Gavin N. Rees

Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics.

Terminal restriction fragment length polymorphism (T-RFLP) is increasingly being used to examine microbial community structure and accordingly, a range of approaches have been used to analyze data sets. A number of published reports have included data and results that were statistically flawed or lacked rigorous statistical testing. A range of simple, yet powerful techniques are available to examine community data, however their use is seldom, if ever, discussed in microbial literature. We describe an approach that overcomes some of the problems associated with analyzing community datasets and offer an approach that makes data interpretation simple and effective. The Bray-Curtis coefficient is suggested as an ideal coefficient to be used for the construction of similarity matrices. Its strengths include its ability to deal with data sets containing multiple blocks of zeros in a meaningful manner. Non-metric multi-dimensional scaling is described as a powerful, yet easily interpreted method to examine community patterns based on T-RFLP data. Importantly, we describe the use of significance testing of data sets to allow quantitative assessment of similarity, removing subjectivity in comparing complex data sets. Finally, we introduce a quantitative measure of sample dispersion and suggest its usefulness in describing site heterogeneity.Terminal restriction fragment length polymorphism (T-RFLP) is increasingly being used to examine microbial community structure and accordingly, a range of approaches have been used to analyze data sets. A number of published reports have included data and results that were statistically flawed or lacked rigorous statistical testing. A range of simple, yet powerful techniques are available to examine community data, however their use is seldom, if ever, discussed in microbial literature. We describe an approach that overcomes some of the problems associated with analyzing community datasets and offer an approach that makes data interpretation simple and effective. The Bray-Curtis coefficient is suggested as an ideal coefficient to be used for the construction of similarity matrices. Its strengths include its ability to deal with data sets containing multiple blocks of zeros in a meaningful manner. Non-metric multi-dimensional scaling is described as a powerful, yet easily interpreted method to examine community patterns based on T-RFLP data. Importantly, we describe the use of significance testing of data sets to allow quantitative assessment of similarity, removing subjectivity in comparing complex data sets. Finally, we introduce a quantitative measure of sample dispersion and suggest its usefulness in describing site heterogeneity.

The short-term effects of salinization on anaerobic nutrient cycling and microbial community structure in sediment from a freshwater wetland

Wetlands in many inland catchments are being subjected to increasing salinity. To expand our limited understanding of how increasing salinity will alter carbon and nutrient dynamics in freshwater sediments, we carried out microcosm experiments to examine the acute effects of increasing salinity on the anaerobic cycling of carbon, nutrients (N, P, and S), metals (Fe and Mn), and microbial community structure in sediments from a non-salt-impacted freshwater wetland. Sediments were collected from a wetland on the River Murray floodplain, south eastern Australia and incubated with NaCl concentrations ranging from 0 to 100 mmol L−1. Increasing NaCl concentration led to the immediate release of between about 80 and 190 μmol L−1 ammonium and 235 to 3300 μmol L−1 Fe(II) from the sediments, the amount released ‘increasing with NaCl concentration. Conversely, net phosphate release decreased with increasing NaCl concentration. The overall microbial community structure, determined from phospholipid fatty acid profiles, changed only at the highest NaCl loadings, with evidence of a decrease in microbial diversity. Bacterial community structure, determined by examining terminal restriction fragment length polymorphism (T-RFLP) of the bacterial 16S rRNA gene, showed little response to increasing NaCl concentration. Conversely, the archaeal (methanogen) population, determined by examining T-RFLP of the archaeal 16S rRNA gene, showed significant changes with increasing NaCl loading. This shift corresponded with a significant decrease in methane production from salt-impacted sediments and therefore shows a linkage between microbial community structure and an ecosystem process.

Impacts of inundation and drought on eukaryote biodiversity in semi-arid floodplain soils

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi‐arid floodplain would transition towards comprising mainly dry‐soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post‐flood) were dominated by persistent rather than either aquatic or dry‐specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post‐flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post‐flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi‐arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.

Variability in sediment microbial communities in a semipermanent stream: impact of drought

Abstract Terminal-restriction fragment length polymorphism (T-RFLP) is a DNA-based technique used to examine microbial community structure. We used this technique to examine variability in microbial community structure in 3 pools and a riffle in a semipermanent stream that becomes a chain of pools during the summer. We examined microbial communities under 3 hydrological conditions: predrought (all sites inundated), drought (all sediments dried for ≥2 mo), and rewet (1 mo after inundation). We used nonmetric multidimensional scaling to ordinate microbial communities and analysis of similarity to test whether communities differed among sites and whether community structure within sites changed in response to drought and rewetting. Microbial communities within sites differed with respect to hydrological condition, with most within-site variability occurring within the riffle section of the stream. Sediment drying significantly changed the microbial community structure at all sites. One month after rewetting at all sites, the microbial communities had not returned to their predrought structures and were significantly different from the predrought and drought microbial communities. Within-site variability in microbial community structure was much lower after the drought than before the drought. T-RFLP proved to be a powerful method for resolving microbial community structure, and its application helped address our limited understanding of microbial community dynamics.

