Sulfur accumulation in eelgrass (Zostera marina) and effect of sulfur on eelgrass growth

Eelgrass (Zostera marina) was grown under exposure to high levels of sediment sulfides to examine their ability to reoxidize sulfides intruding into the plants. The plants were kept under full light (control and high sulfide level) and at 10% of light saturation (high sulfide level) for 3 weeks and growth and accumulation of elemental sulfur (S⁰) in the plants were examined. The growth rate was reduced with ∼75% in the low light treatment, whereas there was no significant difference between the rates at full light saturation. S⁰ was accumulating in the below-ground structures of the plants exposed to high sulfide concentrations with highest concentration in the youngest roots and oldest internodes. There was no accumulation of S⁰ in the leaves, suggesting that the intruding sulfides were reoxidized in the below-ground structures before reaching the leaves. The accumulation of S⁰ was higher in the roots of the low light treatment (up to two times) suggesting a larger intrusion of sulfides. These plants also appeared highly affected by the treatment with rotting meristems and increased mortality after the 3-week growth period. These results are the first to show an accumulation of sulfur compounds internally in seagrasses as a result of reoxidation of sulfides. The reoxidation is facilitated by the internal transport of oxygen and is an example of the advantage of the internal lacunae system in seagrasses.     

Sulphide intrusion in eelgrass (Zostera marina L.)

Sudden events of seagrass die‐off have been suggested to be induced by invasion of the phytotoxin sulphide under environmental stress generating low oxygen supply in seagrass tissues. Laboratory experiments were conducted with eelgrass (Zostera marina L.) to measure intra‐plant changes in oxygen and sulphide by means of microelectrodes at different oxygen concentrations in the water column. The objectives were to examine whether sulphide intrusion into seagrass tissues can be induced, to determine the role of plant oxygen status for sulphide intrusion and to determine how fast internal sulphide pools are depleted after internal oxygen supplies have been restored. Under conditions with oxygen partial pressures (pO₂) above 7.4 kPa (> 35% of air saturation) within eelgrass rhizomes or meristematic tissues no intrusion of sulphide occurred in spite of high sediment concentrations of gaseous sulphide (> 1000 µm ). Lack of sulphide intrusion at high internal pO₂ suggested that oxygen release from the roots ensured complete re‐oxidation of sulphide in the rhizosphere. Under oxygen stress, however, the experiments clearly demonstrated intrusion of sulphide in eelgrass rhizomes and meristematic tissues. Rates of sulphide intrusion were controlled by internal pO₂, which in turn was controlled by water column oxygen concentrations. Maximum internal sulphide content reached 325 µm which by far exceeded the 1–10 µm known to inhibit mitochondrial activity in eukaryotic cells. Sulphide and low levels of oxygen could coexist in the eelgrass tissues reflecting fast internal transport of sulphide and slow rates of sulphide re‐oxidation. Upon re‐establishment of high internal oxygen concentrations the depletion of the sulphide pool was slow (half‐life = 20–30 min) indicating, that sulphide re‐oxidation within the eelgrass tissue was not bacterially or enzymatically facilitated but occurred by simple chemical oxidation. The results of this study are consistent with the proposed detrimental role of sulphide intrusion in events of sudden seagrass die‐off.     

Identification and characterization of 14 polymorphic EST‐derived microsatellites in eelgrass (Zostera marina)

Zostera marina, the dominant seagrass on the Northern Hemisphere, forms the basis of important but threatened marine ecosystems. Here, we report 14 microsatellite DNA markers derived from an expressed sequence tag library corresponding to a wide range of genes. All loci were moderately to highly polymorphic, with allele numbers ranging from three to eight in a single Wadden Sea population of 48 individuals. Observed heterozygosities ranged from 0.082 to 0.837. Reaction conditions for five pooled polymerase chain reactions are given. The markers will advance the population genetics of seagrasses because they allow indirect tests of selection on closely linked genes.     

