Climate research priorities for policy-makers, practitioners, and scientists in Georgia, USA.

Climate change has far-reaching effects on human and ecological systems, requiring collaboration across sectors and disciplines to determine effective responses. To inform regional responses to climate change, decision-makers need credible and relevant information representing a wide swath of knowledge and perspectives. The southeastern U. S. State of Georgia is a valuable focal area for study because it contains multiple ecological zones that vary greatly in land use and economic activities, and it is vulnerable to diverse climate change impacts. We identified 40 important research questions that, if answered, could lay the groundwork for effective, science-based climate action in Georgia. Top research priorities were identified through a broad solicitation of candidate research questions (180 were received). A group of experts across sectors and disciplines gathered for a workshop to categorize, prioritize, and filter the candidate questions, identify missing topics, and rewrite questions. Participants then collectively chose the 40 most important questions. This cross-sectoral effort ensured the inclusion of a diversity of topics and questions (e.g., coastal hazards, agricultural production, ecosystem functioning, urban infrastructure, and human health) likely to be important to Georgia policy-makers, practitioners, and scientists. Several cross-cutting themes emerged, including the need for long-term data collection and consideration of at-risk Georgia citizens and communities. Workshop participants defined effective responses as those that take economic cost, environmental impacts, and social justice into consideration. Our research highlights the importance of collaborators across disciplines and sectors, and discussing challenges and opportunities that will require transdisciplinary solutions.

Microspatial gene expression patterns in the Amazon River Plume.

We investigated expression of genes mediating elemental cycling at the microspatial scale in the ocean's largest river plume using, to our knowledge, the first fully quantitative inventory of genes and transcripts. The bacterial and archaeal communities associated with a phytoplankton bloom in Amazon River Plume waters at the outer continental shelf in June 2010 harbored ∼ 1.0 × 10(13) genes and 4.7 × 10(11) transcripts per liter that mapped to several thousand microbial genomes. Genomes from free-living cells were more abundant than those from particle-associated cells, and they generated more transcripts per liter for carbon fixation, heterotrophy, nitrogen and phosphorus uptake, and iron acquisition, although they had lower expression ratios (transcripts ⋅ gene(-1)) overall. Genomes from particle-associated cells contributed more transcripts for sulfur cycling, aromatic compound degradation, and the synthesis of biologically essential vitamins, with an overall twofold up-regulation of expression compared with free-living cells. Quantitatively, gene regulation differences were more important than genome abundance differences in explaining why microenvironment transcriptomes differed. Taxa contributing genomes to both free-living and particle-associated communities had up to 65% of their expressed genes regulated differently between the two, quantifying the extent of transcriptional plasticity in marine microbes in situ. In response to patchiness in carbon, nutrients, and light at the micrometer scale, Amazon Plume microbes regulated the expression of genes relevant to biogeochemical processes at the ecosystem scale.

Role for urea in nitrification by polar marine Archaea.

Despite the high abundance of Archaea in the global ocean, their metabolism and biogeochemical roles remain largely unresolved. We investigated the population dynamics and metabolic activity of Thaumarchaeota in polar environments, where these microorganisms are particularly abundant and exhibit seasonal growth. Thaumarchaeota were more abundant in deep Arctic and Antarctic waters and grew throughout the winter at surface and deeper Arctic halocline waters. However, in situ single-cell activity measurements revealed a low activity of this group in the uptake of both leucine and bicarbonate (<5% Thaumarchaeota cells active), which is inconsistent with known heterotrophic and autotrophic thaumarchaeal lifestyles. These results suggested the existence of alternative sources of carbon and energy. Our analysis of an environmental metagenome from the Arctic winter revealed that Thaumarchaeota had pathways for ammonia oxidation and, unexpectedly, an abundance of genes involved in urea transport and degradation. Quantitative PCR analysis confirmed that most polar Thaumarchaeota had the potential to oxidize ammonia, and a large fraction of them had urease genes, enabling the use of urea to fuel nitrification. Thaumarchaeota from Arctic deep waters had a higher abundance of urease genes than those near the surface suggesting genetic differences between closely related archaeal populations. In situ measurements of urea uptake and concentration in Arctic waters showed that small-sized prokaryotes incorporated the carbon from urea, and the availability of urea was often higher than that of ammonium. Therefore, the degradation of urea may be a relevant pathway for Thaumarchaeota and other microorganisms exposed to the low-energy conditions of dark polar waters.

