Microbial diversity through an oceanographic lens: refining the concept of ocean provinces through trophic-level analysis and productivity-specific length scales.

In the marine realm, microorganisms are responsible for the bulk of primary production, thereby sustaining marine life across all trophic levels. Longhurst provinces have distinct microbial fingerprints; however, little is known about how microbial diversity and primary productivity change at finer spatial scales. Here, we sampled the Atlantic Ocean from south to north (~50°S-50°N), every ~0.5° latitude. We conducted measurements of primary productivity, chlorophyll-a and relative abundance of 16S and 18S rRNA genes, alongside analyses of the physicochemical and hydrographic environment. We analysed the diversity of autotrophs, mixotrophs and heterotrophs, and noted distinct patterns among these guilds across provinces with high and low chlorophyll-a conditions. Eukaryotic autotrophs and prokaryotic heterotrophs showed a shared inter-province diversity pattern, distinct from the diversity pattern shared by mixotrophs, cyanobacteria and eukaryotic heterotrophs. Additionally, we calculated samplewise productivity-specific length scales, the potential horizontal displacement of microbial communities by surface currents to an intrinsic biological rate (here, specific primary productivity). This scale provides key context for our trophically disaggregated diversity analysis that we could relate to underlying oceanographic features. We integrate this element to provide more nuanced insights into the mosaic-like nature of microbial provincialism, linking diversity patterns to oceanographic transport through primary production.

Machine learning techniques to characterize functional traits of plankton from image data.

Plankton imaging systems supported by automated classification and analysis have improved ecologists' ability to observe aquatic ecosystems. Today, we are on the cusp of reliably tracking plankton populations with a suite of lab-based and in situ tools, collecting imaging data at unprecedentedly fine spatial and temporal scales. But these data have potential well beyond examining the abundances of different taxa; the individual images themselves contain a wealth of information on functional traits. Here, we outline traits that could be measured from image data, suggest machine learning and computer vision approaches to extract functional trait information from the images, and discuss promising avenues for novel studies. The approaches we discuss are data agnostic and are broadly applicable to imagery of other aquatic or terrestrial organisms.

Oceanographic boundaries constrain microbial diversity gradients in the South Pacific Ocean.

Marine microbes along with microeukaryotes are key regulators of oceanic biogeochemical pathways. Here we present a high-resolution (every 0.5° of latitude) dataset describing microbial pro- and eukaryotic richness in the surface and just below the thermocline along a 7,000-km transect from 66°S at the Antarctic ice edge to the equator in the South Pacific Ocean. The transect, conducted in austral winter, covered key oceanographic features including crossing of the polar front (PF), the subtropical front (STF), and the equatorial upwelling region. Our data indicate that temperature does not determine patterns of marine microbial richness, complementing the global model data from Ladau et al. [Ladau J, et al. (2013) ISME J 7:1669-1677]. Rather, NH4+, nanophytoplankton, and primary productivity were the main drivers for archaeal and bacterial richness. Eukaryote richness was highest in the least-productive ocean region, the tropical oligotrophic province. We also observed a unique diversity pattern in the South Pacific Ocean: a regional increase in archaeal and bacterial diversity between 10°S and the equator. Rapoport's rule describes the tendency for the latitudinal ranges of species to increase with latitude. Our data showed that the mean latitudinal ranges of archaea and bacteria decreased with latitude. We show that permanent oceanographic features, such as the STF and the equatorial upwelling, can have a significant influence on both alpha-diversity and beta-diversity of pro- and eukaryotes.

Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and (14)C/(15)N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.

Biogeographic gradients of picoplankton diversity indicate increasing dominance of prokaryotes in warmer Arctic fjords.