Characterization of single nucleotide polymorphism markers for eelgrass (Zostera marina)

We characterized 37 single nucleotide polymorphism (SNP) makers for eelgrass Zostera marina. SNP markers were developed using existing EST (expressed sequence tag)-libraries to locate polymorphic loci and develop primers from the functional expressed genes that are deposited in The ZOSTERA database (V1.2.1). SNP loci were genotyped using a single-base-extension approach which facilitated high-throughput genotyping with minimal optimization time. These markers show a wide range of variability among 25 eelgrass populations and will be useful for population genetic studies including evaluation of population structure, historical demography, and phylogeography. Potential applications include haplotype inference of physically linked SNPs and identification of genes under selection for temperature and desiccation stress.     

Enumeration and characterization of nitrogen-fixing bacteria in a stripmine site

Summary Nitrogen-fixing heterotrophic bacteria were isolated from a stripmine reclamation site in western North Dakota. Of 232 isolates, 164 wereBacillus spp., 38 were members of the Enterobacteriaceae, and 21 wereClostridium spp. Single isolates ofAzotobacter chroococcum andAzospirillum lipoferum were identified, as were several isolates considered to beXanthobacter autotrophicum. Fewer nitrogen-fixing bacteria adhered to the root system of crested wheatgrass (Agropyrum desertorum) in sites with replaced topsoil than in sites with no topsoil. Several media and techniques were compared in enumeration of nitrogen fixers by most probable numbers.     

Decomposition processes of eelgrass,Zostera marina L.

Summary In Lake Grevelingen decomposition of eelgrass was studied in the field with the litter bag method from July 1977 till February 1978. After 6 months only 6% refractory matter remained in the bags. Under aerobic conditions the decomposition of eelgrass is completed within one year. The organic fraction decreased from 80 to 55%. Chlorophyll a was always present in the detritus, but with this parameter no decomposition stages could be distinguished. Fragmentation was mainly physical, and a particle size spectrum showed a maximum towards the small pieces.The POC content was fairly constant, and the N and P content changed during the decomposition. The C/N and C/P ratios increased the first 10 weeks (leaching) and decreased after 10 weeks (microbial colonization). This did not correspond with the three decomposition stages, based on the dry weight loss per day. The C/N ratio does not seem to be a simple index for the decomposition stage in eelgrass.Communication no. 235 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

In situ phytoremediation of PAH- and PCB-contaminated marine sediments with eelgrass (Zostera marina)

In view of the fact that there are presently no cost-effective in situ treatment technologies for contaminated sediments, a 60-week-long phytoremediation feasibility study was conducted in seawater-supplied outdoor ponds to determine whether eelgrass (Zostera marina) is capable of removing polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) from submerged marine sediments. It was determined that all PAHs and PCBs, independent of the number of aromatic rings and degree of chlorination, respectively, were removed to a much larger extent in planted sediments compared to unplanted controls. After 60 weeks of treatment, the concentration of total PAHs decreased by 73% in planted sediments but only 25% in unplanted controls. Similarly, total PCBs declined by 60% in the presence of plants while none were removed in the unplanted sediment. Overall, the apparent PAH and PCB biodegradation was greatest in the sediment layer that contained most of the eelgrass roots. Abiotic desorption tests conducted at week 32 confirmed that the phytoremediation process was not controlled by mass-transfer or bioavailability limitations since all PAHs and PCBs desorbed rapidly and to a large extent from the sediment. PAHs were detected in both roots and shoots, with root and shoot bioaccumulation factors for total PAHs amounting to approximately 3 and 1, respectively, after 60 weeks of phytoremediation treatment. Similarly, the root bioaccumulation factor for total PCBs was around 4, while no PCBs were detected in the eelgrass leaves at the end of the experiment. The total mass fraction of PAHs and PCBs absorbed and translocated by plant biomass during the 60-week period was insignificant, amounting to less than 0.5% of the total mass of PAHs and PCBs which was initially present in the sediment. Finally, the number of total heterotrophic bacteria and hydrocarbon degraders was slightly but not statistically significantly greater in planted sediments than in unplanted controls. After ruling out contaminant loss to the water column or absorption and transformation within plant cells, it is most likely that the presence of eelgrass stimulated the microbial biodegradation of PAHs and PCBs in the rhizosphere by releasing root exudates, plant enzymes, or even oxygen. Additional research is needed to further elucidate these potential phytoremediation mechanisms.     