Pole-to-pole biogeography of surface and deep marine bacterial communities.

The Antarctic and Arctic regions offer a unique opportunity to test factors shaping biogeography of marine microbial communities because these regions are geographically far apart, yet share similar selection pressures. Here, we report a comprehensive comparison of bacterioplankton diversity between polar oceans, using standardized methods for pyrosequencing the V6 region of the small subunit ribosomal (SSU) rRNA gene. Bacterial communities from lower latitude oceans were included, providing a global perspective. A clear difference between Southern and Arctic Ocean surface communities was evident, with 78% of operational taxonomic units (OTUs) unique to the Southern Ocean and 70% unique to the Arctic Ocean. Although polar ocean bacterial communities were more similar to each other than to lower latitude pelagic communities, analyses of depths, seasons, and coastal vs. open waters, the Southern and Arctic Ocean bacterioplankton communities consistently clustered separately from each other. Coastal surface Southern and Arctic Ocean communities were more dissimilar from their respective open ocean communities. In contrast, deep ocean communities differed less between poles and lower latitude deep waters and displayed different diversity patterns compared with the surface. In addition, estimated diversity (Chao1) for surface and deep communities did not correlate significantly with latitude or temperature. Our results suggest differences in environmental conditions at the poles and different selection mechanisms controlling surface and deep ocean community structure and diversity. Surface bacterioplankton may be subjected to more short-term, variable conditions, whereas deep communities appear to be structured by longer water-mass residence time and connectivity through ocean circulation.

Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA).

Diatom diazotroph associations (DDAs) are important components in the world's oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont's nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (>106 cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30-35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (>0.1 µmol L-1) and Si (>1.0 µmol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 × 105nifH copies L-1) than het-2 and limited to regions of oceanic SSS (>36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (>103nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8-34.3), immeasurable DIN, moderate DIP (0.1-0.60 µmol L-1) and higher Si (4.19-22.1 µmol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations.

Microbial Community Response to Terrestrially Derived Dissolved Organic Matter in the Coastal Arctic.

Warming at nearly twice the global rate, higher than average air temperatures are the new 'normal' for Arctic ecosystems. This rise in temperature has triggered hydrological and geochemical changes that increasingly release carbon-rich water into the coastal ocean via increased riverine discharge, coastal erosion, and the thawing of the semi-permanent permafrost ubiquitous in the region. To determine the biogeochemical impacts of terrestrially derived dissolved organic matter (tDOM) on marine ecosystems we compared the nutrient stocks and bacterial communities present under ice-covered and ice-free conditions, assessed the lability of Arctic tDOM to coastal microbial communities from the Chukchi Sea, and identified bacterial taxa that respond to rapid increases in tDOM. Once thought to be predominantly refractory, we found that ∼7% of dissolved organic carbon and ∼38% of dissolved organic nitrogen from tDOM was bioavailable to receiving marine microbial communities on short 4 - 6 day time scales. The addition of tDOM shifted bacterial community structure toward more copiotrophic taxa and away from more oligotrophic taxa. Although no single order was found to respond universally (positively or negatively) to the tDOM addition, this study identified 20 indicator species as possible sentinels for increased tDOM. These data suggest the true ecological impact of tDOM will be widespread across many bacterial taxa and that shifts in coastal microbial community composition should be anticipated.

Expression patterns of elemental cycling genes in the Amazon River Plume.

Metatranscriptomics and metagenomics data sets benchmarked with internal standards were used to characterize the expression patterns for biogeochemically relevant bacterial and archaeal genes mediating carbon, nitrogen, phosphorus and sulfur uptake and metabolism through the salinity gradient of the Amazon River Plume. The genes were identified in 48 metatranscriptomic and metagenomic data sets summing to >500 million quality-controlled reads from six locations in the plume ecosystem. The ratio of transcripts per gene copy (a direct measure of expression made possible by internal standard additions) showed that the free-living bacteria and archaea exhibited only small changes in the expression levels of biogeochemically relevant genes through the salinity and nutrient zones of the plume. In contrast, the expression levels of genes in particle-associated cells varied over orders of magnitude among the stations, with the largest differences measured for genes mediating aspects of nitrogen cycling (nifH, amtB and amoA) and phosphorus acquisition (pstC, phoX and phoU). Taxa varied in their baseline gene expression levels and extent of regulation, and most of the spatial variation in the expression level could be attributed to changes in gene regulation after removing the effect of shifting taxonomic composition. We hypothesize that changes in microbial element cycling along the Amazon River Plume are largely driven by shifting activities of particle-associated cells, with most activities peaking in the mesohaline regions where N2 fixation rates are elevated.