The effects of in situ drying on sediment–phosphate interactions in sediments from an old wetland

We examined the effects of drying on sediment–P interactions for sediments from a shallow wetland ca. 6000 years old. Sediments from this wetland would have been subjected to numerous drying and wetting episodes during the life of the wetland. The factors affecting potential orthophosphate release were compared in surface sediments that had previously been desiccated for 3 months, surface sediments that had remained inundated and wet sediment 25–30 cm below the surface. All sediments released small amounts of orthophosphate once subjected to anaerobic conditions. Sulfate alone stimulated phosphate release from the surface sediment, irrespective of the previous hydrological status, whereas orthophosphate release from deep sediments was co-limited by carbon and sulfate. Decreases in soluble Fe (II) were measured concurrently with sulfate-stimulated P release, which is consistent with formation of iron sulfides. Similar numbers of culturable sulfate-reducing bacteria were obtained from the wet and dry sediments, their tolerance to desiccation explaining why release could occur after extensive drying of the sediment. Phosphate adsorption isotherms of the sediments showed that sediments from all sites had a relatively low affinity for ortho-phosphate. The adsorption data showed reasonable fit to the Freundlich adsorption isotherm. No difference in the maximum amount of ortho-phosphate uptake was observed if the sediment isotherm experiments were done under an inert atmosphere or, following exposure to air, suggesting that even anoxic sediments were not highly reduced. However, significant variations in the value of the Freundlich constants were observed. The results presented in this study are consistent with the hypothesis that repeated wetting/drying cycles select for bacteria that are tolerant to periods of desiccation and/or oxidation. Furthermore, repeated wetting and drying cycles may result in changes to sediment mineralogy.

Provisioning of bioavailable carbon between the wet and dry phases in a semi-arid floodplain.

Ecosystem functioning on arid and semi-arid floodplains may be described by two alternate traditional paradigms. The pulse-reserve model suggests that rainfall is the main driver of plant growth and subsequent carbon and energy reserve formation in the soil of arid and semi-arid regions. The flood pulse concept suggests that periodic flooding facilitates the two-way transfer of materials between a river and its adjacent floodplain, but focuses mainly on the period when the floodplain is inundated. We compared the effects of both rainfall and flooding on soil moisture and carbon in a semi-arid floodplain to determine the relative importance of each for soil moisture recharge and the generation of a bioavailable organic carbon reserve that can potentially be utilised during the dry phase. Flooding, not rainfall, made a substantial contribution to moisture in the soil profile. Furthermore, the growth of aquatic macrophytes during the wet phase produced at least an order of magnitude more organic material than rainfall-induced pulse-reserve responses during the dry phase, and remained as recognizable soil carbon for years following flood recession. These observations have led us to extend existing paradigms to encompass the reciprocal provisioning of carbon between the wet and dry phases on the floodplain, whereby, in addition to carbon fixed during the dry phase being important for driving biogeochemical transformations upon return of the next wet phase, aquatic macrophyte carbon fixed during the wet phase is recognized as an important source of energy for the dry phase. Reciprocal provisioning presents a conceptual framework on which to formulate questions about the resistance and ecosystem resilience of arid and semi-arid floodplains in the face of threats like climate change and alterations to flood regimes.

Spatial and temporal variability of nitrogen dynamics in an upland stream before and after a drought

In the current study, we explore the spatial and temporal variability of ammonia, nitrate and urea dynamics in an upland stream before and after a major drying event, using short-term nitrogen additions to benthic chambers. The potential for an initial flush of mineral nitrogen from re-wetted sediments following a prolonged period of drying was also assessed. The distribution of dissolved nitrogen species at four sites spaced along a 1-km reach of the stream were quite variable over time but, in general, not between sites. Conversely, sediment nitrogen dynamics were spatially variable. For example, in one instance, sediments from the uppermost site were a net sink for ammonia, whereas the sediments immediately downstream (separated from the first site by a small sand bar) were a net source of ammonia; with measured sediment fluxes up to ~2 µg N m−2 s−1. In general, the short-term addition of nitrate, ammonia or urea did not substantially affect the sediment nitrogen dynamics. After ~3 months of in situ drying, upon re-wetting, the sediments from all sites immediately produced pulses of ammonia, nitrate and, to a substantially lesser extent, urea. The rates of release of nitrogen were spatially variable, with up to an order of magnitude difference in the rate of release of ammonia from re-wetted sediments from the same small pool. Some differences were observed between nitrogen dynamics before and after drying but a causal linkage could not be established.

