Search

Conservation efforts for endangered sea turtle species, such as Kemp’s ridley turtles Lepidochelys kempii and green turtles Chelonia mydas, may benefit from information on the microbial communities that contribute to host health. Previous studies examining host-associated microbiomes of these species have been limited in geographic region, life stage, and/or health. Here, we characterized the microbiome of the oral cavity and cloaca from wild-captured Kemp’s ridley and green turtles off the west coast of Florida, USA, by using Illumina sequencing to analyze the 16S rRNA gene. Microbial communities were distinct between body sites as well as between turtle species, suggesting that the turtle species is more important than the local environment in determining the microbiome of sea turtles. We identified the core microbiome for each species at each body site and determined that there were very few bacteria shared among the oral samples of both species, and no taxa co-occurred in the cloaca samples among both species. The core microbiome of the green turtle cloaca was primarily from the order Clostridiales, which plays an important role in digestion for other herbivorous species. Due to high prevalence of fibropapillomatosis in the green turtles (90%), we also investigated the correlation between the microbiome and the severity of fibropapillomatosis, and we identified changes in beta diversity associated with the total number of tumors. This study provides the first glimpse of the microbiome in 2 sympatric species of sea turtle and sheds an important species-specific light on the microbiome of these endangered species.

Background The term holobiont is widely accepted to describe animal hosts and their associated microorganisms. The genomes of all that the holobiont encompasses, are termed the hologenome and it has been proposed as a unit of selection in evolution. To demonstrate that natural selection acts on the hologenome, a significant portion of the associated microbial genomes should be transferred between generations. Using the Sydney Rock Oyster (Saccostrea glomerata) as a model, we tested if the microbes of this broadcast spawning species could be passed down to the next generation by conducting single parent crosses and tracking the microbiome from parent to offspring and throughout early larval stages using 16S rRNA gene amplicon sequencing. From each cross, we sampled adult tissues (mantle, gill, stomach, gonad, eggs or sperm), larvae (D-veliger, umbo, eyed pediveliger, and spat), and the surrounding environment (water and algae feed) for microbial community analysis. Results We found that each larval stage has a distinct microbiome that is partially influenced by their parental microbiome, particularly the maternal egg microbiome. We also demonstrate the presence of core microbes that are consistent across all families, persist throughout early life stages (from eggs to spat), and are not detected in the microbiomes of the surrounding environment. In addition to the core microbiomes that span all life cycle stages, there is also evidence of environmentally acquired microbial communities, with earlier larval stages (D-veliger and umbo), more influenced by seawater microbiomes, and later larval stages (eyed pediveliger and spat) dominated by microbial members that are specific to oysters and not detected in the surrounding environment. Conclusion Our study characterized the succession of oyster larvae microbiomes from gametes to spat and tracked selected members that persisted across multiple life stages. Overall our findings suggest that both horizontal and vertical transmission routes are possible for the complex microbial communities associated with a broadcast spawning marine invertebrate. We demonstrate that not all members of oyster-associated microbiomes are governed by the same ecological dynamics, which is critical for determining what constitutes a hologenome.

In response to current threats to salt marshes, there are increasing efforts to restore these vital coastal ecosystems and promote their resilience to global change drivers. Unfortunately, the economic cost associated with assessing the effectiveness of restoration is prohibitive and more information is needed about the trajectory and timing of restoration outcomes to improve restoration practices. Microbial communities provide essential salt marsh functions so assessing the degree to which microbial communities in restored marshes resemble reference marshes can serve as a proxy indicator for the potential return of microbial function. We studied a recently restored marsh located on Cape Cod, MA, USA, by examining shifts in the microbial community and sediment edaphic properties in three habitats of a degraded oligohaline marsh, both before and after restoration of tidal flooding and in comparison with three nearby S. alterniflora reference marshes that never had flow restrictions. We hypothesized that the microbial community would respond rapidly to the restoration and that over time these communities would be indistinguishable from reference marsh communities. We found that soil edaphic characteristics shifted along a trajectory of recovery toward the reference marshes, with increases in salinity and decreases in soil organic matter, percentage of carbon, and percentage of nitrogen. The microbial communities in all three habitats within the restored marsh were different from reference marshes, and both the prokaryotic and fungal communities within P. australis and Typha sp. habitats became more similar to reference marshes (similarities increasing from an average of 5 to 16% for prokaryotes and 3 to 10% for fungi) during the first 2 years after restoration. In that same time period, by contrast, there was no return of the native marsh vegetation. These results suggest that shifts in microbial community structure occur prior to shifts in marsh vegetation and may facilitate the successful revegetation of restored marshes. Understanding the recovery trajectory of marshes during restoration and the role that microbes play in promoting the long-term sustainability of these habitats is essential; these results suggest that microbial communities respond rapidly and in a positive direction to restoration efforts.

To better understand how complex microbial communities become assembled on eukaryotic hosts, it is essential to disentangle the balance between stochastic and deterministic processes that drive their assembly. Deterministic processes can create consistent patterns of microbiome membership that result in persistent resident communities, while stochastic processes can result in random fluctuation of microbiome members that are transient with regard to their association to the host. We sampled oyster reefs from six different populations across the east coast of the United States. At each site we collected gill tissues for microbial community analysis and additionally collected and shipped live oysters to Northeastern University where they were held in a common garden experiment. We then examined the microbiome shifts in gill tissues weekly for 6 weeks using 16S rRNA gene amplicon sequencing. We found a strong population-specific signal in the microbial community composition of field-sampled oysters. Surprisingly, the oysters sampled during the common garden experiment maintained compositionally distinct gill-associated microbial communities that reflected their wild population of origin, even after rearing them in a common garden for several weeks. This indicates that oyster gill-associated microbiota are predominantly composed of resident microbes specific to host population, rather than being a reflection of their immediate biotic and abiotic surroundings. However, certain bacterial taxa tended to appear more frequently on individuals from different populations than on individuals from the same population, indicating that there is a small portion of the gill microbiome that is transient and is readily exchanged with the environmental pool of microbes. Regardless, the majority of gill-associated microbes were resident members that were specific to each oyster population, suggesting that there are strong deterministic factors that govern a large portion of the gill microbiome. A small portion of the microbial communities, however, was transient and moved among oyster populations, indicating that stochastic assembly also contributes to the oyster gill microbiome. Our results are relevant to the oyster aquaculture industry and oyster conservation efforts because resident members of the oyster microbiome may represent microbes that are important to oyster health and some of these key members vary depending on oyster population.