Virioplankton Assemblage Structure in the Lower River and Ocean Continuum of the Amazon.

The Amazon River watershed and its associated plume comprise a vast continental and oceanic area. The microbial activities along this continuum contribute substantially to global carbon and nutrient cycling, and yet there is a dearth of information on the diversity, abundance, and possible roles of viruses in this globally important river. The aim of this study was to elucidate the diversity and structure of virus assemblages of the Amazon River-ocean continuum. Environmental viral DNA sequences were obtained for 12 locations along the river's lower reach (n = 5) and plume (n = 7). Sequence assembly yielded 29,358 scaffolds, encoding 82,546 viral proteins, with 15 new complete viral genomes. Despite the spatial connectivity mediated by the river, virome analyses and physical-chemical water parameters clearly distinguished river and plume ecosystems. Bacteriophages were ubiquitous in the continuum and were more abundant in the transition region. Eukaryotic viruses occurred mostly in the river, while the plume had more viruses of autotrophic organisms (Prochlorococcus, Synechococcus) and heterotrophic bacteria (Pelagibacter). The viral families Microviridae and Myoviridae were the most abundant and occurred throughout the continuum. The major functions of the genes in the continuum involved viral structures and life cycles, and viruses from plume locations and Tapajós River showed the highest levels of functional diversity. The distribution patterns of the viral assemblages were defined not only by the occurrence of possible hosts but also by water physical and chemical parameters, especially salinity. The findings presented here help to improve understanding of the possible roles of viruses in the organic matter cycle along the river-ocean continuum. IMPORTANCE The Amazon River forms a vast plume in the Atlantic Ocean that can extend for more than 1,000 km. Microbial communities promote a globally relevant carbon sink system in the plume. Despite the importance of viruses for the global carbon cycle, the diversity and the possible roles of viruses in the Amazon are poorly understood. The present work assesses, for the first time, the abundance and diversity of viruses simultaneously in the river and ocean in order to elucidate their possible roles. DNA sequence assembly yielded 29,358 scaffolds, encoding 82,546 viral proteins, with 15 new complete viral genomes from the 12 river and ocean locations. Viral diversity was clearly distinguished by river and ocean. Bacteriophages were the most abundant and occurred throughout the continuum. Viruses that infect eukaryotes were more abundant in the river, whereas phages appeared to have strong control over the host prokaryotic populations in the plume.

Bacterial Biogeography across the Amazon River-Ocean Continuum.

Spatial and temporal patterns in microbial biodiversity across the Amazon river-ocean continuum were investigated along ∼675 km of the lower Amazon River mainstem, in the Tapajós River tributary, and in the plume and coastal ocean during low and high river discharge using amplicon sequencing of 16S rRNA genes in whole water and size-fractionated samples (0.2-2.0 µm and >2.0 µm). River communities varied among tributaries, but mainstem communities were spatially homogeneous and tracked seasonal changes in river discharge and co-varying factors. Co-occurrence network analysis identified strongly interconnected river assemblages during high (May) and low (December) discharge periods, and weakly interconnected transitional assemblages in September, suggesting that this system supports two seasonal microbial communities linked to river discharge. In contrast, plume communities showed little seasonal differences and instead varied spatially tracking salinity. However, salinity explained only a small fraction of community variability, and plume communities in blooms of diatom-diazotroph assemblages were strikingly different than those in other high salinity plume samples. This suggests that while salinity physically structures plumes through buoyancy and mixing, the composition of plume-specific communities is controlled by other factors including nutrients, phytoplankton community composition, and dissolved organic matter chemistry. Co-occurrence networks identified interconnected assemblages associated with the highly productive low salinity near-shore region, diatom-diazotroph blooms, and the plume edge region, and weakly interconnected assemblages in high salinity regions. This suggests that the plume supports a transitional community influenced by immigration of ocean bacteria from the plume edge, and by species sorting as these communities adapt to local environmental conditions. Few studies have explored patterns of microbial diversity in tropical rivers and coastal oceans. Comparison of Amazon continuum microbial communities to those from temperate and arctic systems suggest that river discharge and salinity are master variables structuring a range of environmental conditions that control bacterial communities across the river-ocean continuum.

