Health Risk Assessment during In Situ Remediation of Cr(VI)-Contaminated Groundwater by Permeable Reactive Barriers: A Field-Scale Study.

The presence of residual Cr(VI) in soils causes groundwater contamination in aquifers, affecting the health of exposed populations. Initially, permeable reactive barriers(PRB) effectively removed Cr(VI) from groundwater. However, as PRB clogging increased and Cr(VI) was released from upstream soils, the contamination plume continued to spread downstream. By 2020, the level of contamination in the downstream was nearly identical to that in the upstream. The study results show that during normal operation, the PRB can successfully remove Cr(VI) from contaminated groundwater and reduce the carcinogenic and non-carcinogenic risks to humans from the downstream side of groundwater. However, the remediated groundwater still poses an unacceptable risk to human health. The sensitivity analysis revealed that the concentration of the pollutant was the most sensitive parameter and interacted significantly with other factors. Ultimately, it was determined that the residual Cr(VI) in the soil of the study region continues to contaminate the groundwater and constitutes a serious health danger to residents in the vicinity. As remediated groundwater still poses a severe threat to human health, PRB may not be as effective as people believe.

Do adult eelgrass shoots rule seedling fate in a large seagrass meadow in a eutrophic bay in northern China?

We conducted field sampling over 19 months to investigate eelgrass population reproduction status and ecological interactions in a large seagrass meadow in a eutrophic bay in northern China. The results showed asexual growth played an important role in the maintenance of existing meadows, and sexual reproduction played a critical role in the colonization of new areas. We conclude that adult eelgrass shoots do rule the fate of seedlings in the large seagrass meadow. Additionally, nutrient resources (N and P) at this location were found to meet eelgrass growth demand. The N/P ratios of seawater and seagrass indicated N limitation relative to P in the eutrophic bay based on the seagrass Redfield ratio (25-30). Nutrient uptake by seagrass might be an important factor in reducing the probability of a red tide in the study area. The results of this study provide fundamental information for eelgrass restoration and conservation.

Can the Non-native Salt Marsh Halophyte Spartina alterniflora Threaten Native Seagrass (Zostera japonica) Habitats? A Case Study in the Yellow River Delta, China.

Seagrass meadows are critical ecosystems, and they are among the most threatened habitats on the planet. As an anthropogenic biotic invader, Spartina alterniflora Loisel. competes with native plants, threatens native ecosystems and coastal aquaculture, and may cause local biodiversity to decline. The distribution area of the exotic species S. alterniflora in the Yellow River Delta had been expanding to ca.4,000 ha from 1990 to 2018. In this study, we reported, for the first time, the competitive effects of the exotic plant (S. alterniflora) on seagrass (Zostera japonica Asch. & Graebn.) by field investigation and a transplant experiment in the Yellow River Delta. Within the first 3 months of the field experiment, S. alterniflora had pushed forward 14 m into the Z. japonica distribution region. In the study region, the area of S. alterniflora in 2019 increased by 516 times compared with its initial area in 2015. Inhibition of Z. japonica growth increased with the invasion of S. alterniflora. Z. japonica had been degrading significantly under the pressure of S. alterniflora invasion. S. alterniflora propagates sexually via seeds for long distance invasion and asexually by tillers and rhizomes for short distance invasion. Our results describe the invasion pattern of S. alterniflora and can be used to develop strategies for prevention and control of S. alterniflora invasion.

Relationships Between Annual and Perennial Seagrass (Ruppia sinensis) Populations and Their Sediment Geochemical Characteristics in the Yellow River Delta.

Annual and perennial populations commonly occur for the same submerged aquatic angiosperm species, yet relationships between population types and sediment characteristics are poorly understood. In the current study two Ruppia sinensis habitats with annual and perennial populations were surveyed in the Yellow River Delta (YRD). Biomass and seasonal seed bank size were used to evaluate population status and potential recruitment capacity. Sediment geochemical parameters including moisture, sulfide, Chl a, carbohydrate, OM, TOC, TN, and TP were measured to compare sediment nutrient composition and variability. The results revealed a higher biomass and larger seed bank in the annual R. sinensis population compared with the perennial population. The P levels in sediments between the two R. sinensis populations were similar; while the N level in the sediment of the annual population was significantly higher than the perennial population, which might support the recruitment of vegetative shoots when a large amount of seeds germinated during wet periods. The annual population exhibited greater resilience after habitat desiccation, with the population recovering rapidly once water appeared. The results of this study add to the knowledge of R. sinensis populations and their sediment geochemical characteristics, and can be used as a reference for Ruppia population conservation and management.