Anthropogenic nitrate amendment to coastal marine sediments can increase rates of heterotrophic mineralization and autotrophic dark carbon fixation (DCF). DCF may be favored in sediments where organic matter is biologically unavailable, leading to a microbial community supported by chemoautotrophy. Niche partitioning among DCF communities and adaptations for nitrate metabolism in coastal marine sediments remain poorly characterized, especially within salt marshes. We used genome-resolved metagenomics, phylogenetics, and comparative genomics to characterize the potential niche space, phylogenetic relationships, and adaptations important to microbial communities within nitrate enriched sediment. We found that nitrate enrichment of sediment from discrete depths between 0-25 cm supported both heterotrophs and chemoautotrophs that use sulfur oxidizing denitrification to drive the Calvin-Benson-Bassham (CBB) or reductive TCA (rTCA) DCF pathways. Phylogenetic reconstruction indicated that the nitrate enriched community represented a small fraction of the phylogenetic diversity contained in coastal marine environmental genomes, while pangenomics revealed close evolutionary and functional relationships with DCF microbes in other oligotrophic environments. These results indicate that DCF can support coastal marine microbial communities and should be carefully considered when estimating the impact of nitrate on carbon cycling in these critical habitats.ImportanceSalt marshes store carbon at one of the fastest rates of any blue carbon system and buffer coastal marine waters from eutrophication. Dark carbon fixation (DCF) conducted by microbes within the sediment can influence the carbon storage capacity, but little is known about the ecology or genomic potential of these organisms. Our study identifies a potential niche space for several functionally distinct groups of chemoautotrophs which primarily use sulfur oxidizing denitrification to fuel DCF under high nitrate concentrations. These findings fill an important gap in our understanding of microbial contributions to carbon storage within salt marsh sediments and how this critical blue carbon system responds to anthropogenic nitrate enrichment.

Salt marsh sediments are known hotspots for nitrogen cycling, including the production and consumption of nitrous oxide (N2O), a potent greenhouse gas and ozone-depleting agent. Coastal eutrophication, particularly elevated nitrogen loading from the application of fertilizers, is accelerating nitrogen cycling processes in salt marsh sediments. Here, we examine the impact of long-term fertilization on nitrogen cycling processes with a focus on N2O dynamics in a New England salt marsh. By combining 15N-tracer experiments with numerical modeling, we found that both nitrification and denitrification contribute to net N2O production in fertilized sediments. Long-term fertilization increased the relative importance of nitrification to N2O production, likely a result of increased oxygen penetration from nutrient-induced increases in marsh elevation. Substrate utilization rates of key nitrogen cycling processes revealed links between functions and the corresponding microbial communities. Higher specific substrate utilization rates leading to N2O production from nitrification in fertilized sediments indicate a shift in the community composition of ammonia oxidizers, whereas the lack of change in specific substrate utilization of N2O production from denitrification under long-term fertilization suggests resilience of the denitrifying communities. Both are consistent with previous studies on the functional gene community composition in these experimental plots.

Coastal seagrass, mangrove and salt-marsh ecosystems—also termed blue-carbon ecosystems—play an important role in the global carbon cycle. Much of the organic carbon they store rests in soils that have accumulated over thousands of years. Rapidly changing climate and environmental conditions, including sea-level rise, warming, eutrophication and landscape development, will impact decomposition and thus the global reservoir of blue soil organic carbon. Yet, it remains unclear how these disturbances will affect the key biogeochemical mechanisms controlling decomposition—mineral protection, redox zonation, water content and movement, and plant–microbe interactions. We assess the spatial and temporal scales over which decomposition mechanisms operate and how their effectiveness may change following disturbances. We suggest that better integration of decomposition mechanisms into blue-carbon models may improve predictions of soil organic carbon stores and facilitate incorporation of coastal vegetated ecosystems into global budgets and management tools. Coastal vegetated ecosystems have experienced rapid changes in climate and environmental conditions. These changes have caused disturbances to the amount of carbon they store in soils by altering the decomposition process of organic carbon.

The diversity and environmental distribution of the nosZ gene, which encodes the enzyme responsible for the consumption of nitrous oxide, was investigated in marine and terrestrial environments using a functional gene microarray. The microbial communities represented by the nosZ gene probes showed strong biogeographical separation. Communities from surface ocean waters and agricultural soils differed significantly from each other and from those in oceanic oxygen minimum zones. Atypical nosZ genes, usually associated with incomplete denitrification pathways, were detected in all the environments, including surface ocean waters. The abundance of nosZ genes, as estimated by quantitative PCR, was highest in agricultural soils and lowest in surface ocean waters.

Enrichment of ecosystems with excess nutrients is occurring at an alarming rate and has fundamentally altered ecosystems worldwide. Salt marshes, which lie at the land-sea interface, are highly effective at removing anthropogenic nutrients through the action of macrophytes and through microbial processes in coastal sediments. The response of salt marsh bacteria to excess nitrogen has been documented; however, the role of fungi and their response to excess nitrogen in salt marsh sediments is not fully understood. Here, we document the response of salt marsh fungal communities to long-term excess nitrate in four distinct marsh habitats within a northern temperate marsh complex. We show that salt marsh fungal communities varied as a function of salt marsh habitat, with both fungal abundance and diversity increasing with carbon quantity. Nutrient enrichment altered fungal communities in all habitats through an increase in fungal abundance and the proliferation of putative fungal denitrifiers. Nutrient enrichment also altered marsh carbon quality in low marsh surface sediments where fungal response to nutrient enrichment was most dramatic, suggesting nutrient enrichment can alter organic matter quality in coastal sediments. Our results indicate that fungi, in addition to bacteria, likely play an important role in anaerobic decomposition of salt marsh sediment organic matter.

Aquaculture currently provides half of all fish for human consumption, and this proportion is expected to increase to meet the growing global demand for protein. As aquaculture, including oyster farming, expands, it is increasingly important to understand effects on coastal ecosystems. The broad-scale ecological effects of oyster aquaculture are well documented; however, less is known regarding the influence of oyster aquaculture on sediment bacterial communities. To better understand this relationship, we compared three different oyster farming practices that varied in oyster biomass and proximity of oysters to the sediment. We used high-throughput sequencing and quantitative polymerase chain reaction to examine the effect of oyster farming on sediment bacterial communities. We examined the entire bacterial community and looked specifically at bacteria that support essential estuarine ecosystem services (denitrifiers), as well as bacteria that can be detrimental to human health (members of the Vibrio genus). We found that oyster biomass increased Vibrio richness and sediment carbon content, which influenced bacterial community composition. When compared to reference sites, the overall abundance of bacteria was increased by the bottom planting method, but the associated increases in denitrifiers and Vibrio were not significant. We were unable to detect V. parahaemolyticus, V. vulnificus, or V. cholera, the three most common Vibrio pathogens, in any sample, suggesting that oyster farming did not enhance these potential human pathogens in sediments at the time of sampling. These results highlight how differences in oyster farming practice can affect sediment bacterial communities, and the ecosystem services they provide.

