarations
Experimental set-up of the laboratory treatment. 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.

Figure 2
Photosynthetic parameters of Thalassia hemprichii derived from rapid light curve cultured in different sediment types. The lowercase and uppercase letters indicate signi cant difference at day 6 and day 21, respectively (P<0.05). 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.

Figure 3
Effect of sediment type on leaf nitrogen and the ratio of amino acids to nitrogen in seagrass Thalassia hemprichii. Different letters on column indicate signi cant difference (P<0.05). 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.  Effect of sediment type on the sulfur stable isotope (δ34S) in the belowground tissue of seagrass Thalassia hemprichii. Different letters on column indicate signi cant difference (P<0.05). 1:0, the in-situ sediment without combining with coarse beach sand was added in the tank; 1:1, the combination of half in-situ sediment and half coarse beach sand was added in the tank; 1:2, the combination of 1/3 in-situ sediment and 2/3 coarse beach sand was added in the tank.

Figure 6
Schematic pictures of the effect of sediment on the photosynthesis, stable isotope sulfur, FAA (free amino acid) and avone of seagrasses. Fig. 6a indicated that seagrass growing in sediment in eutrophic bay with lower particle size, showed smaller RTER (relative electron transport rate) and Ek (the minimum saturating irradiance), while higher FAA and Flavone accumulated in the belowground tissue to counteract anoxic stress. Fig. 6b indicated that beach sand addition indirectly enhanced RETR and Ek by improving the growth condition for seagrass with lower avone and FAA.

Supplementary Files
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