Urban stormwater runoff drives denitrifying community composition through changes in sediment texture and carbon content

The export of nitrogen from urban catchments is a global problem, and denitrifying bacteria in stream ecosystems are critical for reducing in-stream N. However, the environmental factors that control the composition of denitrifying communities in streams are not well understood. We determined whether denitrifying community composition in sediments of nine streams on the eastern fringe of Melbourne, Australia was correlated with two measures of catchment urban impact: effective imperviousness (EI, the proportion of a catchment covered by impervious surfaces with direct connection to streams) or septic tank density (which affects stream water chemistry, particularly stream N concentrations). Denitrifying community structure was examined by comparing terminal restriction fragment length polymorphisms of nosZ genes in the sediments, as the nosZ gene codes for nitrous oxide reductase, the last step in the denitrification pathway. We also determined the chemical and physical characteristics of the streams that were best correlated with denitrifying community composition. EI was strongly correlated with community composition and sediment physical and chemical properties, while septic tank density was not. Sites with high EI were sandier, with less fine sediment and lower organic carbon content, higher sediment cations (calcium, sodium and magnesium) and water filterable reactive phosphorus concentrations. These were also the best small-scale environmental variables that explained denitrifying community composition. Among our study streams, which differed in the degree of urban stormwater impact, sediment grain size and carbon content are the most likely drivers of change in community composition. Denitrifying community composition is another in a long list of ecological indicators that suggest the profound degradation of streams is caused by urban stormwater runoff. While the relationships between denitrifying community composition and denitrification rates are yet to be unequivocally established, landscape-scale indices of environmental impact such as EI may prove to be useful indicators of change in microbial communities.

Characterisation of adaptive genetic diversity in environmentally contrasted populations of Eucalyptus camaldulensis Dehnh. (river red gum).

As an increasing number of ecosystems face departures from long standing environmental conditions under climate change, our understanding of the capacity of species to adapt will become important for directing conservation and management of biodiversity. Insights into the potential for genetic adaptation might be gained by assessing genomic signatures of adaptation to historic or prevailing environmental conditions. The river red gum (Eucalyptus camaldulensis Dehnh.) is a widespread Australian eucalypt inhabiting riverine and floodplain habitats which spans strong environmental gradients. We investigated the effects of adaptation to environment on population level genetic diversity of E. camaldulensis, examining SNP variation in candidate gene loci sampled across 20 climatically diverse populations approximating the species natural distribution. Genetic differentiation among populations was high (FST = 17%), exceeding previous estimates based on neutral markers. Complementary statistical approaches identified 6 SNP loci in four genes (COMT, Dehydrin, ERECTA and PIP2) which, after accounting for demographic effects, exhibited higher than expected levels of genetic differentiation among populations and whose allelic variation was associated with local environment. While this study employs but a small proportion of available diversity in the eucalyptus genome, it draws our attention to the potential for application of wide spread eucalypt species to test adaptive hypotheses.

Analysis of denitrifying communities in streams from an urban and non-urban catchment

Urbanization leads to degradation in water quality and has a major effect on the biota of streams, but its effect on microbial communities is not as well understood. DNA-techniques that target functional genes are being used to examine microbial communities, but less frequently applied to freshwater aquatic systems. Our aim was to determine whether terminal restriction fragment length polymorphism and sequence analysis of polymerase chain reaction-amplified (PCR) nosZ gene sequences could be used to show if there were measurable differences in the denitrifying community in two urban streams in catchments with contrasting degrees of catchment urbanization. Community structure in the sediments and associated riparian zones were studied at the contrasting sites. We showed that the denitrifying community in the sediments and riparian soils of the two streams were significantly different. There were also significant differences between the sediment and riparian zone communities within each of the sites. Terminal restriction fragment length polymorphism analysis proved to be a valuable technique that could resolve patterns of the denitrifying community in streams of contrasting degrees of urbanization, but sequence analysis was required to confirm the identity of the amplified products.