The American lobster Homarus americanus supports a valuable commercial fishery in the Northeastern USA and Maritime Canada; however, stocks in the southern portion of the lobster's range have shown declines, in part due to the emergence of shell disease. Epizootic shell disease is a bacterially induced cuticular erosion that renders even mildly affected lobsters unmarketable because of their appearance, and in more severe cases can cause mortality. Despite the importance of this disease, the associated bacterial communities have not yet been fully characterized. We sampled 2 yr old, laboratory-reared lobsters that displayed signs of shell disease at the site of disease as well as at 0.5, 1, and 1.5 cm away from the site of disease to determine how the bacterial community changed over this fine spatial scale. Illumina sequencing of the 16S rRNA gene revealed a distinct bacterial community at the site of disease, with significant reductions in bacterial diversity and richness compared to more distant sampling locations. The bacterial community composition 0.5 cm from the site of disease was also altered, and there was an observable decrease in bacterial diversity and richness, even though there were no signs of disease at that location. Given the distinctiveness of the bacterial community at the site of disease and 0.5 cm from the site of disease, we refer to these communities as affected and transitionary, and suggest that these bacteria, including the previously proposed causative agent, Aquimarina 'homaria', are important for the initiation and progression of this laboratory model of shell disease.

The balance between nitrate respiration pathways, denitrification and dissimilatory nitrate (NO3 - ) reduction to ammonium (DNRA), determines whether bioavailable nitrogen is removed as N2 gas or recycled as ammonium. Saltwater intrusion and organic matter enrichment may increase sulphate reduction leading to sulphide accumulation. We investigated the effects of sulphide on the partitioning of NO3 - between complete denitrification and DNRA and the microbial communities in salt marsh sediments. Complete denitrification significantly decreased with increasing sulphide, resulting in an increase in the contribution of DNRA to NO3 - respiration. Alternative fates of NO3 - became increasingly important at higher sulphide treatments, which could include N2 O production and/or transport into intracellular vacuoles. Higher 16S transcript diversity was observed in the high sulphide treatment, with clear shifts in composition. Generally, low and no sulphide, coupled with high NO3 - , favoured the activity of Campylobacterales, Oceanospirillales and Altermonadales, all of which include opportunistic denitrifiers. High ∑sulphide conditions promoted the activity of potential sulphide oxidizing nitrate reducers (Desulfobulbaceae, Acidiferrobacteraceae and Xanthomonadales) and sulphate reducers (Desulfomonadaceae, Desulfobacteraceae). Our study highlights the tight coupling between N and S cycling, and the implications of these dynamics on the fate of bioavailable N in coastal environments susceptible to intermittent saltwater inundation and organic matter enrichment.

Microbial communities of animals play a role in health and disease, including immunocompromised conditions. In the northeastern United States, cold-stunning events often cause endangered Kemp’s ridley turtles (Lepidochelys kempii) to become stranded on beaches in autumn. These sea turtles are admitted to rehabilitation facilities when rescued alive and are presumed immunocompromised secondary to hypothermia. To better understand the role that microbes play in the health of cold-stunned sea turtles, we characterized the oral and cloacal microbiome from Kemp’s ridley turtles at multiple timepoints during rehabilitation, from admission to pre-release, by using Illumina sequencing to analyze the 16S rRNA gene. Microbial communities were distinct between body sites and among turtles that survived and those that died. We found that clinical parameters such as presence of pneumonia or values for various blood analytes did not correlate with oral or cloacal microbial community composition. We also investigated the effect of antibiotics on the microbiome during rehabilitation and prior to release and found that the type of antibiotic altered the microbial community composition, yet overall taxonomic diversity remained the same. The microbiome of cold-stunned Kemp’s ridley turtles gradually changed through the course of rehabilitation with environment, antibiotics, and disease status all playing a role in those changes and ultimately the release status of the turtles.

Rates of sea level rise associated with climate change are predicted to increase in the future, potentially altering ecosystems at all ecological levels. Sea level rise can increase the extent of brackish water intrusion into freshwater ecosystems, which in turn can affect the structure and function of resident microbial communities. In this study, we performed a year-long mesocosm experiment using intact tidal freshwater marsh sediment cores to examine the effect of a 5-part per thousand (ppt) salinity increase on the diversity and community composition of sulfate-reducing prokaryotes. We used a clone library approach to examine the dsrA gene, which encodes an important catalytic enzyme in sulfate reduction. Our results indicate that tidal freshwater marshes contain extremely diverse communities of sulfate-reducing bacteria. Members of these communities were, on average, only 71 % similar to known cultured sulfate reducers and 81 % similar to previously sequenced environmental clones. Salinity and associated increases in sulfate availability did not significantly affect the diversity or community composition of sulfate-reducing prokaryotes. However, carbon quality and quantity, which correlated with depth, were found to be the strongest drivers of sulfate-reducing community structure. Our study demonstrates that the sulfate-reducing community in tidal freshwater marsh sediments appears resistant to increased salinity in the face of sea level rise. Additionally, the microorganisms that comprise this sulfate-reducing community appear to be unique to tidal freshwater marsh sediments and may represent novel lineages of previously undescribed sulfate reducers.

Bivalves serve an important ecosystem function in delivering organic matter from pelagic to benthic zones and are important in mediating eutrophication. However, the fate of this organic matter (i.e., biodeposits) is an important consideration when assessing the ecological roles of these organisms in coastal ecosystems. In addition to environmental conditions, the processing of biodeposits is dependent on its composition and the metabolic capacity of the associated microbial community. The objectives of this study were to compare the biological reactivity, potential denitrification rates, and microbial communities of biodeposits sourced from different bivalve species: hard clam (Mercenaria mercenaria), eastern oyster (Crassostrea virginica), and ribbed mussel (Geukensia demissa). To our knowledge, this is the first study to investigate and compare the microbiome of bivalve biodeposits using high-throughput sequencing and provide important insight into the mechanisms by which bivalves may alter sediment microbial communities and benthic biogeochemical cycles. We show that clam biodeposits had significantly higher bioreactivity compared to mussel and oyster biodeposits, as reflected in higher dissolved inorganic carbon and ammonium production rates in controlled incubations. Potential denitrification rates were also significantly higher for clam biodeposits compared to oyster and mussel biodeposits. The microbial communities associated with the biodeposits were significantly different across bivalve species, with significantly greater abundances of Alteromonadales, Chitinophagales, Rhodobacterales, and Thiotrichales associated with the clam biodeposits. These bioreactivity and microbial differences across bivalve species are likely due to differences in bivalve physiology and feeding behavior and should be considered when evaluating the effects of bivalves on water quality and ecosystem function.