Patterns of Transcript Abundance of Eukaryotic Biogeochemically-Relevant Genes in the Amazon River Plume.

The Amazon River has the largest discharge of all rivers on Earth, and its complex plume system fuels a wide array of biogeochemical processes, across a large area of the western tropical North Atlantic. The plume thus stimulates microbial processes affecting carbon sequestration and nutrient cycles at a global scale. Chromosomal gene expression patterns of the 2.0 to 156 µm size-fraction eukaryotic microbial community were investigated in the Amazon River Plume, generating a robust dataset (more than 100 million mRNA sequences) that depicts the metabolic capabilities and interactions among the eukaryotic microbes. Combining classical oceanographic field measurements with metatranscriptomics yielded characterization of the hydrographic conditions simultaneous with a quantification of transcriptional activity and identity of the community. We highlight the patterns of eukaryotic gene expression for 31 biogeochemically significant gene targets hypothesized to be valuable within forecasting models. An advantage to this targeted approach is that the database of reference sequences used to identify the target genes was selectively constructed and highly curated optimizing taxonomic coverage, throughput, and the accuracy of annotations. A coastal diatom bloom highly expressed nitrate transporters and carbonic anhydrase presumably to support high growth rates and enhance uptake of low levels of dissolved nitrate and CO2. Diatom-diazotroph association (DDA: diatoms with nitrogen fixing symbionts) blooms were common when surface salinity was mesohaline and dissolved nitrate concentrations were below detection, and hence did not show evidence of nitrate utilization, suggesting they relied on ammonium transporters to aquire recently fixed nitrogen. These DDA blooms in the outer plume had rapid turnover of the photosystem D1 protein presumably caused by photodegradation under increased light penetration in clearer waters, and increased expression of silicon transporters as silicon became limiting. Expression of these genes, including carbonic anhydrase and transporters for nitrate and phosphate, were found to reflect the physiological status and biogeochemistry of river plume environments. These relatively stable patterns of eukaryotic transcript abundance occurred over modest spatiotemporal scales, with similarity observed in sample duplicates collected up to 2.45 km in space and 120 minutes in time. These results confirm the use of metatranscriptomics as a valuable tool to understand and predict microbial community function.

An extensive reef system at the Amazon River mouth.

Large rivers create major gaps in reef distribution along tropical shelves. The Amazon River represents 20% of the global riverine discharge to the ocean, generating up to a 1.3 × 10(6)-km(2) plume, and extensive muddy bottoms in the equatorial margin of South America. As a result, a wide area of the tropical North Atlantic is heavily affected in terms of salinity, pH, light penetration, and sedimentation. Such unfavorable conditions were thought to imprint a major gap in Western Atlantic reefs. We present an extensive carbonate system off the Amazon mouth, underneath the river plume. Significant carbonate sedimentation occurred during lowstand sea level, and still occurs in the outer shelf, resulting in complex hard-bottom topography. A permanent near-bottom wedge of ocean water, together with the seasonal nature of the plume's eastward retroflection, conditions the existence of this extensive (~9500 km(2)) hard-bottom mosaic. The Amazon reefs transition from accretive to erosional structures and encompass extensive rhodolith beds. Carbonate structures function as a connectivity corridor for wide depth-ranging reef-associated species, being heavily colonized by large sponges and other structure-forming filter feeders that dwell under low light and high levels of particulates. The oxycline between the plume and subplume is associated with chemoautotrophic and anaerobic microbial metabolisms. The system described here provides several insights about the responses of tropical reefs to suboptimal and marginal reef-building conditions, which are accelerating worldwide due to global changes.

Metagenomic and metatranscriptomic inventories of the lower Amazon River, May 2011.