Climate change is opening the Arctic Ocean to increasing human impact and ecosystem changes. Arctic fjords, the region's most productive ecosystems, are sustained by a diverse microbial community at the base of the food web. Here we show that Arctic fjords become more prokaryotic in the picoplankton (0.2-3 µm) with increasing water temperatures. Across 21 fjords, we found that Arctic fjords had proportionally more trophically diverse (autotrophic, mixotrophic, and heterotrophic) picoeukaryotes, while subarctic and temperate fjords had relatively more diverse prokaryotic trophic groups. Modeled oceanographic connectivity between fjords suggested that transport alone would create a smooth gradient in beta diversity largely following the North Atlantic Current and East Greenland Current. Deviations from this suggested that picoeukaryotes had some strong regional patterns in beta diversity that reduced the effect of oceanographic connectivity, while prokaryotes were mainly stopped in their dispersal if strong temperature differences between sites were present. Fjords located in high Arctic regions also generally had very low prokaryotic alpha diversity. Ultimately, warming of Arctic fjords could induce a fundamental shift from more trophic diverse eukaryotic- to prokaryotic-dominated communities, with profound implications for Arctic ecosystem dynamics including their productivity patterns.

Marine snow morphology illuminates the evolution of phytoplankton blooms and determines their subsequent vertical export.

The organic carbon produced in the ocean's surface by phytoplankton is either passed through the food web or exported to the ocean interior as marine snow. The rate and efficiency of such vertical export strongly depend on the size, structure and shape of individual particles, but apart from size, other morphological properties are still not quantitatively monitored. With the growing number of in situ imaging technologies, there is now a great possibility to analyze the morphology of individual marine snow. Thus, automated methods for their classification are urgently needed. Consequently, here we present a simple, objective categorization method of marine snow into a few ecologically meaningful functional morphotypes using field data from successive phases of the Arctic phytoplankton bloom. The proposed approach is a promising tool for future studies aiming to integrate the diversity, composition and morphology of marine snow into our understanding of the biological carbon pump.

Metabolic pathways inferred from a bacterial marker gene illuminate ecological changes across South Pacific frontal boundaries.

Global oceanographic monitoring initiatives originally measured abiotic essential ocean variables but are currently incorporating biological and metagenomic sampling programs. There is, however, a large knowledge gap on how to infer bacterial functions, the information sought by biogeochemists, ecologists, and modelers, from the bacterial taxonomic information (produced by bacterial marker gene surveys). Here, we provide a correlative understanding of how a bacterial marker gene (16S rRNA) can be used to infer latitudinal trends for metabolic pathways in global monitoring campaigns. From a transect spanning 7000 km in the South Pacific Ocean we infer ten metabolic pathways from 16S rRNA gene sequences and 11 corresponding metagenome samples, which relate to metabolic processes of primary productivity, temperature-regulated thermodynamic effects, coping strategies for nutrient limitation, energy metabolism, and organic matter degradation. This study demonstrates that low-cost, high-throughput bacterial marker gene data, can be used to infer shifts in the metabolic strategies at the community scale.

Sea ice presence is linked to higher carbon export and vertical microbial connectivity in the Eurasian Arctic Ocean.

Arctic Ocean sea ice cover is shrinking due to warming. Long-term sediment trap data shows higher export efficiency of particulate organic carbon in regions with seasonal sea ice compared to regions without sea ice. To investigate this sea-ice enhanced export, we compared how different early summer phytoplankton communities in seasonally ice-free and ice-covered regions of the Fram Strait affect carbon export and vertical dispersal of microbes. In situ collected aggregates revealed two-fold higher carbon export of diatom-rich aggregates in ice-covered regions, compared to Phaeocystis aggregates in the ice-free region. Using microbial source tracking, we found that ice-covered regions were also associated with more surface-born microbial clades exported to the deep sea. Taken together, our results showed that ice-covered regions are responsible for high export efficiency and provide strong vertical microbial connectivity. Therefore, continuous sea-ice loss may decrease the vertical export efficiency, and thus the pelagic-benthic coupling, with potential repercussions for Arctic deep-sea ecosystems.