Heavy metals in eelgrass (Zostera marina L.) during growth and decomposition

The distributions of cadmium, chromium, lead, and zinc in eelgrass were studied in samples collected from the field, and the loss/accumulation of the metals during decomposition of eelgrass leaves was studied in laboratory experiments.Concentrations of heavy metals in the below grounds parts were greater in the roots than in the different age groups of the rhizomes. In the rhizomes, the highest concentrations of lead were recorded in the oldest parts, whereas highest chromium and zinc concentrations were found in the youngest parts. The concentration of cadmium did not vary. In the above ground parts, the concentrations of cadmium, lead, and zinc increased with age of the leaves, and concentrations in the leaves were greater than in the stem fraction. The concentrations of chromium decreased with age of the leaves.In the laboratory study of decomposition of leaf material, the concentrations of chromium, lead and zinc increased significantly and a net absorption from the surrounding water was recorded. Cadmium concentrations were relatively constant and a loss of cadmium was proportional to the release of soluble organic compounds indicating an association of cadmium with the soluble phase.The investigation demonstrated the utility of compositional analyses and decomposition experiments in assessing the significance of eelgrass in the heavy metal cycling in coastal areas. Furthermore, significant differences in the fate of heavy metals associated with eelgrass detritus are discussed.     

Anthesis and seed production in Zostera marina L. (eelgrass) from the chesepeak by

Anthesis and seed production in Zostera marina L. were studied in three areas of the Chesapeake Bay from January to June 1980. Inflorescence primordia with distinguishable anthers and pistils were first observed in February when water temperature was 3°C. Development of the reproductive shoots in the field continued after February as water temperature rose, with the first evidence of pollen release in mid-April (water temperature 14.3°C). Stigmata loss was first observed in samples taken in late April at all locations by as water temperatures averaged above 16°C. Pollination was complete at all locations by 19 May and anthers were no longer present. Few reproduction shoots were found on 3–5 June and seed release was assumed to be complete by this time (water temperature 25°C). The density of flowering shoots ranged from 11 to 19% of the total number of shoots, producing an estimated 8127 seeds m^?2.Comparison of flowering events with other areas along a latitudinal gradient from North Carolina to Canada indicated that reproductive events occurred earlier in the most southern locations and at successively later dates with increasing latitude.     

Production and decomposition of eelgrass (Zostera marina L.) in saline Lake Grevelingen

Summary During five 28-hours measurements in 1981, the oxygen production and consumption in an eelgrass community in saline Lake Grevelingen were investigated using light plexiglass enclosures. Applying a conversion factor of 0.29 the amount of carbon fixed and the amount of organic carbon mineralized were estimated. Gross and net production were estimated over 24-hours periods.There appeared to be a good correlation between production and insolation on the water surface. For every measurement period the production as a function of light and aboveground eelgrass biomass in the enclosure were calculated. This showed a maximum of 5.10^–6 mg C.J.^–1 g dry weight^–1 in April and minimum of 1.4.10^–6 mg C.J.^–1 g^–1 in August.Using the calculated production coefficients, the insolation and the eelgrass biomass the gross production, net production and consumption during the growing season of 1976 were calculated. Gross production amounted to 340 gC.m^–2, and net production came to 130 g C.m^–2. Approximately 60 gC.m^–2 was respired by the eelgrass plants while the remaining 150 gC.m^–2 was consumed or mineralized by other organisms on the sampling spot. Approximately 120 g C.m^–2.yr^–1 was transported by wind and wave action towards the eastern part of the lake where it became anaerobically degraded. This resulted in the formation of sulfide and methane.Communication no. 236 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands.     

Photoadaptation and growth of Zostera marina L. (eelgrass) transplants along a depth gradient

Photosynthetic and growth responses were assessed in Zostera marina L. transplants within and beyond the natural extent of an eelgrass meadow in Great Harbor, Woods Hole, MA. Transplant survival and rapid growth inshore of the shallow edge of the meadow (0.5 and 0.8 m water depth) indicated a periodic disturbance factor maintaining the shallow edge of the meadow. Transplant mortality, reduced growth, and a negative carbon balance of eelgrass transplanted offshore the deep edge of the meadow (7 and 10m) supported the hypothesis of light-limited eelgrass growth in the deep regions of the Great Harbor meadow. Photoadaptive responses occurred along the water depth gradient, and both photosynthesis and growth responses were used to assess the genetic vs. phenotypic components of eelgrass response to the water depth gradient. Reciprocal transplants between shallow (1.3 m) and deep (5.5 m) areas within the eelgrass meadow indicated photosynthetic and growth responses were primarily a result of growth habitat rather than genetic differentiation within the eelgrass meadow.     