New insights into physiological effects of anoxia under darkness on the iconic seagrass Zostera marina based on a combined analysis of transcriptomics and metabolomics.

Coastal hypoxia/anoxia is a major emerging threat to global coastal ecosystems. Macroalgae blooms of tens of kilometers are often observed in open waters. These blooms not only cause a lack of oxygen, but also benthic light limitation. We explored the physiological responses of Zostera marina L. to anoxia under darkness. After exposing Z. marina to anoxia under darkness for 72 h, we measured the elongation of leaves and the decrease in maximal quantum yield of photosystem II (Fv/Fm), and investigated the transcriptomic and metabolomic responses to anoxic stress based on RNA-sequencing and liquid chromatography-mass spectrometry (LC-MS) technology. The results showed that anoxic stress significantly reduced the leaf Fv/Fm, and had a significant negative effect on the photosynthesis and growth of Z. marina. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of up-regulated differentially expressed genes (DEGs) showed that glycolysis was the most significant enrichment pathway (p < 0.001), and most of the important products in glycolysis were significantly up-regulated. This indicated that the glycolysis process of anaerobic respiration is promoted under anoxia. The metabolite results also showed that glyceraldehyde 3-phosphate in the glycolysis pathway was significantly up-regulated. Moreover, three genes encoding sucrose synthase (gene-ZOSMA_310G00150, gene-ZOSMA_81G00980, and gene-ZOSMA_8G00730) and one gene encoding alpha-amylase (gene-ZOSMA_95G00270) were significantly up-regulated, providing the sugar basis for the subsequent increase in glycolysis. Furthermore, gene-encoding oxoglutarate dehydrogenase, the rate-limiting step of the tricarboxylic acid (TCA) cycle, was significantly down-regulated, indicating that this cycle was inhibited under anoxia. Metabolomic results showed that L-tryptophan, L-phenylalanine, and DL-leucine were significantly up-regulated. Only significantly decreased glutamate and non-significantly decreased glutamine, substances consumed in alanine and γ-aminobutyric acid (GABA) shunt mechanisms, were detected in the leaves, while GABA and alanine were not detected. The results of this study show that anoxic stress induces a programmed transcriptomic and metabolomic response in seagrass, most likely reflecting a complex strategy of acclimation and adaptation in seagrass to resist anoxic stress.

Programmed responses of different life-stages of the seagrass Ruppia sinensis to copper and cadmium exposure.

Seagrass meadows are recognized as crucial and are among the most vulnerable habitats worldwide. The aquatic plant genus Ruppia is tolerant of a wide salinity range, and high concentrations of trace metals. However, the tolerance of its early life stages to such trace metal exposure is unclear. Thus, the current study investigated the trace metal-absorbing capacity of three different life-history stages of Ruppia sinensis, a species that is widely distributed in China, by observing toxic symptoms at the individual, subcellular, and transcription levels. The seedling period was the most vulnerable, with visible toxic effects at the individual level in response to 50 µM copper and 500 µM cadmium after 4 days of exposure. The highest concentrations of trace metals occurred in the vacuoles and cytoplasmic structures of aboveground tissues. Genes related to signal identification and protein processing were significantly downregulated after 4 days of exposure to copper and cadmium. These results provide information relating to the strategies evolved by R. sinensis to absorb and isolate trace elements, and highlight the phytoremediation potential of this species.

Sonar and in situ surveys of eelgrass distribution, reproductive effort, and sexual recruitment contribution in a eutrophic bay with intensive human activities: Implication for seagrass conservation.

