Seasonality and longer-term development generate temporal dynamics in the Populus microbiome.

Temporal variation in community composition is central to our understanding of the assembly and functioning of microbial communities, yet the controls over temporal dynamics for microbiomes of long-lived plants, such as trees, remain unclear. Temporal variation in tree microbiomes could arise primarily from seasonal (i.e., intra-annual) fluctuations in community composition or from longer-term changes across years as host plants age. To test these alternatives, we experimentally isolated temporal variation in plant microbiome composition using a common garden and clonally propagated plants, and we used amplicon sequencing to characterize bacterial/archaeal and fungal communities in the leaf endosphere, root endosphere, and rhizosphere of two Populus spp. over four seasons across two consecutive years. Microbial community composition differed among seasons and years (which accounted for up to 21% of the variation in microbial community composition) and was correlated with seasonal dissimilarity in climatic conditions. However, microbial community dissimilarity was also positively correlated with time, reflecting longer-term compositional shifts as host trees aged. Together, our findings demonstrate that temporal patterns in tree microbiomes arise from both seasonal fluctuations and longer-term changes, which interact to generate unique seasonal patterns each year. In addition to shedding light on two important controls over the assembly of plant microbiomes, our results also suggest future studies of tree microbiomes should account for background temporal dynamics when testing the drivers of spatial patterns in microbial community composition and temporal responses of plant microbiomes to environmental change.IMPORTANCEMicrobiomes are integral to the health of host plants, but we have a limited understanding of the factors that control how the composition of plant microbiomes changes over time. Especially little is known about the microbiome of long-lived trees, relative to annual and non-woody plants. We tested how tree microbiomes changed between seasons and years in poplar (genus Populus), which are widespread and ecologically important tree species that also serve as important biofuel feedstocks. We found the composition of bacterial, archaeal, and fungal communities differed among seasons, but these seasonal differences depended on year. This dependence was driven by longer-term changes in microbial composition as host trees developed across consecutive years. Our findings suggest that temporal variation in tree microbiomes is driven by both seasonal fluctuations and longer-term (i.e., multiyear) development.

Tectonic and geological setting influence hot spring microbiology.

Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA (n = 41; mantle plume setting), Iceland (n = 41, divergent boundary), and Japan (n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.

Trajectories and state changes of a grassland stream and riparian zone after a decade of woody vegetation removal.

Riparian zones and the streams they border provide vital habitat for organisms, water quality protection, and other important ecosystem services. These areas are under pressure from local (land use/land cover change) to global (climate change) processes. Woody vegetation is expanding in grassland riparian zones worldwide. Here we report on a decade-long watershed-scale mechanical removal of woody riparian vegetation along 4.5 km of stream channel in a before-after control impact experiment. Prior to this removal, woody plants had expanded into grassy riparian areas, associated with a decline in streamflow, loss of grassy plant species, and other ecosystem-scale impacts. We confirmed some expected responses, including rapid increases in stream nutrients and sediments, disappearance of stream mosses, and decreased organic inputs to streams via riparian leaves. We were surprised that nutrient and sediment increases were transient for 3 years, that there was no recovery of stream discharge, and that areas with woody removal did not shift back to a grassland state, even when reseeded with grassland species. Rapid expansion of shrubs (Cornus drummondii, Prunus americana) in the areas where trees were removed allowed woody vegetation to remain dominant despite repeating the cutting every 2 years. Our results suggest woody expansion can fundamentally alter terrestrial and aquatic habitat connections in grasslands, resulting in inexorable movement toward a new ecosystem state. Human pressures, such as climate change, atmospheric CO2 increases, and elevated atmospheric nitrogen deposition, could continue to push the ecosystem along a trajectory that is difficult to change. Our results suggest that predicting relationships between riparian zones and the streams they border could be difficult in the face of global change in all biomes, even in well-studied sites.

Cultivating the Bacterial Microbiota of Populus Roots.