Seed germination and seedling growth of Zostera marina L. (eelgrass) in the chesapeake bay

Seed germination and seedling growth of Zostera marina L. were monitored in the Chesapeake Bay in 1979 and 1980. Harvested seeds were placed in small acrylic tubes at several sites representing the salinity range of Z. marina distribution. Seed germination was observed first in late September and continued through May, with peaks in the fall and spring. The majority of seeds that germinated (66%) did so between December and March when water temperatures ranged from 0–10°C. There was no correlation between sites (different salinity regimes) and frequency of germination rates, indicating that salinity was not a major factor in the germination process in this study. Additional information on seed germination was available for seeds collected in 1977 and 1980 and subsequently monitored for germination at only one site. These data were similar to germination frequency recorded in 1979–1980.Seedling growth was measured from individuals collected from an existing Zostera marina bed. Seedlings were collected from November through May, at which time we could no longer distinguish seedlings from existing vegetative stock. Growth was characterized by the increased length of the primary shoot, number of leaves per shoot and numbers of shoots per plant. Seedling growth was slow during the winter months (water temperature ? 10°C) but rapidly increased in the spring (temperatures > 10°C). The size range of the harvested seedlings indicated that seed germination in the field probably occurred from October through April, corroborating evidence from the seed germination experiments.     

Reproduction of annual eelgrass: Variation among habitats and comparison with perennial eelgrass (Zostera marina L.)

The variation in reproductive potential of annual eelgrass was examined along a continuous gradient on an extensive mudflat bordering on and sloping down from the shore (Eastern River) and in a habitat mosaic in a salt marsh (Petpeswick Inlet) in Nova Scotia. Spathe and flower production as well as plant density were compared among habitats. Of the four habitats investigated in Petpeswick Inlet, the largest numbers of spathes and flowers per spathe were produced by plants in ponds on raised flats of Spartina alterniflora Loisel. The highest number of seeds per unit area was produced by plants in depressions on these flats which drained with each low tide. Seed production of annual eelgrass in drained depressions (4 889 seeds per 625 cm²) was seven times that of perennial shoots in creeks and higher than any records in the literature for perennial eelgrass. On average, seed production of annual eelgrass in this study was higher than values reported for other locations. Along the gradient, both annual and perennial eelgrass showed peaks in reproductive potential, but the annual peaked further up the gradient where there was greater exposure to air at low tide. Transplanting studies indicated that the among-habitat differences in reproductive potential were largely controlled by environmental as opposed to genetic factors. The possible effect of inter- and intraspecific competition on the reproductive potential of annual eelgrass was investigated experimentally in two habitats where co-existing species were abundant. In creeks the presence of perennial eelgrass significantly reduced the reproductive potential of annual eelgrass, but in a drained depression, the removal of Ruppia maritima L. s.l. had no effect. The upper distribution limit of annual eelgrass is likely determined by desiccation while the lower limit is probably determined by a combination of light availability (to some extent affected by perennial eelgrass) and exposure to spring rains which would significantly enhance seed germination.     

Means of rapid eelgrass (Zostera marina L.) recolonisation in former dieback areas