Salt marshes sequester carbon at rates more than an order of magnitude greater than their terrestrial counterparts, helping to mitigate climate change. As nitrogen loading to coastal waters continues, primarily in the form of nitrate, it is unclear what effect it will have on carbon storage capacity of these highly productive systems. This uncertainty is largely driven by the dual role nitrate can play in biological processes, where it can serve as a nutrient-stimulating primary production or a thermodynamically favorable electron acceptor fueling heterotrophic metabolism. Here, we used a controlled flow-through reactor experiment to test the role of nitrate as an electron acceptor, and its effect on organic matter decomposition and the associated microbial community in salt marsh sediments. Organic matter decomposition significantly increased in response to nitrate, even at sediment depths typically considered resistant to decomposition. The use of isotope tracers suggests that this pattern was largely driven by stimulated denitrification. Nitrate addition also significantly altered the microbial community and decreased alpha diversity, selecting for taxa belonging to groups known to reduce nitrate and oxidize more complex forms of organic matter. Fourier Transform-Infrared Spectroscopy further supported these results, suggesting that nitrate facilitated decomposition of complex organic matter compounds into more bioavailable forms. Taken together, these results suggest the existence of organic matter pools that only become accessible with nitrate and would otherwise remain stabilized in the sediment. The existence of such pools could have important implications for carbon storage, since greater decomposition rates as N loading increases may result in less overall burial of organic-rich sediment. Given the extent of nitrogen loading along our coastlines, it is imperative that we better understand the resilience of salt marsh systems to nutrient enrichment, especially if we hope to rely on salt marshes, and other blue carbon systems, for long-term carbon storage.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Salt marshes provide many key ecosystem services that have tremendous ecological and economic value. One critical service is the removal of fixed nitrogen from coastal waters, which limits the negative effects of eutrophication resulting from increased nutrient supply. Nutrient enrichment of salt marsh sediments results in higher rates of nitrogen cycling and, commonly, a concurrent increase in the flux of nitrous oxide, an important greenhouse gas. Little is known, however, regarding controls on the microbial communities that contribute to nitrous oxide fluxes in marsh sediments. To address this disconnect, we generated profiles of microbial communities and communities of micro-organisms containing specific nitrogen cycling genes that encode several enzymes (amoA, norB, nosZ) related to nitrous oxide flux from salt marsh sediments. We hypothesized that communities of microbes responsible for nitrogen transformations will be structured by nitrogen availability. Taxa that respond positively to high nitrogen inputs may be responsible for the elevated rates of nitrogen cycling processes measured in fertilized sediments. Our data show that, with the exception of ammonia-oxidizing archaea, the community composition of organisms involved in the production and consumption of nitrous oxide was altered under nutrient enrichment. These results suggest that previously measured rates of nitrous oxide production and consumption are likely the result of changes in community structure, not simply changes in microbial activity.

Nitrous oxide (N2O) is important to Earth's climate because it is a strong absorber of radiation and an important ozone depletion agent. Increasing anthropogenic nitrogen input into the marine environment, especially to coastal waters, has led to increasing N2O emissions. Identifying the nitrogen compounds that serve as substrates for N2O production in coastal waters reveals important pathways and helps us understand their control by environmental factors. In this study, sediments were collected from a long-term fertilization site in Great Sippewissett Marsh, Falmouth, Massachusetts. The 15N tracer incubation time course experiments were conducted and analyzed for potential N2O production and consumption rates. The two nitrogen substrates of N2O production, ammonium and nitrate, correspond to the two production pathways, nitrification and denitrification, respectively. When measurable nitrate was present, despite ambient high ammonium concentrations, denitrification was the major N2O production pathway. When nitrate was absent, ammonium became the dominant substrate for N2O production, via nitrification and coupled nitrification-denitrification. Net N2O consumption was enhanced under low oxygen and nitrate conditions. N2O production and consumption rates increased with increasing levels of nitrogen fertilization in long-term experimental plots. These results indicate that increasing anthropogenic nitrogen input to salt marshes can stimulate sedimentary N2O production via both nitrification and denitrification, whereas episodic oxygen depletion results in net N2O consumption.

Enrichment of natural waters, soils, and sediments by inorganic nutrients, including nitrogen, is occurring at an increasing rate and has fundamentally altered global biogeochemical cycles. Salt marshes are critical for the removal of land-derived nitrogen before it enters coastal waters. This is accomplished via multiple microbially mediated pathways, including denitrification. Many of these pathways, however, are also a source of the greenhouse gas nitrous oxide (N2O). We used clone libraries and quantative PCR (qPCR) to examine the effect of fertilization on the diversity and abundance of two functional genes associated with denitrification and N2O production (norB and nosZ) in experimental plots at the Great Sippewissett Salt Marsh (Falmouth, MA, USA) that have been enriched with nutrients for over 40 years. Our data showed distinct nosZ and norB community structures at different nitrogen loads, especially at the highest level of fertilization. Furthermore, calculations of the Shannon Diversity Index and Chao1 Richness Estimator indicated that nosZ gene diversity and richness increased with increased nitrogen supply, however no such relationship existed with regard to richness and diversity of the norB gene. Results from qPCR demonstrated that nosZ gene abundance was an order of magnitude lower in the extra-highly fertilized plots compared to the other plots, but the abundance of norB was not affected by fertilization. The majority of sequences obtained from the marsh plots had no close cultured relatives and they were divergent from previously sequenced norB and nosZ fragments. Despite their divergence from any cultured representatives, most of the norB and nosZ sequences appeared to be from members of the Alpha- and Betaproteobacteria, suggesting that these classes are particularly important in salt marsh nitrogen cycling. Our results suggest that both norB and nosZ containing microbes are affected by fertilization and that the Great Sippewissett Marsh may harbor distinct clades of novel denitrifying microorganisms that are responsible for both the production and removal of N2O.

© 2017 Kearns, Fischer, Fernández-Beaskoetxea, Gabor, Bosch, Bowen, Tlusty and Woodhams, Emerging infectious diseases caused by fungal taxa are increasing and are placing a substantial burden on economies and ecosystems worldwide. Of the emerging fungal diseases, chytridomycosis caused by the fungus Batrachochytrium dendrobatidis (hereafter Bd) is linked to global amphibian declines. Amphibians have innate immunity, as well as additional resistance through cutaneous microbial communities. Despite the targeting of bacteria as potential probiotics, the role of fungi in the protection against Bd infection in unknown. We used a four-part approach, including high-throughput sequencing of bacterial and fungal communities, cultivation of fungi, Bd challenge assays, and experimental additions of probiotic to Midwife Toads (Altyes obstetricans), to examine the overlapping roles of bacterial and fungal microbiota in pathogen defense in captive bred poison arrow frogs (Dendrobates sp.). Our results revealed that cutaneous fungal taxa differed from environmental microbiota across three species and a subspecies of Dendrobates spp. frogs. Cultivation of host-associated and environmental fungi realved numerous taxa with the ability to inhibit or facilitate the growth of Bd. The abundance of cutaneous fungi contributed more to Bd defense (∼45% of the fungal community), than did bacteria (∼10%) and frog species harbored distinct inhibitory communities that were distinct from the environment. Further, we demonstrated that a fungal probiotic therapy did not induce an endocrine-immune reaction, in contrast to bacterial probiotics that stressed amphibian hosts and suppressed antimicrobial peptide responses, limiting their long-term colonization potential. Our results suggest that probiotic strategies against amphibian fungal pathogens should, in addition to bacterial probiotics, focus on host-associated and environmental fungi such as Penicillium and members of the families Chaetomiaceae and Lasiosphaeriaceae., This work was conducted on behalf of the New England Aquarium in Boston MA, a not-for profit institution (501c3).