Seagrass beds are recognized as pivotal and among the most vulnerable coastal marine ecosystems globally. The eelgrass Zostera marina L. is the most widely distributed seagrass species and dominates the temperate northern hemisphere. However, an alarming decline in seagrass has been occurring worldwide due to multiple stressors. Seagrass meadow degradation is particularly serious in the Bohai Sea, in temperate China; however, large areas (> 500 ha) of seagrass meadows and population recruitment have rarely been reported in this area. In the present study, we report on a large eelgrass bed in a eutrophic bay of the Bohai Sea. Sonar and field survey methods were used to investigate the distribution of seagrass and its population recruitment. We also analyzed the major threats to this large seagrass bed. Results showed that a large Z. marina bed with an area of 694.36 ha occurred in this area of the Bohai Sea, with a peripheral area of ~25 km2. Seagrass canopy height and plant coverage had a significant correlation with water depth. Asexual reproduction principally occurred in autumn and played a dominant role in population recruitment in vegetated areas, where no seedlings successfully colonized. In contrast, a considerable number of seedlings survived in the seagrass meadow gaps, and thus played a critical role in the recruitment in these areas. The maximum reproductive shoot densities were about 100 and 70 shoots m-2 at sampling site (S)-1 and S-2 in 2018, respectively, which was about two times more than in 2019 (50 and 20 reproductive shoots m-2 at S-1 and S-2, respectively). The potential seed output per unit area in 2019 was about 1020 seeds m-2 at S-1 and 830 seeds m-2 at S-2, and the seed output in the study area was at a low level compared with global values. Overall, high spring and summer water temperature appeared to induce sexual reproduction of Z. marina in the study area, including reproductive effort, reproductive investment, and seedling development. Furthermore, eelgrass height, aboveground biomass, and density were significantly related to water temperature. Among the potential threatening factors to seagrass in this area, the activities of clam harvesting were intense with daily clam catches >2000 kg, leading to patchy seagrass meadows, especially in the fringe areas. The seagrass bed was also threatened by marine pollution (nutrient loading) and land reclamation. Therefore, the protection and restoration of this seagrass bed are strongly recommended. Our study will provide fundamental information for the conservation and management strategies of large eelgrass beds in the Bohai Sea.

Sheaths of Zostera marina L. as ecological indicators of shoot length and the elemental stoichiometry of aboveground tissues.

Given a large quantity of epiphytes and other material attached on eelgrass leaf blades, we explored the relationship between eelgrass sheaths and different-aged leaf blades (1st, 2nd, 3rd, and 4th leaf blade) on nutrient content and their ratios (C, N, P, C/N, C/P, and N/P) to identify whether eelgrass sheaths could be used to instead of leaf blades in terms of nutrient content. In addition, we explored the relationship between eelgrass sheath length and shoot length. Results showed that there were significant relationships between the sheath and leaf blades in terms of N and P content and their ratios. For length analysis, there was a significant relationship between sheath length and shoot length, and shoot length was approximately four to five times (mean 4.4659) longer than sheath length, such that shoot length can be estimated by sheath length. These significant relationships suggest that eelgrass sheath could be used as a suitable predictor of leaf blade in length and nutrient stoichiometry, thus eelgrass sheath could be used as an indicator for further eelgrass nutrient monitoring and research.

Seed selection and storage with nano-silver and copper as potential antibacterial agents for the seagrass Zostera marina: implications for habitat restoration.

Globally, seagrass meadows are extremely important marine ecosystems that are disappearing at an alarming rate. Therefore, research into seagrass restoration has become increasingly important. Various strategies have been used in Zostera marina L. (eelgrass) restoration, including planting seeds. To improve the efficiency of restoration by planting seeds, it is necessary to select high-quality seeds. In addition, a suitable antibacterial agent is necessary for wet storage of desiccation sensitive seeds to reduce or inhibit microorganism infection and seed decay. In the present study, an efficient method for selecting for high-quality eelgrass seeds using different specific gravities of salt water was developed, and potential antibacterial agents (nano-silver and copper sulfate) for seed storage were assessed. The results showed that the highest proportion of intact seeds (72.91 ± 0.50%) was recorded at specific gravities greater than 1.20. Therefore, specific gravities greater than 1.20 can be used for selecting high-quality eelgrass seeds. During seed storage at 0 °C, the proportion of intact seeds after storage with nano-silver agent was over 90%, and also higher than 80% with copper sulfate agent, which was significantly higher than control treatments. The findings revealed a potential selection method for high-quality seeds and long-term seed storage conditions for Z. marina, which could facilitate conservation and habitat restoration.

Single beam sonar reveals the distribution of the eelgrass Zostera marina L. and threats from the green tide algae Chaetomorpha linum K. in Swan-Lake lagoon (China).

