Multiple stressors threaten the imperiled coastal foundation species eelgrass (Zostera marina) in Chesapeake Bay,USA

Interactions among global change stressors and their effects at large scales are often proposed, but seldom evaluated. This situation is primarily due to lack of comprehensive, sufficiently long‐term, and spatially extensive datasets. Seagrasses, which provide nursery habitat, improve water quality, and constitute a globally important carbon sink, are among the most vulnerable habitats on the planet. Here, we unite 31 years of high‐resolution aerial monitoring and water quality data to elucidate the patterns and drivers of eelgrass (Zostera marina) abundance in Chesapeake Bay, USA, one of the largest and most valuable estuaries in the world, with an unparalleled history of regulatory efforts. We show that eelgrass area has declined 29% in total since 1991, with wide‐ranging and severe ecological and economic consequences. We go on to identify an interaction between decreasing water clarity and warming temperatures as the primary drivers of this trend. Declining clarity has gradually reduced eelgrass cover the past two decades, primarily in deeper beds where light is already limiting. In shallow beds, however, reduced visibility exacerbates the physiological stress of acute warming, leading to recent instances of decline approaching 80%. While degraded water quality has long been known to influence underwater grasses worldwide, we demonstrate a clear and rapidly emerging interaction with climate change. We highlight the urgent need to integrate a broader perspective into local water quality management, in the Chesapeake Bay and in the many other coastal systems facing similar stressors.     

Seed size variation within Zostera marina L. (Zosteraceae)

In 1997 and 1998 seeds were collected from the seagrass, Zostera marina L. (Zosteraceae) from Long Island, New York, USA (var. typica ), Shaw Island, Washington, USA (var. latifolia ) and Izembek Lagoon, Alaska, USA (var. izembekensis ) to determine if seed size varied between the varieties, if seed size varied within the same variety, and if the relationship between seed length and seed weight was linear. Measurements of seed length revealed statistically significant differences in this metric between varieties associated with smaller intertidal plants (var. typica and var. izembekensis ) and larger plants from the subtidal region (var. latifolia ). It was also found that seed length varied within the same variety and verified that the cross-sectional shape of these seeds was primarily elliptical. Using seed cross-sectional area and weight from a randomly selected batch of seeds from the three locations sampled in 1998, a linear model was created to determine seed weight and seeds from var. latifolia were found to be heavier than seeds from var. typica . The potential advantage of heavy seeds for subtidal seeds in search of safe sites is discussed, together with the merit of using the model to calculate the weight of other elliptical seeds within the Zosteraceae and other seagrass families.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 281–288.     

Seasonal and year-to-year variations in the growth of Zostera marina L. (eelgrass) in the lower Chesapeake Bay

Seasonal and year-to-year variations in the growth of Zostera marina L. were measured at three sites in two locations in the lower Chesapeake Bay between 1978 and 1980. The maximum values for the 1979 above- and belowground standing crop ranged from 161–336 g dry wt m⁻² and 61–155 g dry wt m⁻², respectively, leaf length was 19.6–59.7 cm and shoot density 1418–2576 shoot m⁻². Values for 1980 tended to be greater and may be related to climatical differences between the two years. Maximum values were usually recorded in the months of June and July when water temperatures were between 20 and 25°C. Significant loss of leaves occurred in July and August, when water temperatures ranged between 25 and 30°C, while new shoots began to appear more rapidly in late September as water temperatures dropped below 20°C. The greatest increase in all growth parameters occurred from April to June during which time reproductive shoots were present, and accounted for up to 25% of the total number of shoots.     

Large‐Scale Zostera marina (eelgrass) Restoration in Chesapeake Bay,Maryland, USA. Part I: A Comparison of Techniques and Associated Costs

