Dynamics of nitrogen genes in intertidal sediments of Darwin Harbour and their connection to N-biogeochemistry.

Microbial mediated nitrogen (N) transformation is subject to multiple controlling factors such as prevailing physical and chemical conditions, and little is known about these processes in sediments of wet-dry tropical macrotidal systems such as Darwin Harbour in North Australia. To understand key transformations, we assessed the association between the relative abundance of nitrogen cycling genes with trophic status, sediment partition and benthic nitrogen fluxes in Darwin Harbour. We analysed nitrogen cycling gene abundance using a functional gene microarray and quantitative PCRs targeting the denitrification gene (nosZ) and archaeal ammonia oxidation (AOA.1). We found a significant negative correlation between archaeal ammonia oxidation and silicate flux (P = 0.004), an indicator for diatom and benthic microalgal activity. It is suggested that the degradation of the diatomaceous organic matter generates localised anoxic conditions and inhibition of nitrification. Abundance of the nosZ gene was negatively correlated with nutrient load. The lowest nosZ gene levels were in hyper-eutrophic tidal creeks with anoxic conditions and increased levels of sulphide limiting the coupling of nitrification-denitrification (P = 0.016). Significantly higher levels of nosZ genes were measured in the surface (top 2 cm) compared to bulk sediment (top 10 cm) and there was a positive association with di-nitrogen flux (N2) in surface (P = 0.024) but not bulk sediment. This suggests that denitrifiers are most active in surficial sediment at the sediment-water interface. Elevated levels of nosZ genes also occurred in the sediments of tidal creek mouths and mudflats with these depositional zones combining the diffuse and seaward supply of nitrogen and carbon supporting denitrifiers. N-cycle molecular assays using surface sediments show promise as a rapid monitoring technique for impact assessment and measuring ecosystem function. This is particularly pertinent for tropical macrotidal systems where systematic monitoring is sparse and in many cases challenged by climatic extremes and remoteness.

Metabolomics as a tool for in situ study of chronic metal exposure in estuarine invertebrates.

Estuaries are subject to intense human use globally, with impacts from multiple stressors, such as metal contaminants. A key challenge is extending beyond traditional monitoring approaches to understand effects to biota and system function. To explore the metabolic effects of complex metal contaminants to sediment dwelling (benthic) fauna, we apply a multiple-lines-of-evidence approach, coupling environmental monitoring, benthic sampling, total metals analysis and targeted metabolomics. We characterise metabolic signatures of metal exposure in three benthic invertebrate taxa, which differed in distribution across sites and severity of metal exposure: sipunculid (very high), amphipod (high), maldanid polychaete (moderate). We observed sediment and tissue metal loads far exceeding sediment guidelines where toxicity-related adverse effects may be expected, for metals including, As, Cd, Pb, Zn and Hg. Change in site- and taxa-specific metabolite profiles was highly correlated with natural environmental drivers (sediment total organic carbon and water temperature). At the most metal influenced sites, metabolite variation was also correlated with sediment metal loads. Using supervised multivariate regression, taxa-specific metabolic signatures of increased exposure and possibility of toxic effects were characterised against multiple reference sites. Metabolic signatures varied according to each taxon and degree of metal exposure, but primarily indicated altered cysteine and methionine metabolism, metal-binding and elimination (lysosomal) activity, coupled to change in complex biosynthesis pathways, responses to oxidative stress, and cellular damage. This novel multiple-lines-of-evidence approach combining metabolomics with traditional environmental monitoring, enabled detection and characterisation of chronic metal exposure effects in situ in multiple invertebrate taxa. With capacity for application to rapid and effective monitoring of non-model species in complex environments, these approaches are critical for improved assessment and management of systems that are increasingly subject to anthropogenic drivers of change.

Can We Use Functional Genetics to Predict the Fate of Nitrogen in Estuaries?

Increasing nitrogen (N) loads present a threat to estuaries, which are among the most heavily populated and perturbed parts of the world. N removal is largely mediated by the sediment microbial process of denitrification, in direct competition to dissimilatory nitrate reduction to ammonium (DNRA), which recycles nitrate to ammonium. Molecular proxies for N pathways are increasingly measured and analyzed, a major question in microbial ecology, however, is whether these proxies can add predictive power around the fate of N. We analyzed the diversity and community composition of sediment nirS and nrfA genes in 11 temperate estuaries, covering four types of land use in Australia, and analyzed how these might be used to predict N removal. Our data suggest that sediment microbiomes play a central role in controlling the magnitude of the individual N removal rates in the 11 estuaries. Inclusion, however, of relative gene abundances of 16S, nirS, nrfA, including their ratios did not improve physicochemical measurement-based regression models to predict rates of denitrification or DNRA. Co-occurrence network analyses of nirS showed a greater modularity and a lower number of keystone OTUs in pristine sites compared to urban estuaries, suggesting a higher degree of niche partitioning in pristine estuaries. The distinctive differences between the urban and pristine network structures suggest that the nirS gene could be a likely gene candidate to understand the mechanisms by which these denitrifying communities form and respond to anthropogenic pressures.