Sea-ice derived meltwater stratification slows the biological carbon pump: results from continuous observations.

The ocean moderates the world's climate through absorption of heat and carbon, but how much carbon the ocean will continue to absorb remains unknown. The North Atlantic Ocean west (Baffin Bay/Labrador Sea) and east (Fram Strait/Greenland Sea) of Greenland features the most intense absorption of anthropogenic carbon globally; the biological carbon pump (BCP) contributes substantially. As Arctic sea-ice melts, the BCP changes, impacting global climate and other critical ocean attributes (e.g. biodiversity). Full understanding requires year-round observations across a range of ice conditions. Here we present such observations: autonomously collected Eulerian continuous 24-month time-series in Fram Strait. We show that, compared to ice-unaffected conditions, sea-ice derived meltwater stratification slows the BCP by 4 months, a shift from an export to a retention system, with measurable impacts on benthic communities. This has implications for ecosystem dynamics in the future warmer Arctic where the seasonal ice zone is expected to expand.

A database of chlorophyll a in Australian waters.

Chlorophyll a is the most commonly used indicator of phytoplankton biomass in the marine environment. It is relatively simple and cost effective to measure when compared to phytoplankton abundance and is thus routinely included in many surveys. Here we collate 173, 333 records of chlorophyll a collected since 1965 from Australian waters gathered from researchers on regular coastal monitoring surveys and ocean voyages into a single repository. This dataset includes the chlorophyll a values as measured from samples analysed using spectrophotometry, fluorometry and high performance liquid chromatography (HPLC). The Australian Chlorophyll a database is freely available through the Australian Ocean Data Network portal (https://portal.aodn.org.au/). These data can be used in isolation as an index of phytoplankton biomass or in combination with other data to provide insight into water quality, ecosystem state, and relationships with other trophic levels such as zooplankton or fish.

Reduction of the Powerful Greenhouse Gas N2O in the South-Eastern Indian Ocean.

Nitrous oxide (N2O) is a powerful greenhouse gas and a key catalyst of stratospheric ozone depletion. Yet, little data exist about the sink and source terms of the production and reduction of N2O outside the well-known oxygen minimum zones (OMZ). Here we show the presence of functional marker genes for the reduction of N2O in the last step of the denitrification process (nitrous oxide reductase genes; nosZ) in oxygenated surface waters (180-250 O2 µmol.kg(-1)) in the south-eastern Indian Ocean. Overall copy numbers indicated that nosZ genes represented a significant proportion of the microbial community, which is unexpected in these oxygenated waters. Our data show strong temperature sensitivity for nosZ genes and reaction rates along a vast latitudinal gradient (32°S-12°S). These data suggest a large N2O sink in the warmer Tropical waters of the south-eastern Indian Ocean. Clone sequencing from PCR products revealed that most denitrification genes belonged to Rhodobacteraceae. Our work highlights the need to investigate the feedback and tight linkages between nitrification and denitrification (both sources of N2O, but the latter also a source of bioavailable N losses) in the understudied yet strategic Indian Ocean and other oligotrophic systems.

A database of marine phytoplankton abundance, biomass and species composition in Australian waters.

There have been many individual phytoplankton datasets collected across Australia since the mid 1900s, but most are unavailable to the research community. We have searched archives, contacted researchers, and scanned the primary and grey literature to collate 3,621,847 records of marine phytoplankton species from Australian waters from 1844 to the present. Many of these are small datasets collected for local questions, but combined they provide over 170 years of data on phytoplankton communities in Australian waters. Units and taxonomy have been standardised, obviously erroneous data removed, and all metadata included. We have lodged this dataset with the Australian Ocean Data Network (http://portal.aodn.org.au/) allowing public access. The Australian Phytoplankton Database will be invaluable for global change studies, as it allows analysis of ecological indicators of climate change and eutrophication (e.g., changes in distribution; diatom:dinoflagellate ratios). In addition, the standardised conversion of abundance records to biomass provides modellers with quantifiable data to initialise and validate ecosystem models of lower marine trophic levels.