The integral role of microbial communities in plant growth and health is now widely recognized, and, increasingly, the constituents of the microbiome are being defined. While phylogenetic surveys have revealed the taxa present in a microbiome and show that this composition can depend on, and respond to, environmental perturbations, the challenge shifts to determining why particular microbes are selected and how they collectively function in concert with their host. In this study, we targeted the isolation of representative bacterial strains from environmental samples of Populus roots using a direct plating approach and compared them to amplicon-based sequencing analysis of root samples. The resulting culture collection contains 3,211 unique isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla, based on 16S rRNA gene sequence analysis. The collection accounts for ∼50% of the natural community of plant-associated bacteria as determined by phylogenetic analysis. Additionally, a representative set of 553 had their genomes sequenced to facilitate functional analyses. The top sequence variants in the amplicon data, identified as Pseudomonas, had multiple representatives within the culture collection. We then explore a simplified microbiome, comprised of 10 strains representing abundant taxa from environmental samples, and tested for their ability to reproducibly colonize Populus root tissue. The 10-member simplified community was able to reproducibly colonize on Populus roots after 21 days, with some taxa found in surface-sterilized aboveground tissue. This study presents a comprehensive collection of bacteria isolated from Populus for use in exploring microbial function and community inoculation experiments to understand basic concepts of plant and environmental selection. IMPORTANCE Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives. Here, we present a bacterial culture collection comprising 3,211 isolates that is representative of the root community of Populus. We then demonstrate the ability to examine underlying microbe-microbe interactions using a synthetic community approach. This culture collection will allow for the greater exploration of the microbial community function through targeted experimentation and manipulation.

Assembly of the Populus Microbiome Is Temporally Dynamic and Determined by Selective and Stochastic Factors.

Recent work shows that the plant microbiome, particularly the initial assembly of this microbiome, influences plant health, survival, and fitness. Here, we characterize the initial assembly of the Populus microbiome across ten genotypes belonging to two poplar species in a common garden using 16S rRNA gene and ITS2 region amplicon sequencing of the leaf endosphere, leaf surface, root endosphere, and rhizosphere. We sampled these microbiomes three times throughout the first growing season and found that the composition of the microbiome changed dramatically over time across all plant-associated habitats and host genotypes. For archaea and bacteria, these changes were dominated by strong homogenizing selection (accounting for 29 to 62% of pairwise comparisons). However, fungal assembly was generally characterized by multiple ecological assembly processes (i.e., a mix of weak selective and dispersal processes). Interestingly, genotype, while a significant moderator of microbiome composition, generally explained less variation than sample date across plant-associated habitats. We defined a set of core genera that accounted for, on average, 36% of the microbiome. The relative abundance of this core community was consistent over time. Additionally, using source tracking modeling, we determined that new microbial taxa colonize from both aboveground and belowground sources, and combined with our ecological assembly null models, we found that both selective and dispersal processes explained the differences between exo- (i.e., leaf surface and rhizosphere) and endospheric microbiomes. Taken together, our results suggest that the initial assembly of the Populus microbiome is time-, genotype-, and habitat-dependent and is moderated by both selective and stochastic factors. IMPORTANCE The initial assembly of the plant microbiome may establish the trajectory of forthcoming microbiome states, which could determine the overall future health of the plant. However, while much is known about the initial microbiome assembly of grasses and agricultural crops, less is known about the initial microbiome of long-lived trees, such as poplar (Populus spp.). Thus, a greater understanding of initial plant microbiome assembly in an ecologically and economically important plant such as Populus is highly desirable. Here, we show that the initial microbiome community composition and assembly in the first growing season of Populus is temporally dynamic and is determined by a combination of both selective and stochastic factors. Our findings could be used to prescribe ecologically informed microbial inoculations and better predict the composition of the Populus microbiome into the future and to better understand its influence on plant health.

Plant Hosts Modify Belowground Microbial Community Response to Extreme Drought.