The Chesapeake Bay, like many other temperate estuaries, has exhibited dramatic declines in the abundance of submerged aquatic vegetation (SAV) during the later half of the twentieth century. Because of the functions SAV serve in maintaining a healthy estuarine ecosystem, SAV restoration has become an important component of Chesapeake Bay restoration. Specifically, recent water quality improvements in areas from which populations of Zostera marina (eelgrass) have been extirpated have suggested that Z. marina restoration could succeed. Early restoration efforts involved transplanting Z. marina plants from healthy source beds to restoration locations, but this was labor intensive, time consuming, expensive, and potentially detrimental to donor beds. This multi‐year project investigated new techniques for large‐scale Z. marina seed collection and processing and compared two seed dispersal methods to evaluate cost effectiveness. Tens of millions of mature Z. marina seeds were collected through snorkeling, SCUBA, or with a mechanical harvester. Seed storage conditions and processing techniques were manipulated in order to maximize seed yield. Seeds were dispersed using two methods: spring seed buoys and fall seed broadcasts. Our costs for planting 1 ha of bottom with Z. marina seeds ranged from $6,674 to $165,699 depending on seeding density and dispersal method used. The average cost per Z. marina seed was $0.17. Interannual variations in seed collection yield and seed viability after summer storage had great impact on final costs. Our results suggest that the use of seeds for large‐scale Z. marina restoration offers a competitive advantage to more traditional transplanting methods.     

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.     

Evidence of Eelgrass (Zostera marina) Seed Dispersal by Northern Diamondback Terrapin (Malaclemys terrapin terrapin) in Lower Chesapeake Bay

The initial discovery in May 2009 of eelgrass (Zostera marina) seeds in fecal samples of wild-caught northern diamondback terrapins (Malaclemys terrapin terrapin) was the first field evidence of eelgrass seed ingestion in this species. This finding suggested the potential of terrapins as seed dispersers in eelgrass beds, which we sampled for two additional years (2010 and 2011). Seeds were only found in feces of terrapins captured prior to June 8 in all three years, coinciding with eelgrass seed maturation and release. Numbers of seeds in terrapin feces varied annually and decreased greatly in 2011 after an eelgrass die off in late 2010. The condition of seeds in terrapin feces was viable-mature, germinated, damaged, or immature. Of terrapins captured during time of seed release, 97% were males and juvenile females, both of which had head widths <30 mm. The fraction of individuals with ingested seeds was 33% for males, 35% for small females, and only 6% for large (mature) females. Probability of seed ingestion decreased exponentially with increasing terrapin head width; only males and small females (head width <30 mm) were likely to be vectors of seed dispersal. The characteristic that diamondback terrapins have well-defined home ranges allowed us to estimate the number of terrapins potentially dispersing eelgrass seeds annually. In seagrass beds of the Goodwin Islands region (lower York River, Virginia), there were 559 to 799 terrapins, which could disperse between 1,341 and 1,677 eelgrass seeds annually. These would represent a small proportion of total seed production within a single seagrass bed. However, based on probable home range distances, terrapins can easily traverse eelgrass meadow boundaries, thereby dispersing seeds beyond the bed of origin. Given the relatively short dispersion distance of eelgrass seeds, the diamondback terrapin may be a major source of inter-bed seed dispersal and genetic diversity.     

Large‐Scale Zostera marina (eelgrass) Restoration in Chesapeake Bay,Maryland, USA. Part II: A Comparison of Restoration Methods in the Patuxent and Potomac Rivers

In response to systemic losses of submerged aquatic vegetation (SAV) in the Chesapeake Bay (east coast of North America), the U.S. Environmental Protection Agency's (EPA) Chesapeake Bay Program (CBP) and Maryland Department of Natural Resources (MD DNR) have considered SAV restoration a critical component in Bay restoration programs. In 2003, the CBP created the “Strategy to Accelerate the Protection and Restoration of Submerged Aquatic Vegetation in the Chesapeake Bay” in an effort to increase SAV area. As part of this strategy, large‐scale eelgrass (Zostera marina) restoration efforts were initiated in the Patuxent and Potomac Rivers in Maryland. From 2004 to 2007, nearly 4 million Z. marina seeds were dispersed over 10 ha on the Patuxent River and almost 9 million seeds over 16 ha on the Potomac River. Z. marina seedling establishment was consistent throughout the project (<4%); however, restored eelgrass survival was highly dependent on restoration site. Restoration locations on the Patuxent River experienced initial Z. marina seedling germination, but no long‐term plant survival. Restored Z. marina on the Potomac River has persisted and expanded, both vegetatively and sexually, beyond initial seeding areas. Healthy Z. marina beds now cover approximately five acres of the Potomac River bottom for the first time in decades. The differential success of Z. marina restoration efforts in the two rivers is evidence for the necessity of carefully considering site‐specific characteristics when using large‐scale seeding methods to achieve successful SAV restoration.     

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.     

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.     

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.     