Seagrass meadows are declining at alarming rates globally due to both anthropogenic activities and natural threats. Seagrasses play key ecological roles in coastal ecosystems as primary producers and providers of habitat and environmental structure. Therefore, mapping seagrass beds is indispensable for the effective monitoring and management of coastal vegetated habitats. In contrast to direct sampling techniques and optical remote sensing, active hydroacoustic techniques are relatively inexpensive and efficient for the detection of seagrass. We used a single beam echosounder to detect the spatial and temporal distribution characteristics of the eelgrass Zostera marina L. in an important overwintering habitat for the whooper swan Cygnus cygnus (Swan-Lake lagoon), northern China. We also distinguished echograms of the macroalgae Chaetomorpha linum K. and outlined its threat to seagrass. We also propose a method for calculating the accuracy of interpolation analyses. Results showed that: (1) The distribution of seagrass in Swan Lake varies with seasons, with maximum distribution area in summer. The maximum distribution area of seagrass beds in Swan Lake was 199.09 ha-231.67 ha, accounting for 41.48%-48.26% of the area of Swan Lake; (2) C. linum is a growing threat for seagrass beds of Swan-lake, with distribution area as high as 129.28 ha in May 2018. The invasion and competition by C. linum against seagrass beds could be one of the reasons for the decline in seagrass beds in Swan-Lake; (3) Topo to Raster has the highest interpolation accuracy and is the most conservative among three interpolation methods. Topo to Raster was the most suitable interpolation method for the sonar detection of seagrass beds. The findings may facilitate the application of sonar technology in seagrass monitoring and provide data for the formulation of appropriate seagrass bed management and restoration strategies and policies.

Effects of salinity and temperature on seed germination and seedling establishment in the endangered seagrass Zostera japonica Asch. & Graebn. in northern China.

Seagrass meadows are recognized as critical and among the most vulnerable habitats on the planet. As a worldwide concern, there is an urgent need to develop techniques to restore and preserve these vital coastal ecosystems due to their alarming decline rate. To effectively preserve and restore seagrasses, more research is required on the germination ecology of seeds. The seagrass Zostera japonica Asch. & Graebn is an endangered species in its native range, the Northwestern Pacific Coast. The present study investigated the germination and seedling establishment in Z. japonica seeds (collected from northern China) under different temperature and salinity conditions to explore suitable seed germination and establishment conditions, as well as the seedling formation process. Results showed that salinity had a more significant influence on seed germination rate. Germination rate decreased with an increase in salinity, and seeds did not germinate when salinity was higher than 40 psu. Temperature was more likely to influence germination speed, which increased with an increase in temperature, with high germination rates and the most rapid germination speed observed at 30 °C. Therefore, the optimal culture conditions were 10 psu salinity at 30 °C for germination and 10-20 psu salinity at 20 °C for seedling establishment, with a seed germination rate of 45.6% after 6 days of germination culture and a seedling establishment rate of 14.3% after 6 days of seedling culture, respectively. A new seedling raising method with low salinity (5 psu) germination and high salinity (20 psu) seedling establishment was proposed and a flow chart of seedling formation of Z. japonica was created. The results provide insight into the seed germination and seedling establishment in Z. japonica, and will facilitate future large-scale seedling culture and field restoration activities for this seagrass species.

A unique meadow of the marine angiosperm Zostera japonica, covering a large area in the turbid intertidal Yellow River Delta, China.

Marine submerged aquatic angiosperms (seagrasses) are declining globally. The species Zostera japonica Asch. & Graebn. is endangered in its native range in Asia, but has been successfully introduced to North America. A large area (1031.8 ha) of Z. japonica meadow has recently been discovered in the intertidal zone of Yellow River Delta, China. This seagrass occurs along both sides of the river mouth, forming dense meadows in turbid water conditions. Seasonal investigations over two years were conducted to examine the distribution, biomass, seed reproduction, seed bank, and population recruitment of the seagrass meadows at three sites in the intertidal zone. The meadows generally showed relatively high coverage, biomass, reproductive effort, and seed production in August. The seed bank was found to be large and contributed to population recruitment. There were significant inter-annual variations overall, and at individual sites. These variations are likely due to winter temperatures, which determine the abundance of overwintering shoots and seedling success. Differences in micro-topography may also play a role in producing variations in seedling success between sites. Microsatellite analysis revealed a high genetic exchange between the two sides of the river mouth. The results indicate that the seagrass bed in the Yellow River Delta shallow waters is in good condition, which can be attributed to its location within a national nature reserve. Establishment of protected areas might act as an effective way to mitigate the anthropogenic disturbance, conserve the seagrass meadows, and then enhance critical ecosystem functions.