Drought stress negatively impacts microbial activity, but the magnitude of stress responses is likely dependent on a diversity of belowground interactions. Populus trichocarpa individuals and no-plant bulk soils were exposed to extended drought (∼0.03% gravimetric water content [GWC] after 12 days), rewet, and a 12-day "recovery" period to determine the effects of plant presence in mediating soil microbiome stability to water stress. Plant metabolomic analyses indicated that drought exposure increased host investment in C and N metabolic pathways (amino acids, fatty acids, phenolic glycosides) regardless of recovery. Several metabolites positively correlated with root-associated microbial alpha-diversity, but not those of soil communities. Soil bacterial community composition shifted with P. trichocarpa presence and with drought relative to irrigated controls, whereas soil fungal composition shifted only with plant presence. However, root fungal communities strongly shifted with drought, whereas root bacterial communities changed to a lesser degree. The proportion of bacterial water-stress opportunistic operational taxonomic units (OTUs) (enriched counts in drought) was high (∼11%) at the end of drying phases and maintained after rewet and recovery phases in bulk soils, but it declined over time in soils with plants present. For root fungi, opportunistic OTUs were high at the end of recovery in drought treatments (∼17% abundance), although relatively not responsive in soils, particularly planted soils (<0.5% abundance for sensitive or opportunistic). These data indicate that plants modulate soil and root-associated microbial drought responses via tight plant-microbe linkages during extreme drought scenarios, but trajectories after extreme drought vary with plant habitat and microbial functional groups.IMPORTANCE Climate change causes significant alterations in precipitation and temperature regimes that are predicted to become more extreme throughout the next century. Microorganisms are important members within ecosystems, and how they respond to these changing abiotic stressors has large implications for the functioning of ecosystems, the recycling of nutrients, and the health of the aboveground plant community. Drought stress negatively impacts microbial activity, but the magnitude of this stress response may be dependent on above- and belowground interactions. This study demonstrates that beneficial associations between plants and microbes can enhance tolerance to abiotic stress.

Historical Drought Affects Microbial Population Dynamics and Activity During Soil Drying and Re-Wet.

A history of drought exposure promoted by variable precipitation regimes can select for drought-tolerant soil microbial taxa, but the mechanisms of survival and death of microbial populations through the selective stresses of soil drying and re-wet are not well understood. We subjected soils collected from a 15-year field drought experiment ("Altered" precipitation history with extended dry periods, versus the "Ambient" field control) to a laboratory drying/re-wetting experiment, to learn whether selective population survival, death, or maintenance of protein synthesis potential and microbial respiration through variable soil water conditions was affected by field drought legacy. Microbial community composition, as measured by Illumina MiSeq sequencing of the 16S rRNA and 16S rRNA gene, shifted with laboratory drying/re-wet and field drought treatments. In Ambient soils, there was a higher proportion of reduced OTU abundance (indicative of mortality) during re-wet, whereas Altered soils had a greater proportion of stable OTU populations that did not change in abundance (indicative of survival) through drying/re-wet. Altered soils also had a lower proportion of rRNA:rRNA genes (lower protein synthesis potential) during dry-down, a greater weighted mean rRNA operon number (potential growth rate and r-selection) which was associated with higher abundance of Firmicutes (order Bacillales), and lower average microbial respiration rates. These data demonstrate that soils with a weaker historical drought legacy exhibit a higher prevalence of microbial water-stress mortality and differential survival and death at OTU levels following short-term dryingand re-wetting, concurrent with higher carbon loss potential. This work provides novel insight into the mechanisms and consequences of soil microbial changes resulting from extended drought conditions.

Rhizosphere microbiomes diverge among Populus trichocarpa plant-host genotypes and chemotypes, but it depends on soil origin.