Fungi and Bacteria in or on Leaves of Eelgrass (Zostera marina L.) from Chesapeake Bay

Samples of green and brown leaves of eelgrass (Zostera marina L.) were incubated in seawater without an additional carbon source. Parallel leaf samples were used for acridine orange bacterial counting and water-soluble aniline blue estimation of fungal biovolume. The incubations produced no evidence that there is an eelgrass counterpart for the chytridialean symbiont which is very common in turtlegrass (Thalassia testudinum König). Sterile mycelium (i.e., living mycelium without identifiable propagules) was the most prevalent fungal form on incubated samples from submerged sites, whereas Dendryphiella salina and Sigmoidea sp. (marina?) were prevalent on brown leaves from the wrack line. Attempts to assay fungal biovolume in field samples indicated that the sterile mycelium observed after incubation represented the outgrowth of formerly dormant propagules or weakly established microcolonies. It was calculated that fungal biomass could not account for more than 0.5% of leaf mass, and it was probably much smaller than this, for no fungal structures were observed even in concentrated leaf homogenates. Bacterial densities fell within the range reported for other particulate substrates. A speculative estimate of bacterial productivity was 1.4× the standing stock per day.     

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.     

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.     

Effects of edge/interior and patch structure on reproduction in Zostera marina L. in Chesapeake Bay,USA

We examined the effects of location (patch edge vs. interior) and shoot density (individual, patchy, continuous) on reproduction in three natural and two transplanted Chesapeake Bay (USA) stands of the submerged marine angiosperm Zostera marina L. (eelgrass; Zosteraceae). There were no edge effects on demographic-based reproductive effort or reproductive output (propagule production), and patch structure (individual, patchy, continuous) alone never accounted for the majority of variability in any metric. Transplant site was the most important predictor of eelgrass reproduction response, and relationships among metrics were predictable within sites. Our results suggest that, in Chesapeake Bay eelgrass, environmental factors acting at a regional scale (km) have a stronger impact on reproductive investment than those at a patch scale (1–10 m). Since tradeoffs between clonal and sexual production are mediated primarily by exogenous environmental factors, site selection may be more critical than transplant configuration for producing self sustaining stands, and achieving long-term restoration success.     

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10² to 4.6×10²/ml for seawater, 4.0×10⁴ to 4.3×10⁵/g wet wt for eelgrass-bed sediment, and 2.1 × 10⁵ to 1.2 × 10⁷/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.     

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.     

Growing Zostera marina (eelgrass) from Seeds in Land‐Based Culture Systems for Use in Restoration Projects

The use of aquaculture systems to grow the seagrass Zostera marina (eelgrass) from seeds for restoration projects was evaluated through laboratory and mesocosm studies. Along the mid‐Atlantic coast of North America Z. marina seeds are shed from late spring through early summer, but seeds typically do not begin to germinate until the late fall. Fall is the optimal season to plant both seeds and shoots in this region. We conducted studies to determine if Z. marina seeds can be induced to germinate in the summer and seedlings grown in mesocosms to a size sufficiently large enough for out‐planting in the fall. Seeds in soil‐less culture germinated in the summer when held at 14°C, with percent germination increasing with lower salinities. Cold storage (4°C) of seeds prior to planting in sediments enhanced germination and seedling survival. Growth rates of seedlings were significantly higher in nutrient enriched estuarine sediments. Results from preliminary studies were used in designing a large‐scale culture project in which 15,000 shoots were grown and out‐planted into the Potomac River estuary in the Chesapeake Bay and compared with an equal number of transplanted shoots. These studies demonstrate that growing Z. marina from seeds is an alternative approach to harvesting plants from donor beds when vegetative shoots are required for restoration projects.     

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.     

Effectiveness of pectin extracted from the eelgrass Zostera marina for alleviating lead-induced liver injury

The effects of pectin from the eelgrass Zostera marina on toxic liver injury induced by enteral administration of lead acetate are examined in experiments on rats. The results show that pectin helps to rapidly reduce lead concentration in liver, to decrease lipid peroxidation, and to normalize the levels of total cholesterol and triglycerides in blood serum and liver.     

Characterization of microsatellite loci in the dwarf eelgrass Zostera noltii (Zosteraceae) and cross‐reactivity with Z. japonica

Zostera noltii is an important species of eelgrass occurring along European, north African, Mediterranean, Black Sea and Azov Sea coasts. Nine microsatellite loci were developed and no linkage disequilibrium was observed. Cross‐amplification was observed for all loci (polymorphic) in Z. japonica; only four loci amplified (monomorphic) in Z. marina.