Growth of a deep-water, predatory fish is influenced by the productivity of a boundary current system.

The effects of climate change on predatory fishes in deep shelf areas are difficult to predict because complex processes may govern food availability and temperature at depth. We characterised the net impact of recent environmental changes on hapuku (Polyprion oxygeneios), an apex predator found in continental slope habitats (>200 m depth) by using dendrochronology techniques to develop a multi-decadal record of growth from otoliths. Fish were sampled off temperate south-western Australia, a region strongly influenced by the Leeuwin Current, a poleward-flowing, eastern boundary current. The common variance among individual growth records was relatively low (3.4%), but the otolith chronology was positively correlated (r = 0.61, p < 0.02) with sea level at Fremantle, a proxy for the strength of the Leeuwin Current. The Leeuwin Current influences the primary productivity of shelf ecosystems, with a strong current favouring growth in hapuku. Leeuwin Current strength is predicted to decline under climate change models and this study provides evidence that associated productivity changes may flow through to higher trophic levels even in deep water habitats.

Temporal variations in metabolic and autotrophic indices for Acropora digitifera and Acropora spicifera--implications for monitoring projects.

Coral health indices are important components of the management assessments of coral reefs, providing insight into local variation in reef condition, as well as tools for comparisons between reefs and across various time scales. Understanding how such health indices vary in space and time is critical to their successful implementation as management tools. Here we compare autotrophic and heterotrophic coral health indices, examining specifically the temporal variation driven by the local environmental variation, at three scales (diel, daily and seasonal). We compared metabolic indices (RNA/DNA ratio, protein concentration) and autotrophic indices (Chlorophyll a (Chl a), zooxanthellae density, effective quantum yield (yield) and relative electron transport rate (rETR)) for two dominant Acropora species, A. digitifera and A. spicifera at Ningaloo Reef (north-western Australia) in August 2010 (austral winter) and February 2011 (austral summer). Clear seasonal patterns were documented for metabolic indices, zooxanthellae density and rETR, while cyclic diel patterns only occurred for yield and rETR, and RNA/DNA ratio. Significant daily variation was observed for RNA/DNA ratio, Chl a concentration, yield and rETR. Results suggest that zooxanthellae density and protein concentrations are good long-term indicators of coral health whose variance is largely seasonal, while RNA/DNA ratio and rETR can be used for both long-term (seasonal) and short-term (diel) coral monitoring. Chl a can be used to describe changes between days and yield for both diel and daily variations. Correlations between health indices and light history showed that short-term changes in irradiance had the strongest impact on all health indices except zooxanthellae density for A. digitifera; for A. spicifera no correlation was observed at all. However, cumulative irradiance over the several days before sampling showed significant correlations with most health indices suggesting that a time-lag effect has to be taken into account when interpreting diel variations in coral condition.

Which environmental factors predict seasonal variation in the coral health of Acropora digitifera and Acropora spicifera at Ningaloo Reef?

The impact of physico-chemical factors on percent coral cover and coral health was examined on a spatial basis for two dominant Acropora species, A. digitifera and A. spicifera, at Ningaloo Reef (north-western Australia) in the southeast Indian Ocean. Coral health was investigated by measuring metabolic indices (RNA/DNA ratio and protein concentration), energy levels (lipid ratio) and autotrophic indices (chlorophyll a (chl a) and zooxanthellae density) at six stations during typical seasons (austral autumn 2010 (March and April), austral winter 2010 (August)) and during an extreme La Niña event in summer 2011 (February). These indices were correlated with 15 physico-chemical factors (measured immediately following coral sampling) to identify predictors for health indices. Variations in metabolic indices (protein concentration and RNA/DNA ratio) for A. spicifera were mainly explained by nitrogen, temperature and zooplankton concentrations under typical conditions, while for A. digitifera, light as well as phytoplankton, in particular picoeukaryotes, were important, possibly due to higher energy requirement for lipid synthesis and storage in A. digitifera. Optimum metabolic values occurred for both Acropora species at 26-28°C when autotrophic indices (chl a and zooxanthellae density) were lowest. The extreme temperature during the La Niña event resulted in a shift of feeding modes, with an increased importance of water column plankton concentrations for metabolic rates of A. digitifera and light and plankton for A. spicifera. Our results suggest that impacts of high sea surface temperatures during extreme events such as La Niña may be mitigated via reduction on metabolic rates in coral host. The high water column plankton concentrations and associated low light levels resulted in a shift towards high symbiont densities, with lower metabolic rates and energy levels than the seasonal norm for the coral host.