Plant–microbe interactions play crucial roles in species invasions but are rarely investigated at the intraspecific level. Here, we study these interactions in three lineages of a globally distributed plant, Phragmites australis. We use field surveys and a common garden experiment to analyze bacterial communities in the rhizosphere of P. australis stands from native, introduced, and Gulf lineages to determine lineage-specific controls on rhizosphere bacteria. We show that within-lineage bacterial communities are similar, but are distinct among lineages, which is consistent with our results in a complementary common garden experiment. Introduced P. australis rhizosphere bacterial communities have lower abundances of pathways involved in antimicrobial biosynthesis and degradation, suggesting a lower exposure to enemy attack than native and Gulf lineages. However, lineage and not rhizosphere bacterial communities dictate individual plant growth in the common garden experiment. We conclude that lineage is crucial for determination of both rhizosphere bacterial communities and plant fitness., Environmental factors often outweigh host heritable factors in structuring host-associated microbiomes. Here, Bowen et al. show that host lineage is crucial for determination of rhizosphere bacterial communities in Phragmites australis, a globally distributed invasive plant.

Emerging infectious diseases caused by fungal taxa are increasing and are placing a substantial burden on economies and ecosystems worldwide. Of the emerging fungal diseases, chytridomycosis caused by the fungus Batrachochytrium dendrobatidis (hereafter Bd) is causing a global amphibian extinction. The host frog does have come internal innate immunity, as well as additional resistance through cutaneous microbial communities, leading to the development of probiotic bacterial therapies with mixed results. Unknown is the role of fungi in the protection against Bd infection, and as such, we examined the overlapping roles of bacterial and fungal microbiota in pathogen defense with a combination of high-throughput sequencing and culturing of symbiotic fungi from poison arrow frogs (Dendrobates sp.). Our analyses revealed that abundance of cutaneous fungi contributed more to pathogen defense (~45%), than bacteria (~10%) and these differed from environmental microbiota. Further, we demonstrated that a fungal probiotic therapy did not induce an endocrine-immune reaction in contrast to bacterial probiotics that stressed amphibian hosts and suppressed antimicrobial peptide responses, limiting their long-term colonization potential. Our results suggest that probiotic strategies against amphibian fungal pathogens should refocus on host-associated and environmental fungi such as Penicillium and member of the families Chaetomiaceae and Lasiosphaeriaceae.

Boston Harbor, an urban harbor located in Massachusetts, USA, has a long history of use and environmental degradation, followed by extensive restoration efforts. The coastal resources of the region sustained native tribes for thousands of years prior to European settlement, and those resources sustained early settlers, allowing them to expand throughout the region. By the late 1600s, the city of Boston was the most influential shipping and trading city in the new colonies. Continuous growth of the region throughout the 18 t h and 19 t h centuries, however, strained those resources. Extensive areas of coastal wetlands were filled for urban development, resident wastewater was disposed of in the Harbor, and industrialization of the waterfront resulted in additional point source contamination. By the 1980s, Boston Harbor was considered among the most polluted harbors in the country. Extensive efforts in recent years, however, have restored the water quality in the Harbor and revitalized the waterfront region. Below we describe the socio-economic and ecological resources of the region, explore the current threats Boston Harbor faces, and highlight two examples of actions that helped restore the Harbor to its current conditions.

Increasing atmospheric CO2 concentra- tions are causing decreased pH over vast expanses of the ocean. This decreasing pH may alter bio - geochemical cycling of carbon and nitrogen via the microbial process of nitrification, a key process that couples these cycles in the ocean, but which is often sensitive to acidic conditions. Recent reports have indicated a decrease in oceanic nitrification rates under experimentally lowered pH. How the compo- sition and abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) assemblages respond to decreasing oceanic pH is unknown. We sampled microbes from 2 different acidification experiments and used a combination of qPCR and functional gene microarrays for the ammonia monooxygenase gene (amoA) to assess how acidification alters the structure of ammonia oxidizer assemblages. We show that despite widely different experimental conditions, acidification consistently altered the com - munity composition of AOB by increasing the rela- tive abundance of taxa related to the Nitrosomonas ureae clade. In one experiment, this increase was sufficient to cause an increase in the overall abun- dance of AOB. There were no systematic shifts in the community structure or abundance of AOA in either experiment. These different responses to acidification underscore the important role of micro- bial community structure in the resiliency of marine ecosystems.

Public aquaria offer numerous educational opportunities for visitors while touch-tank exhibits offer guests the ability to directly interact with marine life via physical contact. Despite the popularity of touch-tanks, there is a paucity of research about animal health in these exhibits and, in particular, there is little research on the microbial communities in these highly interactive exhibits. Microbial community structure can have implications for both host health and habitat function. To better understand the microbiome of a touch-tank we used high-throughput sequencing of the 16S rRNA gene to analyze the microbial community on the dorsal and ventral surfaces of cow-nose rays (Rhinoptera bonasus) as well as their environment in a frequently visited touch-tank exhibit at the New England Aquarium. Our analyses revealed a distinct microbial community associated with the skin of the ray that had lower diversity than the surrounding habitat. The ray skin was dominated by three orders: Burkholderiales (∼55%), Flavobacteriales (∼19%), and Pseudomonadales (∼12%), taxonomic groups commonly associated with other fish species. Our results provide a survey of ray-associated bacterial communities in a touch-tank environment, thereby laying the foundation for future studies examining the role of potential challenges to ray microbiota and their associated health.

Microorganisms control key biogeochemical pathways, thus changes in microbial diversity, community structure and activity can affect ecosystem response to environmental drivers. Understanding factors that control the proportion of active microbes in the environment and how they vary when perturbed is critical to anticipating ecosystem response to global change. Increasing supplies of anthropogenic nitrogen to ecosystems globally makes it imperative that we understand how nutrient supply alters active microbial communities. Here we show that nitrogen additions to salt marshes cause a shift in the active microbial community despite no change in the total community. The active community shift causes the proportion of dormant microbial taxa to double, from 45 to 90%, and induces diversity loss in the active portion of the community. Our results suggest that perturbations to salt marshes can drastically alter active microbial communities, however these communities may remain resilient by protecting total diversity through increased dormancy., Increased anthropogenic nitrogen inputs into the biosphere are fundamentally altering ecosystems worldwide. Here, Kearns et al. show that a decade of nitrogen additions to salt marshes reduces the proportion of active microorganisms, despite no net change to the total microbial community.

Salt marshes provide numerous valuable ecological services. In particular, nitrogen (N) removal in salt marsh sediments alleviates N loading to the coastal ocean. N removal reduces the threat of eutrophication caused by increased N inputs from anthropogenic sources. It is unclear, however, whether chronic nutrient over-enrichment alters the capacity of salt marshes to remove anthropogenic N. To assess the effect of nutrient enrichment on N cycling in salt marsh sediments, we examined important N cycle pathways in experimental fertilization plots in a New England salt marsh. We determined rates of nitrification, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) using sediment slurry incubations with 15 N labeled ammonium or nitrate tracers under oxic headspace (20% oxygen / 80% helium). Nitrification and denitrification rates were more than ten-fold higher in fertilized plots compared to control plots. By contrast, DNRA, which retains N in the system, was high in control plots but not detected in fertilized plots. The relative contribution of DNRA to total nitrate reduction largely depends on the carbon/nitrate ratio in the sediment. These results suggest that long-term fertilization shifts N cycling in salt marsh sediments from predominantly retention to removal. Long-term fertilization alters the relative importance of nitrate reduction pathways in salt marsh sediments: NO 3 - reduction in salt marsh sediments (PDF Download Available). Available from: https://www.researchgate.net/publication/305480944_Long-term_fertilization_alters_the_relative_importance_of_nitrate_reduction_pathways_in_salt_marsh_sediments_NO_3_-_reduction_in_salt_marsh_sediments [accessed Jun 6, 2017].