Recolonisation of eelgrass (Zostera marina L.) was studied in a Danish estuary during summer 2001 following an anoxia event the previous summer. Leaf bundles had detached from the rhizomes while healthy-looking roots and rhizomes remained in the sediment. We hypothesise that the stabilising effect of remaining belowground biomass, the presence of rhizomes with buds, the presence of a large seed bank and the potential surviving shoots from the previous population may stimulate and speed up recovery in previously colonised areas compared to bare areas.A large seed bank containing more than 11,000 seeds m⁻² was found in the dieback area. Seeds were found in the upper 14 cm of the sediment but judging from the length of the hypocotyle of the seedlings, only seeds from the upper 5.5 cm of sediment germinated successfully. The upper 5.5 cm represented a seed pool of approximately 1000 seeds m⁻². Germination of these seeds was the primary mode of recolonisation in the estuary, since 96% of the plants in the investigated plot were seedlings. Only 4% of the plants were survivors from the previous year.Although densities of seedlings may exceed densities of surviving shoots, we argue that plants surviving oxygen depletion may still contribute considerably to the recolonisation of a former dieback area as these plants have faster elongation and branching rates and lower mortality rates relative to seedlings.There was no indication of recolonisation from dormant buds on rhizomes. This finding was confirmed in laboratory experiments where buds failed to germinate in the absence of the apical shoot. We examined the structure and ageing of buds and found general withering with age, indicating that buds should germinate shortly after the dieback if at all. Our results, therefore, suggest that rhizome buds are not dormant buds but simply side shoots that have failed to grow.     

HPLC determination of phenolic acids in the water-soluble extract of Zostera marina L. (eelgrass)

Six phenolic acids were quantitatively assessed in the water-soluble extract of Zostera marina L. (eelgrass) from Puget Sound, Washington, using high-performance liquid chromatography (HPLC). Ferulic (4-hydroxy, 3-methoxycinnamic), vanillic (4-hydroxy, 3-methoxybenzoic), p-hydroxybenzoic (4-hydroxybenzoic), caffeic (3,4-dihydroxycinnamic), gallic (3,4,5-trihydroxybenzoic) and protocatechuic (3,4-dihydroxybenzoic) acids were in suceedingly lower quantities. Gentisic (2,5-dihydroxybenzoic) acid was also found to be present in the extract. However, its quantification was not possible using this method.     

Effects of the herbicide atrazine on adenine nucleotide levels in Zostera marina L. (eelgrass)

Response of adenine nucleotides (ATP, ADP, AMP) and adenylate energy charge (EC) to atrazine, a triazine herbicide, was evaluated as an indicator of metabolic state in Zostera marina L. (eelgrass), a submerged marine angiosperm. Short-term (6 h) atrazine stress reduced ATP and total adenylates (AT) at both 10 and 100 ppb, but EC remained constant. Net productivity decreased at 100, but not at 10 ppb atrazine over 6 h. Long-term (21 day) atrazine stress was evidenced by growth inhibition and 50% mortality near 100 ppb. EC was reduced at 0.1, 1.0 and 10 ppb atrazine, but ATP and EC increased with physiological response to severe stress (100 ppb) after 21 days. Apparently, ATP and AT decrease over the short-term but rebound over the long-term with severe atrazine stress, increasing beyond control levels before plant death results. Supplementing adenine nucleotide and EC results with more conventional quantitative analyses should afford greater knowledge of physiological response to environmental variation.     

Innovative Techniques for Large‐scale Seagrass Restoration Using Zostera marina (eelgrass) Seeds

The use of Zostera marina (eelgrass) seeds for seagrass restoration is increasingly recognized as an alternative to transplanting shoots as losses of seagrass habitat generate interest in large‐scale restoration. We explored new techniques for efficient large‐scale restoration of Z. marina using seeds by addressing the factors limiting seed collection, processing, survival, and distribution. We tested an existing mechanical harvesting system for expanding the scale of seed collections, and developed and evaluated two new experimental systems. A seeding technique using buoys holding reproductive shoots at restoration sites to eliminate seed storage was tested along with new techniques for reducing seed‐processing labor. A series of experiments evaluated storage conditions that maintain viability of seeds during summer storage for fall planting. Finally, a new mechanical seed‐planting technique appropriate for large scales was developed and tested. Mechanical harvesting was an effective approach for collecting seeds, and impacts on donor beds were low. Deploying seed‐bearing shoots in buoys produced fewer seedlings and required more effort than isolating, storing, and hand‐broadcasting seeds in the fall. We show that viable seeds can be separated from grass wrack based on seed fall velocity and that seed survival during storage can be high (92–95% survival over 3 months). Mechanical seed‐planting did not enhance seedling establishment at our sites, but may be a useful tool for evaluating restoration sites. Our work demonstrates the potential for expanding the scale of seed‐based Z. marina restoration but the limiting factor remains the low rate of initial seedling establishment from broadcast seeds.     