Systematic, continental scale temporal monitoring of marine pelagic microbiota by the Australian Marine Microbial Biodiversity Initiative.

Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and processed fashion. Currently 952 samples are publically available for bacteria and archaea which include 88,951,761 bacterial (72,435 unique) and 70,463,079 archaeal (24,205 unique) 16 S rRNA v1-3 gene sequences, and 388 samples are available for eukaryotes which include 39,801,050 (78,463 unique) 18 S rRNA v4 gene sequences.

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.

Water addition regulates the metabolic activity of ammonia oxidizers responding to environmental perturbations in dry subhumid ecosystems.

Terrestrial arid and semi-arid ecosystems (drylands) constitute about 41% of the Earth's land surface and are predicted to experience increasing fluctuations in water and nitrogen availability. Mounting evidence has confirmed the significant importance of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in nitrification, plant nitrogen availability and atmospheric N2 O emissions, but their responses to environmental perturbations in drylands remain largely unknown. Here we evaluate how the factorial combinations of irrigation and fertilization in forests and land-use change from grassland to forest affects the dynamics of AOA and AOB following a 6-year dryland field study. Potential nitrification rates and AOA and AOB abundances were significantly higher in the irrigated plots, accompanied by considerable changes in community compositions, but their responses to fertilization alone were not significant. DNA-stable isotope probing results showed increased (13) CO2 incorporation into the amoA gene of AOA, but not of AOB, in plots receiving water addition, coupled with significantly higher net mineralization and nitrification rates. High-throughput microarray analysis revealed that active AOA assemblages belonging to Nitrosopumilus and Nitrosotalea were increasingly labelled by (13) CO2 following irrigation. However, no obvious effects of land-use changes on nitrification rates or metabolic activity of AOA and AOB could be observed under dry conditions. We provide evidence that water addition had more important roles than nitrogen fertilization in influencing the autotrophic nitrification in dryland ecosystems, and AOA are increasingly involved in ammonia oxidation when dry soils become wetted.

DNA barcoding reveals overlooked marine fishes.

With more than 15 000 described marine species, fishes are a conspicuous, diverse and increasingly threatened component of marine life. It is generally accepted that most large-bodied fishes have been described, but this conclusion presumes that current taxonomic systems are robust. DNA barcoding, the analysis of a standardized region of the cytochrome c oxidase 1 gene (COI), was used to examine patterns of sequence divergence between populations of 35 fish species from opposite sides of the Indian Ocean, chosen to represent differing lifestyles from inshore to offshore. A substantial proportion of inshore species showed deep divergences between populations from South African and Australian waters (mean = 5.10%), a pattern which also emerged in a few inshore/offshore species (mean = 0.84%), but not within strictly offshore species (mean = 0.26%). Such deep divergences, detected within certain inshore and inshore/offshore taxa, are typical of divergences between congeneric species rather than between populations of a single species, suggesting that current taxonomic systems substantially underestimate species diversity. We estimate that about one third of the 1000 fish species thought to bridge South African and Australian waters actually represent two taxa.

DNA barcoding discriminates echinoderm species.

DNA barcode sequences (a 657-bp segment of the mtDNA cytochrome oxidase I gene, COI) were collected from 191 species (503 specimens) of Echinodermata. All five classes were represented: Ophiuroidea, Asteroidea, Echinoidea, Holothuroidea and Crinoidea. About 30% of sequences were collected specifically for this study, the remainder came from GenBank. Fifty-one species were represented by multiple samples, with a mean intraspecific divergence of 0.62%. Several possible instances of cryptic speciation were noted. Thirty-two genera were represented by multiple species, with a mean congeneric divergence of 15.33%. One hundred and eighty-seven of the 191 species (97.9%) could be distinguished by their COI barcodes. Those that could not were from the echinoid genus Amblypneustes. Neighbour-joining trees of COI sequences generally showed low bootstrap support for anything other than shallow splits, although with very rare exceptions, members of the same class clustered together. Two ophiuran species, in both nucleotide and amino acid neighbour-joining trees, grouped loosely as sister taxa to Crinoidea rather than Ophiuroidea; sequences of these two species appear to have evolved very quickly. Results suggest that DNA barcoding is likely to be an effective, accurate and useful method of species diagnosis for all five classes of Echinodermata.