BACKGROUND: Plants have developed defense strategies for phytopathogen and herbivore protection via coordinated metabolic mechanisms. Low-molecular weight metabolites produced within plant tissues, such as salicylic acid, represent one such mechanism which likely mediates plant - microbe interactions above and below ground. Salicylic acid is a ubiquitous phytohormone at low levels in most plants, yet are concentrated defense compounds in Populus, likely acting as a selective filter for rhizosphere microbiomes. We propagated twelve Populus trichocarpa genotypes which varied an order of magnitude in salicylic acid (SA)-related secondary metabolites, in contrasting soils from two different origins. After four months of growth, plant properties (leaf growth, chlorophyll content, and net photosynthetic rate) and plant root metabolomics specifically targeting SA metabolites were measured via GC-MS. In addition, rhizosphere microbiome composition was measured via Illumina MiSeq sequencing of 16S and ITS2 rRNA-genes. RESULTS: Soil origin was the primary filter causing divergence in bacterial/archaeal and fungal communities with plant genotype secondarily influential. Both bacterial/archaeal and fungal evenness varied between soil origins and bacterial/archaeal diversity and evenness correlated with at least one SA metabolite (diversity: populin; evenness: total phenolics). The production of individual salicylic acid derivatives that varied by host genotype resulted in compositional differences for bacteria /archaea (tremuloidin) and fungi (salicylic acid) within one soil origin (Clatskanie) whereas soils from Corvallis did not illicit microbial compositional changes due to salicylic acid derivatives. Several dominant bacterial (e.g., Betaproteobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadete, Firmicutes) and one fungal phyla (Mortierellomycota) also correlated with specific SA secondary metabolites; bacterial phyla exhibited more negative interactions (declining abundance with increasing metabolite concentration) than positive interactions. CONCLUSIONS: These results indicate microbial communities diverge most among soil origin. However, within a soil origin, bacterial/archaeal communities are responsive to plant SA production within greenhouse-based rhizosphere microbiomes. Fungal microbiomes are impacted by root SA-metabolites, but overall to a lesser degree within this experimental context. These results suggest plant defense strategies, such as SA and its secondary metabolites, may partially drive patterns of both bacterial/archaeal and fungal taxa-specific colonization and assembly.

Methanogenic Archaea dominate mature heartwood habitats of Eastern Cottonwood (Populus deltoides).

While recent reports demonstrate that the direct emission of methane from living tree trunks may be a significant terrestrial emission source, there has been debate whether tree emissions are due to transport from soils or produced in the wood environment itself. Reports of methanogens from wood of trees were prominent in the literature 40 years ago but have not been revisited with molecular ecology approaches. We examined communities associated with Populus deltoides using rRNA gene sequence analyses and how these vary with tree and wood properties. Our data indicate that wood environments are dominated by anaerobic microbiomes. Methanogens are prominent in heartwood (mean 34% relative abundance) compared to sapwood environments (13%), and dominant operational taxonomic units (OTUs) were classified as the Methanobacterium sp. Members of the Firmicutes phylum comprised 39% of total sequences and were in 42% greater abundance in sapwood over heartwood niches. Tree diameter was the strongest predictor of methanogen abundance, but wood moisture content and pH were also significant predictors of taxon abundance and overall community composition. Unlike microbiomes of the soil, rhizosphere and phyllosphere, wood associated communities are shaped by unique environmental conditions and may be prominent and overlooked sources of methane emissions in temperate forest systems.

Testing the light:nutrient hypothesis: Insights into biofilm structure and function using metatranscriptomics.

Aquatic biofilms are hotspots of biogeochemical activity due to concentrated microbial biomass (Battin, Kaplan, Newbold, & Hansen, ). However, biofilms are often considered a single entity when their role in biogeochemical transformations is assessed, even though these biofilms harbour functionally diverse microbial communities (Battin, Besemer, Bengtsson, Romani, & Packmann, ; Veach, Stegen, Brown, Dodds, & Jumpponen, ). Often overlooked are the biotic interactions among biofilm components that can affect ecosystem-scale processes such as primary production and nutrient cycling. These interactions are likely to be especially important under resource limitation. Light is a primary resource mediating algal photosynthesis and both phototrophic and heterotrophic production due to bacterial reliance on C-rich algal exudates (Cole, ). However, current understanding of function-structure linkages in streams has yet to unravel the relative degree of these microbial feedbacks under resource availability gradients. In this issue of Molecular Ecology, Bengtsson, Wagner, Schwab, Urich, and Battin () studied stream biofilm responses to light availability to understand its impact across three domains of life. By integrating biogeochemical rate estimation and metatranscriptomics within a microcosm experiment, they were able to link primary production and nutrient uptake rates to algal and bacterial metabolic processes and specify what taxa contributed to gene expression. Under low light, diatoms and cyanobacteria upregulated photosynthetic machinery and diatom-specific chloroplast rRNA suggesting heightened transcriptional activity under light limitation to maintain phototrophic energy demands. Under high light, heterotrophic bacteria upregulated mRNAs related to phosphorous (P) metabolism while biofilm P uptake increased indicating high bacterial-specific P demand when algal biomass was high. Together, these results indicate that biogeochemical function is mediated by complex microbial interactions across trophic levels.