Optimal long-term seed storage conditions for the endangered seagrass Zostera japonica: implications for habitat conservation and restoration.

BACKGROUND: Seagrass meadows are recognized as critical and among the most vulnerable habitats on the planet. The alarming rates of decline in seagrass meadows have attracted the attention globally. There is an urgent need to develop techniques to restore and preserve these vital coastal ecosystems. So far little work has been done to develop effective long-term storage method for seagrass seeds. The seagrass Zostera japonica Asch. & Graebn is an endangered species in its native range. Here we utilized combinations of different storage times, salinities, and temperature to determine the most appropriate conditions for optimal seed storage. RESULTS: Zostera japonica seeds were strongly desiccation sensitive, with a complete loss of viability after 24 h of desiccation. Therefore, long periods of exposure to air should be avoided to minimize seed mortality. In addition, Z. japonica seeds could not endure freezing conditions such as - 5 °C. However, our results indicated that reduced storage temperature to 0 °C could effectively prolong the duration of dormancy of Z. japonica seeds. Seeds stored at 0 °C under a salinity of 40-60 psu showed relatively low seed loss, high seed vigor and fast seed germination, suggesting these to be optimal seed storage conditions. For example, after storage for 540 days (ca. 600 days since the seed collection from reproductive shoots in early October, 2016) at 0 °C under a salinity of 50 psu, seeds still had a considerable vigor, i.e. 57.8 ± 16.8%. CONCLUSION: Our experiments demonstrated that seeds stored at 0 °C under a salinity of 40-60 psu could effectively prolong the duration of dormancy of Z. japonica seeds. The proposed technique is a simple and effective long-term storage method for Z. japonica seeds, which can then be used to aid future conservation, restoration and management of these sensitive and ecologically important habitat formers. The findings may also serve as useful reference for seed storage of other threatened seagrass species and facilitate their ex situ conservation and habitat restoration.

Which Genes in a Typical Intertidal Seagrass (Zostera japonica) Indicate Copper-, Lead-, and Cadmium Pollution?

Healthy seagrasses are considered a prime indicator of estuarine and coastal ecosystem function; however, as the only group of flowering plants recolonizing the sea, seagrasses are frequently exposed to anthropogenic heavy metal pollutants, which are associated with high levels of molecular damage. To determine whether biologically relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene expression study using transcriptome analyses (RNA-seq). We exposed the typical intertidal seagrass Zostera japonica to 0 and 50 µM of copper (Cu), lead (Pb), and cadmium (Cd) under laboratory conditions. A total of 18,266 differentially expressed genes (DEGs) were identified, of which 2001 co-expressed genes directly related by Cu, Pb, and Cd stress. We also examined the effects of short-term heavy metal Cu, Pb, and Cd pulses on the accumulation of metals in Z. japonica and showed metal concentrations were higher in the shoots than in roots. Twelve differentially expressed genes were further analyzed for expression differences using real-time quantitative polymerase chain reaction (RT-qPCR). Our data suggest that as coastal seawater pollution worsens, the sensitive genes identified in this study may be useful biomarkers of sublethal effects and provide fundamental information for Z. japonica resistant gene engineering.

Tolerance of Ruppia sinensis Seeds to Desiccation, Low Temperature, and High Salinity With Special Reference to Long-Term Seed Storage.

Seeds are important materials for the restoration of globally-threatened marine angiosperm (seagrass) populations. In this study, we investigated the differences between different Ruppia sinensis seed types and developed two feasible long-term R. sinensis seed storage methods. The ability of R. sinensis seeds to tolerate the short-term desiccation and extreme cold had been investigated. The tolerance of R. sinensis seeds to long-term exposure of high salinity, cold temperature, and desiccation had been considered as potential methods for long-term seed storage. Also, three morphological and nine physiological indices were measured and compared between two types of seeds: Shape L and Shape S. We found that: (1) wet storage at a salinity of 30-40 psu and 0°C were the optimal long-term storage conditions, and the proportion of viable seeds reached over 90% after a storage period of 11 months since the seeds were collected from the reproductive shoots; (2) dry condition was not the optimal choice for long-term storage of R. sinensis seeds; however, storing seeds in a dry condition at 5°C and 33 ± 10% relative humidity for 9 months had a relatively high percentage (74.44 ± 2.22%) of viable seeds, consequently desiccation exposure could also be an acceptable seed storage method; (3) R. sinensis seeds would lose vigor in the interaction of extreme cold (-27°C) and desiccation; (4) there were significant differences in seed weight, seed curvature, and endocarp thickness between the two types of seeds. These findings provided fundamental physiological information for R. sinensis seeds and supported the long-term storage of its seeds. Our results may also serve as useful reference for seed storage of other threatened seagrass species and facilitate their ex situ conservation and habitat restoration.