Much of the phylogenetic diversity in microbial systems arises from rare taxa that comprise the long tail of taxon rank distribution curves. This vast diversity presents a challenge to testing hypotheses about the effects of perturbations on microbial community composition because variability of rare taxa among environmental replicates may be sufficiently large that it would require a prohibitive degree of sequencing to discern differences between samples. In this study we used pyrosequencing of 16S rRNA tags to examine the diversity and within-site variability of salt marsh sediment bacteria. Our goal was to determine whether pyrosequencing could produce similar patterns in community composition among replicate environmental samples from the same location. We hypothesized that repeated sampling from the same location would produce different snapshots of the rare community due to incomplete sequencing of the taxonomically rich rare biosphere. We demonstrate that the salt marsh sediments we sampled contain a remarkably diverse array of bacterial taxa and, in contrast to our hypothesis, repeated sampling from within the same site produces reliably similar patterns in bacterial community composition, even among rare organisms. These results demonstrate that deep sequencing of 16s tags is well suited to distinguish site-specific similarities and differences among rare taxa and is a valuable tool for hypothesis testing in microbial ecology.

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation. The ISME Journal advance online publication, 17 March 2011; doi:10.1038/ismej.2011.22 Subject Category: microbial ecology and functional diversity of natural habitats

We examined the role of chronic low-level nutrient enrichment on the productivity of heterotrophic marsh bacteria via a marsh fertilization experiment in which we mimicked the conditions of widespread coastal eutrophication by enriching entire salt marshes to approximately 153 background nutrient concentrations. We measured the uptake of tritiated leucine, as a proxy for bacterial production, in both low and high marsh habitats in four salt marshes, two of which were enriched with nutrients. We hypothesized that adding nitrogen in these detritus-rich systems would directly stimulate bacterial decomposition of marsh peat. Contrary to our expectations, we found no response to added nutrients in high marsh habitats, where there is a significant supply of organic matter from marsh vegetation. Bacterial production did increase in the low marsh habitats, where fertilization increased the standing stock of benthic chlorophyll. Fertilization did not directly increase bacterial production by providing added nutrients that could be used to decompose organic matter derived from nutrient-poor marsh grasses. Rather, bacterial productivity was indirectly stimulated by the concomitant increase in labile benthic microalgae in low marsh habitats. Decomposition of salt marshes may therefore have a greater resilience to the threat of chronic eutrophication than has been previously recognized.

Hydrological coupling between watersheds and estuaries is an important element in establishing eutrophic conditions in coastal waters. In coupled systems, nutrients derived on land enter estuaries through groundwater flow or stream runoff, resulting in increased rates of primary production. The extent of coupling is determined by precipitation, evapotransporation, and water use practices. We use stable nitrogen isotopes, salinity, and nutrient data to assess coupling in two temperate and two tropical ecosystems. In each region, we selected two estuaries, one pristine and one with considerable agricultural or urban development. In pristine systems freshwater entering estuaries had low nitrogen concentrations and low nitrogen isotopic signatures. In receiving waters of the pristine systems, there were similarly low nutrient concentrations, and the isotopic signatures in the estuary were similar to the signatures of incoming groundwater. In both disturbed sites freshwater on the watershed had high n...

Salt marsh ecosystems have been considered not susceptible to nitrogen overloading because early studies suggested that salt marshes adsorbed excess nutrients in plant growth. However, the possible effect of nutrient loading on species composition, and the combined effects of nutrients and altered species composition on structure and function, was largely ignored. Failure to understand interactions between nutrient loading and species composition may lead to severe underestimates of the impacts of stresses. We altered whole salt marsh ecosystems (;60 000 m 2 /treatment) by addition of nutrients in flooding waters and by reduction of a key predatory fish, the mummichog. We added nutrients (N and P; 15-fold increase over ambient conditions) directly to the flooding tide to mimic the way anthropogenic nutrients are delivered to marsh ecosystems. Despite the high concentrations (70 mmol N/L) achieved in the water column, our annual N loadings (15-60 g Nm � 2 � yr � 1 ) were an order of magnitude less than most plot-level fertilization experiments, yet we detected responses at several trophic levels. Preliminary calculations suggest that 30-40% of the added N was removed by the marsh during each tidal cycle. Creek bank Spartina alterniflora and high marsh S. patens production increased, but not stunted high marsh S. alterniflora. Microbial production increased in the fertilized creek bank S. alterniflora habitat where benthic microalgae also increased. We found top-down control of benthic microalgae by killifish, but only under nutrient addition and in the opposite direction (increase) than that predicted by a fish-invertebrate-microalgae trophic cascade. Surprisingly, infauna declined in abundance during the first season of fertilization and with fish removal. Our results demonstrate ecological effects of both nutrient addition and mummichog reduction at the whole-system level, including evidence for synergistic interactions.

Eutrophication of estuaries is an increasing global concern that requires development of new tools to identify causes, quantify conditions, and propose management options that address this environmental problem. Since eutrophication is often associated with increased inputs of land-derived nitrogen to estuaries, we developed NLOAD, a user-friendly, web-based tool that brings together six different published models that predict nitrogen loading to estuaries and two models that estimate nitrogen concentrations in coastal waters. Here we describe each of the models, demonstrate how NLOAD is designed to function, and then use the models in NLOAD to predict nitrogen loads to Barnegat Bay, New Jersey (USA). The four models that we used to estimate nitrogen loads to Barnegat Bay, when adjusted, all had similar results that matched well with measured values and indicated that Barnegat Bay receives roughly 26 kg N-ha^-yr"1. Atmospheric deposition was the dominant source of nitrogen to Barnegat Bay, followed by fertilizer nitrogen. Wastewater in Barnegat Bay is diverted to an offshore outfall and contributes no nitrogen to the system. The NLOAD tool has an additional feature that allows managers to assess the effectiveness of a variety of management options to reduce nitrogen loads. We demonstrate this feature of NLOAD through simulations in which fertilizer inputs to the Barnegat Bay watershed are reduced. Even modest cutbacks in the use of fertilizers on agricultural fields and lawns can be shown to reduce the amount of N entering Barnegat Bay.