Toxicity of reduced nitrogen in eelgrass (Zostera marina) is highly dependent on shoot density and pH

In sheltered, eutrophicated estuaries, reduced nitrogen (NHx), and pH levels in the water layer can be greatly enhanced. In laboratory experiments, we studied the interactive effects of NHx, pH, and shoot density on the physiology and survival of eelgrass (Zostera marina). We tested long-term tolerance to NHx at pH 8 in a 5-week experiment. Short-term tolerance was tested for two shoot densities at both pH 8 and 9 in a 5-day experiment. At pH 8, eelgrass accumulated nitrogen as free amino acids when exposed to high loads of NHx, but showed no signs of necrosis. Low shoot density treatments became necrotic within days when exposed to NHx at pH 9. Increased NH3 intrusion and carbon limitation seemed to be the cause of this, as intracellular NHx could no longer be assimilated. Remarkably, experiments with high shoot densities at pH 9 showed hardly any necrosis, as the plants seemed to be able to alleviate the toxic effects of high NHx loads through joint NHx uptake. Our results suggest that NHx toxicity can be important in worldwide observed seagrass mass mortalities. We argue that the mitigating effect of high seagrass biomass on NHx toxicity is a positive feedback mechanism, potentially leading to alternative stable states in field conditions.     

Population structure and genetic diversity among eelgrass (Zostera marina) beds and depths in San Francisco Bay

The seagrass Zostera marina is widely distributed in coastal regions throughout much of the northern hemisphere, forms the foundation of an important ecological habitat, and is suffering population declines. Studies in the Atlantic and Pacific oceans indicate that the degree of population genetic differentiation is location dependent. San Francisco Bay, California, USA, is a high-current, high-wind environment where rafting of seed-bearing shoots has the potential to enhance genetic connectivity among Z. marina populations. We tested Z. marina from six locations, including one annual population, within the bay to assess population differentiation and to compare levels of within-population genetic diversity. Using 7 microsatellite loci, we found significant differentiation among all populations. The annual population had significantly higher clonal diversity than the others but showed no detectible differences in heterozygosity or allelic richness. There appears to be sufficient input of genetic variation through sexual reproduction or immigration into the perennial populations to prevent significant declines in the number and frequency of alleles. In additional depth comparisons, we found differentiation among deep and shallow portions in 1 of 3 beds evaluated. Genetic drift, sweepstakes recruitment, dispersal limitation, and possibly natural selection may have combined to produce genetic differentiation over a spatial scale of 3-30 km in Z. marina. This implies that the scale of genetic differentiation may be smaller than expected for seagrasses in other locations too. We suggest that populations in close proximity may not be interchangeable for use as restoration material.     

Modifications of the specific diversity and feeding guilds in an intertidal sediment colonized by an eelgrass meadow (Zostera marina) (Brittany,France)

A sandy beach located in a bay of north Brittany (France) is partly colonized by an eelgrass meadow. The overall macrobenthic fauna, i.e. epifauna, endofauna and fish, living in this seagrass and in non-vegetated sands was compared in terms of species, abundance and biomass in the different feeding guilds. Seagrass makes this habitat more complex by increasing both its surface by 5.45–fold and volume by 4.79–fold. Eighteen species among the 23 living in the non-vegetated sands were observed in the seagrass assemblage. Their abundance and biomass were increased by 7.33–fold and 3.54–fold, respectively. The occurrence of Zostera was associated with the presence of 124 new species and 34 new feeding guilds. In the Zostera meadow, a spatial segregation exists among the taxa from the different microhabitats explored for food-quest. The balanced distribution of biomass within the various microhabitats may suggest that an optimization in the exploitation of feeding resources goes hand in hand with a higher complexity of the trophic web. It looks as if the architecturally increasing complexity of the habitat resulting from the development of a seagrass meadow on sediment both stimulates the abundance and biomass of the indigenous macrobenthic assemblage, increases the specific and functional biodiversity and favours the specialization of feeding guilds.