Seasonal dynamics of trace elements in sediment and seagrass tissues in the largest Zostera japonica habitat, the Yellow River Estuary, northern China.

Trace element accumulation is an anthropogenic threat to seagrass ecosystems, which in turn may affect the health of humans who depend on these ecosystems. Trace element accumulation in seagrass meadows may vary temporally due to, e.g., seasonal patterns in sediment discharge from upstream areas. In addition, when several trace elements are present in sufficiently high concentrations, the risk of seagrass loss due to the cumulative impact of these trace elements is increased. To assess the seasonal variation and cumulative risk of trace element contamination to seagrass meadows, trace element (As, Cd, Cr, Cu, Pb, Hg, Mn and Zn) levels in surface sediment and seagrass tissues were measured in the largest Chinese Zostera japonica habitat, located in the Yellow River Estuary, at three sites and three seasons (fall, spring and summer) in 2014-2015. In all three seasons, trace element accumulation in the sediment exceeded background levels for Cd and Hg. Cumulative risk to Z. japonica habitat in the Yellow River Estuary, from all trace elements together, was assessed as "moderate" in all three seasons examined. Bioaccumulation of trace elements by seagrass tissues was highly variable between seasons and between above-ground and below-ground biomass. The variation in trace element concentration of seagrass tissues was much higher than the variation in trace element concentration of the sediment. In addition, for trace elements which tended to accumulate more in above-ground biomass than below-ground biomass (Cd and Mn), the ratio of above-ground to below-ground trace element concentration peaked at times corresponding to high water discharge and high sediment loads in the Yellow River Estuary. Overall, our results suggest that trace element accumulation in the sediment may not vary between seasons, but bioaccumulation in seagrass tissues is highly variable and may respond directly to trace elements in the water column.

New Insights into Different Reproductive Effort and Sexual Recruitment Contribution between Two Geographic Zostera marina L. Populations in Temperate China.

Seagrasses are important components of global coastal ecosystems, and the eelgrass Zostera marina L. is widely distributed along the Atlantic and Pacific coasts in the temperate northern hemisphere, but limited datum related to the contribution of sexual reproduction to population recruitment have been reported. This study aimed to understand eelgrass sexual reproduction and population recruitment in Swan Lake (SLL), and Huiquan Bay (HQB) was included for comparison. Random sampling, permanent quadrats or cores and laboratory seed germination-based experimental methods were employed. The flowering, seed production, seed banks, seed germination, seedling survival, and seedling growth of eelgrass were investigated from July 2014 to December 2015 to evaluate the contribution of sexual reproduction to population recruitment. Results indicated a dominant role of asexual reproduction in HQB, while sexual reproduction played a relatively important role in SLL. The highest flowering shoot density in SLL was 517.27 ± 504.29 shoots m-2 (June) and represented 53.34% of the total shoots at the center site. The potential seed output per reproductive shoot and per unit area in SLL were 103.67 ± 37.95 seeds shoot-1 and 53,623.66 ± 19,628.11 seeds m-2, respectively. The maximum seed bank density in SLL was 552.21 ± 204.94 seeds m-2 (October). Seed germination mainly occurred from the middle of March to the end of May, and the highest seedling density was 296.88 ± 274.27 seedlings m-2 in April. The recruitment from seedlings accounted for 41.36% of the Z. marina population recruitment at the center site, while the sexual recruitment contribution at the patch site (50.52%) was greater than that at the center site. Seeds in SLL were acclimated to spring germination, while in HQB, they were acclimated to autumn germination (early October-late November). Seed bank density in HQB was very low, with a value of 254.35 ± 613.34 seeds m-2 (early October). However, seeds in HQB were significantly larger and heavier than those in SLL (size: P = 0.004; weight: P < 0.001). The recruitment from seedlings accounted for as low as 2.53% of the Z. marina population recruitment in HQB. Our laboratory seed germination experiment, which was conducted in autumn, showed that the seed germination percent in HQB was significantly greater than in SLL at optimal germination temperatures (10 and 15°C; P < 0.001). A laboratory seed germination test at suitable temperature may be a potential novel approach to identify the ecological differences among different geographic populations. It is suggested that the Z. marina population recruitment may have different strategies and adapt to specific local conditions, such as in SLL and HQB, and the temperature regime may control morphological and phonological variations.