Nitrogen enrichment suppresses other environmental drivers and homogenizes salt marsh leaf microbiome.

Microbial community assembly is affected by a combination of forces that act simultaneously, but the mechanisms underpinning their relative influences remain elusive. This gap strongly limits our ability to predict human impacts on microbial communities and the processes they regulate. Here, we experimentally demonstrate that increased salinity stress, food web alteration and nutrient loading interact to drive outcomes in salt marsh fungal leaf communities. Both salinity stress and food web alterations drove communities to deterministically diverge, resulting in distinct fungal communities. Increased nutrient loads, nevertheless, partially suppressed the influence of other factors as determinants of fungal assembly. Using a null model approach, we found that increased nutrient loads enhanced the relative importance of stochastic over deterministic divergent processes; without increased nutrient loads, samples from different treatments showed a relatively (deterministic) divergent community assembly whereas increased nutrient loads drove the system to more stochastic assemblies, suppressing the effect of other treatments. These results demonstrate that common anthropogenic modifications can interact to control fungal community assembly. Furthermore, our results suggest that when the environmental conditions are spatially heterogeneous (as in our case, caused by specific combinations of experimental treatments), increased stochasticity caused by greater nutrient inputs can reduce the importance of deterministic filters that otherwise caused divergence, thus driving to microbial community homogenization.

Use of in-field bioreactors demonstrate groundwater filtration influences planktonic bacterial community assembly, but not biofilm composition.

Using in-field bioreactors, we investigated the influence of exogenous microorganisms in groundwater planktonic and biofilm microbial communities as part of the Integrated Field Research Challenge (IFRC). After an acclimation period with source groundwater, bioreactors received either filtered (0.22 µM filter) or unfiltered well groundwater in triplicate and communities were tracked routinely for 23 days after filtration was initiated. To address geochemical influences, the planktonic phase was assayed periodically for protein, organic acids, physico-/geochemical measurements and bacterial community (via 16S rRNA gene sequencing), while biofilms (i.e. microbial growth on sediment coupons) were targeted for bacterial community composition at the completion of the experiment (23 d). Based on Bray-Curtis distance, planktonic bacterial community composition varied temporally and between treatments (filtered, unfiltered bioreactors). Notably, filtration led to an increase in the dominant genus, Zoogloea relative abundance over time within the planktonic community, while remaining relatively constant when unfiltered. At day 23, biofilm communities were more taxonomically and phylogenetically diverse and substantially different from planktonic bacterial communities; however, the biofilm bacterial communities were similar regardless of filtration. These results suggest that although planktonic communities were sensitive to groundwater filtration, bacterial biofilm communities were stable and resistant to filtration. Bioreactors are useful tools in addressing questions pertaining to microbial community assembly and succession. These data provide a first step in understanding how an extrinsic factor, such as a groundwater inoculation and flux of microbial colonizers, impact how microbial communities assemble in environmental systems.

Fungal Communities and Functional Guilds Shift Along an Elevational Gradient in the Southern Appalachian Mountains.

Nitrogen deposition alters forest ecosystems particularly in high elevation, montane habitats where nitrogen deposition is greatest and continues to increase. We collected soils across an elevational (788-1940 m) gradient, encompassing both abiotic (soil chemistry) and biotic (vegetation community) gradients, at eight locations in the southern Appalachian Mountains of southwestern North Carolina and eastern Tennessee. We measured soil chemistry (total N, C, extractable PO4, soil pH, cation exchange capacity [ECEC], percent base saturation [% BS]) and dissected soil fungal communities using ITS2 metabarcode Illumina MiSeq sequencing. Total soil N, C, PO4, % BS, and pH increased with elevation and plateaued at approximately 1400 m, whereas ECEC linearly increased and C/N decreased with elevation. Fungal communities differed among locations and were correlated with all chemical variables, except PO4, whereas OTU richness increased with total N. Several ecological guilds (i.e., ectomycorrhizae, saprotrophs, plant pathogens) differed in abundance among locations; specifically, saprotroph abundance, primarily attributable to genus Mortierella, was positively correlated with elevation. Ectomycorrhizae declined with total N and soil pH and increased with total C and PO4 where plant pathogens increased with total N and decreased with total C. Our results demonstrate significant turnover in taxonomic and functional fungal groups across elevational gradients which facilitate future predictions on forest ecosystem change in the southern Appalachians as nitrogen deposition rates increase and regional temperature and precipitation regimes shift.