Hydrologists have long been concerned with the interface of groundwater flow into estuaries, but not until the end of the last century did other disciplines realize the major role played by groundwater transport of nutrients to estuaries. Mass balance and stable isotopic data suggest that land-derived NO3, NH4, and dissolved organic N do enter estuaries in amounts likely to affect the function of the receiving ecosystem. Because of increasing human occupancy of the coastal zone, the nutrient loads borne by groundwater have increased in recent decades, in spite of substantial interception of nutrients within the land and aquifer components of watersheds. Groundwater-borne nutrient loads have increased the N content of receiving estuaries, increased phytoplankton and macroalgal production and biomass, decreased the area of seagrasses, and created a cascade of associated ecological changes. This linkage between land use and eutrophication of estuaries occurs in spite of mechanisms, including uptake of land-derived N by riparian vegetation and fringing wetlands, “unloading” by rapid water removal, and direct N inputs to estuaries, that tend to uncouple the effects of land use on receiving estuaries. It can be expected that as human activity on coastal watersheds continues to increase, the role of groundwater-borne nutrients to the receiving estuary will also increase.

Summary: Pyrosequencing of 16S rDNA is widely used to study microbial communities, and a rich set of software tools support this analysis. Pyrosequencing of protein-coding genes, which can help elucidate functional differences among microbial communities, significantly lags behind 16S rDNA in availability of sequence analysis software. In both settings, frequent homopolymer read errors inflate the estimation of microbial diversity, and de-noising is required to reduce that bias. Here we describe FunFrame, an R-based data-analysis pipeline that uses recently described algorithms to de-noise functional gene pyrosequences and performs ecological analysis on de-noised sequence data. The novelty of this pipeline is that it provides users a unified set of tools, adapted from disparate sources and designed for different applications, that can be used to examine a particular protein coding gene of interest. We evaluated FunFrame on functional genes from four PCR-amplified clones with sequence depths ranging from 9084 to 14494 sequences. FunFrame produced from one to nine Operational Taxanomic Units for each clone, resulting in an error rate ranging from 0 to 0.18%. Importantly, FunFrame reduced spurious diversity while retaining more sequences than a commonly used de-noising method that discards sequences with frameshift errors. Availability: Software, documentation and a complete set of sample data files are available at http://faculty.www.umb.edu/jennifer.bowen/software/FunFrame.zip. Contact: Jennifer.Bowen@umb.edu Supplementary information: Supplementary data are available at Bioinformatics online.

ELM is an Estuarine Loading Model that calculates mean annual concentration of dissolved inorganic nitrogen (DIN) available to producers in shallow estuaries by considering how different processes modify pools of nitrogen provided by inputs (streams, groundwater flow, atmospheric deposition, N2 fixation, and regeneration), and losses (burial and denitrification), within components of the estuarine system (bare sediments, seagrass meadows, salt marshes, water column). ELM also considers the effect of flushing rate within an estuary. Its formulation was constrained to minimize demands of data needed to run the model. In spite of simplifications such as the use of loss coefficients instead of functional formulations of processes, and uncertainties in all the terms included in ELM, predictions of mean annual DIN in water were not significantly different than field measurements done in estuaries in Cape Cod, Massachusetts, subject to different rates of nitrogen (N) loading. This verification suggests that, in spite of its simple formulation, ELM captures the functioning of nutrient dynamics within estuaries. ELM may therefore be a reasonable tool for use in basic studies in nutrient dynamics and land/estuary coupling. Because of its simplicity and comprehensiveness in inclusion of components and processes, ELM may also be useful in efforts to manage N loads to estuaries and related management issues.

Nitrogen (N) loading to estuaries has become a major concern for coastal planners. As urban development on coastal watershed continues, estuaries and bays are becoming more eutrophic, and cascading effects are being felt at every trophic level. Managers and stakeholders need to have a suite of effective management tools that can be applied to coastal watersheds to minimize the effects of eutrophication. We applied an N loading model and an estuarine loading model to examine the effectiveness of a suite of potential management options that could be implemented in Waquoit Bay, Cape Cod, Massachusetts. This estuarine system is a case study in which we can explore the relative potential effectiveness of decreasing inputs from wastewater and fertilizer-derived N, diverting nitrogenous runoff from impervious surfaces, altering zoning ordinances, preserving forested tracts of land as well as freshwater and saltwater wetlands, harvesting macroalgae, dredging estuary channels, and exterminating waterfowl. From a combination of simulation results, assessment of the magnitude of loads from different sources, and through different land covers, and the additional consideration of feasibility we identified management options with high, intermediate, and low potential effectiveness. Improvement of septic system performance, use of zoning regulations, preservation of forested tracts and freshwater bodies, and conservation of salt marshes emerged as the most promising avenues to manage N loads in our system. Installation of wastewater treatment plants, controlling fertilizer use, and harvesting macroalgae would potentially have intermediate success. Diversion of runoff from impervious surfaces, dredging, and extermination of waterfowl show little promise at reducing N loads. These conclusions potentially set priorities for decision-makers charged with the management of Waquoit Bay. The same procedures applied to another watershed-estuary system with different land covers and different estuarine features may differ. Evaluation studies like this need to be done for any particular site, since the watershed-estuary coupling and the loads delivered to the receiving estuary could differ. The Waquoit Bay case study provides an example of a protocol that leads to identification of the most promising management options.

The performance of several models used to estimate land-derived N loads to shallow receiving estuaries are compared. Models included in the comparison differed in complexity and approach, and predicted either loads or concentrations in estuary water. In all cases, model predictions were compared to measured loads or concentrations, as appropriate. Measured N loads to 9 estuaries on Cape Cod, MA, were obtained as the product of mean concentrations in groundwater about to seep into estuaries multiplied by the annual recharge of groundwater. Measured annual mean N concentrations in estuaries were obtained by extensive sampling surveys. The validity of this procedure to measure loads was verified by comparison against seepage meter data. Responsiveness of model predictions was generally good: predictions increased significantly as measured values increased in 8 of the 10 models evaluated. Precision of predictions was significant for all models. Three models provided highly accurate predictions; correction terms were calculated that could be applied to predictions from the other models to improve accuracy. Four of the models provided reasonable predictive ability. Simulations were run with somewhat different versions of two of the models; in both cases, the modified versions yielded improved predictions. The more complex models tended to be more responsive and precise, but not necessarily more accurate or predictive. Simpler models are attractive because they demand less information for use, but models with more comprehensive formulations, and emphasis on processes tended to perform better. Different models predicted widely different partitioning of land-derived N loads from wastewater, fertilizers, and atmospheric deposition. This is of concern, because mitigation options would be based on such partitioning of predictions. Choice of model to be used in management decisions or for research purposes therefore is not a trivial decision.