Effects of temperature and salinity on Ruppia sinensis seed germination, seedling establishment, and seedling growth.

As typical submerged aquatic vegetation, Ruppia species are facing population reductions due to anthropogenic impacts. In this study, we investigated the effects of temperature and salinity on seed germination and seedling establishment of Ruppia sinensis seeds collected from northern China. The effects of seven salinities (0-50) and six water temperatures (0-30°C) on seed germination were investigated to identify the environmental conditions that could potentially limit survival and growth. We found that: 1) optimum seed germination was salinity 5 at 30°C; 2) high salinity (salinity 40-50) and low temperature (0°C) significantly inhibited seed germination; 3) seed germination with increasing temperature showed a bimodal pattern at suitable salinities (5-10); 4) storing seeds at high salinities (40-50) or low temperature (0°C) promoted germination after transferal to optimal germination conditions. These findings may serve as useful information for R. sinensis habitat establishment and restoration programs.

Salinity and temperature significantly influence seed germination, seedling establishment, and seedling growth of eelgrass Zostera marina L.

Globally, seagrass beds have been recognized as critical yet declining coastal habitats. To mitigate seagrass losses, seagrass restorations have been conducted in worldwide over the past two decades. Seed utilization is considered to be an important approach in seagrass restoration efforts. In this study, we investigated the effects of salinity and temperature on seed germination, seedling establishment, and seedling growth of eelgrass Zostera marina L. (Swan Lake, northern China). We initially tested the effects of salinity (0, 5, 10, 15, 20, 25, 30, 35, and 40 ppt) and water temperature (5, 10, 15, and 20 °C) on seed germination to identify optimal levels. To identify levels of salinity that could potentially limit survival and growth, and, consequently, the spatial distribution of seedlings in temperate estuaries, we then examined the effect of freshwater and other salinity levels (10, 20, and 30 ppt) on seedling growth and establishment to confirm suitable conditions for seedling development. Finally, we examined the effect of transferring germinated seeds from freshwater or low salinity levels (1, 5, and 15 ppt) to natural seawater (32 ppt) on seedling establishment rate (SER) at 15 °C. In our research, we found that: (1) Mature seeds had a considerably lower moisture content than immature seeds; therefore, moisture content may be a potential indicator of Z. marina seed maturity; (2) Seed germination significantly increased at low salinity (p < 0.001) and high temperature (p < 0.001). Salinity had a much stronger influence on seed germination than temperature. Maximum seed germination (88.67 ± 5.77%) was recorded in freshwater at 15 °C; (3) Freshwater and low salinity levels (< 20 ppt) increased germination but had a strong negative effect on seedling morphology (number of leaves per seedling reduced from 2 to 0, and maximum seedling leaf length reduced from 4.48 to 0 cm) and growth (seedling biomass reduced by 46.15-66.67% and maximum seedling length reduced by 21.16-69.50%). However, Z. marina performed almost equally well at salinities of 20 and 30 ppt. Very few germinated seeds completed leaf differentiation and seedling establishment in freshwater or at low salinity, implying that freshwater and low salinity may potentially limit the distribution of this species in coastal and estuarine waters. Therefore, the optimum salinity for Z. marina seedling establishment and colonization appears to be above 20 ppt in natural beds; (4) Seeds germinated in freshwater or at low salinity levels could be transferred to natural seawater to accomplish seedling establishment and colonization. This may be the optimal method for the adoption of seed utilization in seagrass restoration. We also identified seven stages of seed germination and seedling metamorphosis in order to characterize growth and developmental characteristics. Our results may serve as useful information for Z. marina habitat establishment and restoration programs.