Fussy feeders: phyllosoma larvae of the Western Rocklobster (Panulirus cygnus) demonstrate prey preference.

The Western Rocklobster (Panulirus cygnus) is the most valuable single species fishery in Australia and the largest single country spiny lobster fishery in the world. In recent years a well-known relationship between oceanographic conditions and lobster recruitment has become uncoupled, with significantly lower recruitment than expected, generating interest in the factors influencing survival and development of the planktonic larval stages. The nutritional requirements and wild prey of the planktotrophic larval stage (phyllosoma) of P. cygnus were previously unknown, hampering both management and aquaculture efforts for this species. Ship-board feeding trials of wild-caught mid-late stage P. cygnus phyllosoma in the eastern Indian Ocean, off the coast of Western Australia, were conducted in July 2010 and August-September 2011. In a series of experiments, phyllosoma were fed single and mixed species diets of relatively abundant potential prey items (chaetognaths, salps, and krill). Chaetognaths were consumed in 2-8 times higher numbers than the other prey, and the rate of consumption of chaetognaths increased with increasing concentration of prey. The highly variable lipid content of the phyllosoma, and the fatty acid profiles of the phyllosoma and chaetognaths, indicated they were from an oligotrophic oceanic food chain where food resources for macrozooplankton were likely to be constrained. Phyllosoma fed chaetognaths over 6 days showed significant changes in some fatty acids and tended to accumulate lipid, indicating an improvement in overall nutritional condition. The discovery of a preferred prey for P. cygnus will provide a basis for future oceanographic, management and aquaculture research for this economically and ecologically valuable species.

Determining the diet of larvae of western rock lobster (Panulirus cygnus) using high-throughput DNA sequencing techniques.

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

Variability in isotope discrimination factors in coral reef fishes: implications for diet and food web reconstruction.

Interpretation of stable isotope ratios of carbon and nitrogen (δ(13)C and δ(15)N) is generally based on the assumption that with each trophic level there is a constant enrichment in the heavier isotope, leading to diet-tissue discrimination factors of 3.4‰ for (15)N (ΔN) and ∼0.5‰ for (13)C (ΔC). Diet-tissue discrimination factors determined from paired tissue and gut samples taken from 152 individuals from 26 fish species at Ningaloo Reef, Western Australia demonstrate a large amount of variability around constant values. While caution is necessary in using gut contents to represent diet due to the potential for high temporal variability, there were significant effects of trophic position and season that may also lead to variability in ΔN under natural conditions. Nitrogen enrichment increased significantly at higher trophic levels (higher tissue δ(15)N), with significantly higher ΔN in carnivorous species. Changes in diet led to significant changes in ΔN, but not tissue δ(15)N, between seasons for several species: Acanthurus triostegus, Chromis viridis, Parupeneus signatus and Pomacentrus moluccensis. These results confirm that the use of meta-analysis averages for ΔN is likely to be inappropriate for accurately determining diets and trophic relationships using tissue stable isotope ratios. Where feasible, discrimination factors should be directly quantified for each species and trophic link in question, acknowledging the potential for significant variation away from meta-analysis averages and, perhaps, controlled laboratory diets and conditions.