Across most of the World's coastal zone there has been a geographic transition from naturally vegetated to human-altered land covers, both agricultural and urban. This transition has increased the nitrogen loads to coastal watersheds, and from watersheds to receiving estuaries. We modeled the nitrogen entering the watershed of Waquoit Bay, Massachusetts, and found that as the transition took place, nitrogen loads to watersheds increased from 1938 to 1990. The relative magnitude of the contribution by wastewater, fertilizers, and atmospheric deposition depends on the land cover mosaics of a watershed. Atmospheric deposition was the major input to the watershed surface during this period, but because of different rates of loss within the watershed, wastewater became the major source of nitrogen flowing from the watershed to the receiving estuaries. Atmospheric deposition prevails in watersheds dominated by natural vegetation such as forests, but wastewater may become a dominant source in watersheds where urbanization increases. Increased nitrogen loads resulting from conversion of natural to human-altered watershed surfaces create eutrophication of receiving waters, with attendant changes in water quality, and marked shifts in the flora and food webs of the affected estuaries. Management efforts for restoration of eutrophied estuaries require maintenance of forested land, and control of wastewater and fertilizer inputs, the major terms in most affected places subject to local management. Wastewater and fertilizer nitrogen derive from within the watershed, which means local measures may effectively be used to control eutrophication of receiving waters.

Denitrification is a critically important biogeochemical pathway that removes fixed nitrogen from ecosystems and thus ultimately controls the rate of primary production in nitrogen-limited systems. We examined the community structure of bacteria containing the nirS gene, a signature gene in the denitrification pathway, from estuarine and salt marsh sediments and from the water column of two of the world’s largest marine oxygen-deficient zones (ODZs). We generated over 125,000 nirS gene sequences, revealing a large degree of genetic diversity including 1,815 unique taxa, the vast majority of which formed clades that contain no cultured representatives. These results underscore how little we know about the genetic diversity of metabolisms underlying this critical biogeochemical pathway. Marine sediments yielded 1,776 unique taxa when clustered at 95 % sequence identity, and there was no single nirS denitrifier that was a competitive dominant; different samples had different highly abundant taxa. By contrast, there were only 39 unique taxa identified in samples from the two ODZs, and 99 % of the sequences belonged to 5 or fewer taxa. The ODZ samples were often dominated by nirS sequences that shared a 92 % sequence identity to a nirS found in the anaerobic ammonium-oxidizing (anammox) genus Scalindua. This sequence was abundant in both ODZs, accounting for 38 and 59 % of all sequences, but it was virtually absent in marine sediments. Our data indicate that ODZs are remarkably depauperate in nirS genes compared to the remarkable genetic richness found in coastal sediments.

Some decades ago Margalef speculated that study of the exchanges across boundaries that separate different types of ecological systems would provide significant insights about properties and processes within the units that make up ecological mosaics. Although such boundaries might be difficult to define, it seemed likely that such exchanges among units would influence the function and structure of the adjoined systems. In this paper we explore exchanges across such ecological boundaries in coastal ecosystems in Cape Cod, Massachusetts, and elsewhere. We find that, indeed, definition of such boundaries is ambiguous, but study of the exchanges is more useful. In the Cape Cod system, water transport down-gradient is the dominant mechanism exerting influence on down-gradient systems. The direction of ecological control across such boundaries is largely asymmetrical, and properties of up-gradient units exert significant influence on down-gradient units. General properties of donor and receptor parcels are hard to discern, but clearly, parcels making up an ecological mosaic are not independent units, but are coupled by transfers from upgradient tesserae. Studies of controls of ecological systems need to include inter-unit influences as well as internal mechanisms., No disponible

Historical changes in land use on coastal watersheds have increased rates of land-derived nitrogen loading to estuaries and altered their biogeochemistry and food webs. We used information on human populations and land uses within the watershed of Waquoit Bay, Cape Cod, Massachusetts, U.S.A., to model how nitrogen loads derived from atmospheric deposition, fertilizer use, and wastewater disposal have changed since the 1930s. Nitrogen loading into Waquoit Bay more than doubled between 1938 and 1990. The predominant source of nitrogen added to the bay changed from atmospheric deposition to wastewater disposal during the 1980s, reflecting the increasing urbanization of Cape Cod. Larger nitrogen loads increased nitrogen concentrations in the water, altering the assemblage of primary producers and resulting in eutrophication of the estuary. Biomass of phytoplankton and macroalgae increased, and areal cover of eelgrass (Zostera marina) decreased, with increasing nitrogen load. An increase in nitrogen load from 15 to 30 kg N·ha–1·year–1 virtually eliminated eelgrass meadows. Land-use changes prompted by urban sprawl can therefore be linked to marked changes in water quality and eutrophication of receiving waters.

Functional gene pyrosequencing is emerging as a useful tool to examine the diversity and abundance of microbes that facilitate key biogeochemical processes. One such process, denitrification, is of particular importance because it converts fixed nitrate (NO3-) to N2 gas, which returns to the atmosphere. In N limited salt marshes, removal of NO3- prior to entering adjacent waters helps prevent eutrophication. Understanding the dynamics of salt marsh microbial denitrification is thus imperative for the maintenance of healthy coastal ecosystems. We used pyrosequencing of the nirS gene to examine the denitrifying community response to fertilization in experimentally enriched marsh plots. A key challenge in the analysis of sequence data derived from pyrosequencing is understanding whether small differences in gene sequences are ecologically meaningful. We apply a novel approach from information theory that determined that the optimal similarity level for clustering DNA sequences into OTUs, while still capturing the ecological complexity of the system, was 88% similarity. With this clustering, phylogenetic analysis yielded 6 dominant clades of denitrifiers, the largest of which, accounting for more than half of all the sequences collected, had no close cultured representatives. Of the 638 OTUs identified, only 11 were present in all plots and no single OTU was dominant. We did, however, find a large number of specialist OTUs that were present only in a single plot. The high degree of endemic OTUs, while accounting for a large proportion of the nirS diversity in the plots, were found in lower abundance than the generalist taxa. The proportion of specialist taxa increased with increasing supply of nutrients, suggesting that addition of fertilizer may create conditions that expand the niche space for denitrifying organisms and may enhance the genetic capacity for denitrification.

A primary focus among microbial ecologists in recent years has been to understand controls on the distribution of microorganisms in various habitats. Much less attention has been paid to the way that environmental disturbance interacts with processes that regulate bacterial community composition. We determined how human disturbance affected the distribution and community structure of salt marsh sediment bacteria by using denaturing gradient gel electrophoresis of 16S rRNA in five different habitats in each of four salt marshes located in northeastern Massachusetts, USA. Two of the four marsh creeks were experimentally enriched 15 � above background by the addition of nitrogen and phosphorus fertilizers for two or more growing seasons. Our results indicate that extrinsic factors acting at broad scales do not influence the distribution of salt marsh sediment bacteria. Intrinsic factors, controlled by local-scale environmental heterogeneity, do play a role in structuring these sediment microbial communities, although nutrient enrichment did not have a consequential effect on the microbial community in most marsh habitats. Only in one habitat, a region of the marsh creek wall that is heavily colonized by filamentous algae, did we see any effect of fertilization on the microbial community structure. When similar habitats were compared among marshes, there was considerable convergence in the microbial community composition during the growing season. Environmental factors that correlated best with microbial community composition varied with habitat, suggesting that habitat-specific intrinsic forces are primarily responsible for maintaining microbial diversity in salt marsh sediments.