BACKGROUND: The Amazon River runs nearly 6500 km across the South American continent before emptying into the western tropical North Atlantic Ocean. In terms of both volume and watershed area, it is the world's largest riverine system, affecting elemental cycling on a global scale. RESULTS: A quantitative inventory of genes and transcripts benchmarked with internal standards was obtained at five stations in the lower Amazon River during May 2011. At each station, metagenomes and metatranscriptomes were obtained in duplicate for two microbial size fractions (free-living, 0.2 to 2.0 µm; particle-associated, 2.0 to 297 µm) using 150 × 150 paired-end Illumina sequencing. Forty eight sample datasets were obtained, averaging 15 × 10(6) potential protein-encoding reads each (730 × 10(6) total). Prokaryotic metagenomes and metatranscriptomes were dominated by members of the phyla Actinobacteria, Planctomycetes, Betaproteobacteria, Verrucomicrobia, Nitrospirae, and Acidobacteria. The actinobacterium SCGC AAA027-L06 reference genome recruited the greatest number of reads overall, with this single bin contributing an average of 50 billion genes and 500 million transcripts per liter of river water. Several dominant taxa were unevenly distributed between the free-living and particle-associated size fractions, such as a particle-associated bias for reads binning to planctomycete Schlesneria paludicola and a free-living bias for actinobacterium SCGC AAA027-L06. Gene expression ratios (transcripts to gene copy ratio) increased downstream from Óbidos to Macapá and Belém, indicating higher per cell activity of Amazon River bacteria and archaea as river water approached the ocean. CONCLUSION: This inventory of riverine microbial genes and transcripts, benchmarked with internal standards for full quantitation, provides an unparalleled window into microbial taxa and functions in the globally important Amazon River ecosystem.

Phaeocystis antarctica blooms strongly influence bacterial community structures in the Amundsen Sea polynya.

Rising temperatures and changing winds drive the expansion of the highly productive polynyas (open water areas surrounded by sea ice) abutting the Antarctic continent. Phytoplankton blooms in polynyas are often dominated by the haptophyte Phaeocystis antarctica, and they generate the organic carbon that enters the resident microbial food web. Yet, little is known about how Phaeocystis blooms shape bacterial community structures and carbon fluxes in these systems. We identified the bacterial communities that accompanied a Phaeocystis bloom in the Amundsen Sea polynya during the austral summers of 2007-2008 and 2010-2011. These communities are distinct from those determined for the Antarctic Circumpolar Current (ACC) and off the Palmer Peninsula. Diversity patterns for most microbial taxa in the Amundsen Sea depended on location (e.g., waters abutting the pack ice near the shelf break and at the edge of the Dotson glacier) and depth, reflecting different niche adaptations within the confines of this isolated ecosystem. Inside the polynya, P. antarctica coexisted with the bacterial taxa Polaribacter sensu lato, a cryptic Oceanospirillum, SAR92 and Pelagibacter. These taxa were dominated by a single oligotype (genotypes partitioned by Shannon entropy analysis) and together contributed up to 73% of the bacterial community. Size fractionation of the bacterial community [<3 µm (free-living bacteria) vs. >3 µm (particle-associated bacteria)] identified several taxa (especially SAR92) that were preferentially associated with Phaeocystis colonies, indicative of a distinct role in Phaeocystis bloom ecology. In contrast, particle-associated bacteria at 250 m depth were enriched in Colwellia and members of the Cryomorphaceae suggesting that they play important roles in the decay of Phaeocystis blooms.

Role of biogenic silica in the removal of iron from the Antarctic seas.

Iron has a key role in controlling biological production in the Southern Ocean, yet the mechanisms regulating iron availability in this and other ocean regions are not completely understood. Here, based on analysis of living phytoplankton in the coastal seas of West Antarctica, we present a new pathway for iron removal from marine systems involving structural incorporation of reduced, organic iron into biogenic silica. Export of iron incorporated into biogenic silica may represent a substantial unaccounted loss of iron from marine systems. For example, in the Ross Sea, burial of iron incorporated into biogenic silica is conservatively estimated as 11 µmol m⁻² per year, which is in the same range as the major bioavailable iron inputs to this region. As a major sink of bioavailable iron, incorporation of iron into biogenic silica may shift microbial population structure towards taxa with relatively lower iron requirements, and may reduce ecosystem productivity and associated carbon sequestration.

Sizing up metatranscriptomics.

A typical marine bacterial cell in coastal seawater contains only ∼200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium's responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.