Spatial and successional dynamics of microbial biofilm communities in a grassland stream ecosystem.

Biofilms represent a metabolically active and structurally complex component of freshwater ecosystems. Ephemeral prairie streams are hydrologically harsh and prone to frequent perturbation. Elucidating both functional and structural community changes over time within prairie streams provides a general understanding of microbial responses to environmental disturbance. We examined microbial succession of biofilm communities at three sites in a third-order stream at Konza Prairie over a 2- to 64-day period. Microbial abundance (bacterial abundance, chlorophyll a concentrations) increased and never plateaued during the experiment. Net primary productivity (net balance of oxygen consumption and production) of the developing biofilms did not differ statistically from zero until 64 days suggesting a balance of the use of autochthonous and allochthonous energy sources until late succession. Bacterial communities (MiSeq analyses of the V4 region of 16S rRNA) established quickly. Bacterial richness, diversity and evenness were high after 2 days and increased over time. Several dominant bacterial phyla (Beta-, Alphaproteobacteria, Bacteroidetes, Gemmatimonadetes, Acidobacteria, Chloroflexi) and genera (Luteolibacter, Flavobacterium, Gemmatimonas, Hydrogenophaga) differed in relative abundance over space and time. Bacterial community composition differed across both space and successional time. Pairwise comparisons of phylogenetic turnover in bacterial community composition indicated that early-stage succession (≤16 days) was driven by stochastic processes, whereas later stages were driven by deterministic selection regardless of site. Our data suggest that microbial biofilms predictably develop both functionally and structurally indicating distinct successional trajectories of bacterial communities in this ecosystem.

Woody plant encroachment, and its removal, impact bacterial and fungal communities across stream and terrestrial habitats in a tallgrass prairie ecosystem.

Woody plant encroachment has become a global threat to grasslands and has caused declines in aboveground richness and changes in ecosystem function; yet we have a limited understanding on the effects of these phenomena on belowground microbial communities. We completed riparian woody plant removals at Konza Prairie Biological Station, Kansas and collected soils spanning land-water interfaces in removal and woody vegetation impacted areas. We measured stream sediments and soils for edaphic variables (C and N pools, soil water content, pH) and bacterial (16S rRNA genes) and fungal (ITS2 rRNA gene repeat) communities using Illumina MiSeq metabarcoding. Bacterial richness and diversity decreased with distance from streams. Fungal richness decreased with distance from the stream in wooded areas, but was similar across landscape position while Planctomycetes and Basidiomycota relative abundance was lower in removal areas. Cyanobacteria, Ascomycota, Chytridiomycota and Glomeromycota relative abundance was greater in removal areas. Ordination analyses indicated that bacterial community composition shifted more across land-water interfaces than fungi yet both were marginally influenced by treatment. This study highlights the impacts of woody encroachment restoration on grassland bacterial and fungal communities which likely subsequently affects belowground processes and plant health in this ecosystem.

Fire and grazing influences on rates of riparian woody plant expansion along grassland streams.

Grasslands are threatened globally due to the expansion of woody plants. The few remaining headwater streams within tallgrass prairies are becoming more like typical forested streams due to rapid conversion of riparian zones from grassy to wooded. Forestation can alter stream hydrology and biogeochemistry. We estimated the rate of riparian woody plant expansion within a 30 m buffer zone surrounding the stream bed across whole watersheds at Konza Prairie Biological Station over 25 years from aerial photographs. Watersheds varied with respect to experimentally-controlled fire and bison grazing. Fire frequency, presence or absence of grazing bison, and the historical presence of woody vegetation prior to the study time period (a proxy for proximity of propagule sources) were used as independent variables to predict the rate of riparian woody plant expansion between 1985 and 2010. Water yield was estimated across these years for a subset of watersheds. Riparian woody encroachment rates increased as burning became less frequent than every two years. However, a higher fire frequency (1-2 years) did not reverse riparian woody encroachment regardless of whether woody vegetation was present or not before burning regimes were initiated. Although riparian woody vegetation cover increased over time, annual total precipitation and average annual temperature were variable. So, water yield over 4 watersheds under differing burn frequencies was quite variable and with no statistically significant detected temporal trends. Overall, burning regimes with a frequency of every 1-2 years will slow the conversion of tallgrass prairie stream ecosystems to forested ones, yet over long time periods, riparian woody plant encroachment may not be prevented by fire alone, regardless of fire frequency.

The influence of six pharmaceuticals on freshwater sediment microbial growth incubated at different temperatures and UV exposures.

Pharmaceutical compounds have been detected in freshwater for several decades. Once they enter the aquatic ecosystem, they may be transformed abiotically (i.e., photolysis) or biotically (i.e., microbial activity). To assess the influence of pharmaceuticals on microbial growth, basal salt media amended with seven pharmaceutical treatments (acetaminophen, caffeine, carbamazepine, cotinine, ibuprofen, sulfamethoxazole, and a no pharmaceutical control) were inoculated with stream sediment. The seven pharmaceutical treatments were then placed in five different culture environments that included both temperature treatments of 4, 25, 37°C and light treatments of continuous UV-A or UV-B exposure. Microbial growth in the basal salt media was quantified as absorbance (OD(550)) at 7, 14, 21, 31, and 48d following inoculation. Microbial growth was significantly influenced by pharmaceutical treatments (P < 0.01) and incubation treatments (P < 0.01). Colonial morphology of the microbial communities post-incubation identified selection of microbial and fungal species with exposure to caffeine, cotinine, and ibuprofen at 37°C; acetaminophen, caffeine, and cotinine at 25°C; and carbamazepine exposed to continuous UV-A. Bacillus and coccus cellular arrangements (1000X magnification) were consistently observed across incubation treatments for each pharmaceutical treatment although carbamazepine and ibuprofen exposures incubated at 25°C also selected spiral-shaped bacteria. These data indicate stream sediment microbial communities are influenced by pharmaceuticals though physiochemical characteristics of the environment may dictate microbial response.

Temporal variation of pharmaceuticals in an urban and agriculturally influenced stream.

Pharmaceuticals have become ubiquitous in the aquatic environment. Previous studies consistently demonstrate the prevalence of pharmaceuticals in freshwater but we do not yet know how concentrations vary over time within a given system. Two sites in central Indiana with varying land use in the surrounding watershed (suburban and agricultural) were sampled monthly for pharmaceutical concentrations and stream physiochemical parameters. Sediment samples were also collected at each sampling event for measurement of δ(15)N natural abundance and sediment organic content. Across sites and sampling events, twelve pharmaceuticals were detected including acetaminophen, caffeine, carbamazepine, cotinine, N,N-diethyl-meta-toluamide (DEET), gemfibrozil, ibuprofen, sulfadimethoxine, sulfamethazine, sulfamethoxazole, triclosan, and trimethoprim. Sulfathiazole, lincomycin, and tylosin were not detected at either site at any time. The agriculturally-influenced site had comparable pharmaceutical concentrations to the urban-influenced site. In general, pharmaceutical concentrations increased during winter at both sites and decreased during spring and summer. Multiple regression analyses indicated that water column dissolved oxygen, the number of days since precipitation, and solar radiation influenced total pharmaceutical concentration in the urban-influenced site; whereas pH, chlorophyll a concentration, and total amount of rainfall in the previous 10 days influenced total pharmaceutical concentrations in the agriculturally-influenced site. Pharmaceutical concentrations were not correlated with sediment δ(15)N across or within sites. However, sediment in the urban-influenced site had higher mean δ(15)N signatures relative to sediment in the agriculturally-influenced site. These data indicate pharmaceuticals are persistent in aquatic ecosystems influenced by both agricultural and suburban activity. Pharmaceuticals are designed to have a physiological effect; therefore, it is likely that they may also influence aquatic organisms, potentially threatening freshwater ecosystem health.