WIDESPREAD CAPACITY FOR DENITRIFICATION ACROSS A BOREAL FOREST LANDSCAPE.

A warming climate combined with frequent and severe fires cause permafrost to thaw, especially in the region of discontinuous permafrost, where soil temperatures may only be a few degrees below 0 °C. Soil thaw releases carbon (C) and nitrogen (N) into the actively cycling pools, and whereas C emissions following permafrost thaw are well documented, the fates of N remain unclear. Denitrification could release N from ecosystems as nitrous oxide (N2O) or nitrogen gas (N2), but the contributions of these processes to the high-latitude N cycle remain uncertain. We quantified microbial capacity for denitrification and N2O production in boreal soils, lakes, and streams using anoxic C- and N-amended assays, and assessed correlates of denitrifying enzyme activity (DEA) in Interior Alaska. Riparian soils and stream sediments supported the highest potential rates of denitrification, upland soils were intermediate, and lakes supported lower rates, whereas deep permafrost soils supported little denitrification. Time since fire had no effect on denitrification potential in upland soils. Across all landscape positions, DEA was negatively correlated with ammonium pools. Within each landscape position, potential rate of denitrification increased with soil or sediment organic matter content. Widespread N loss to denitrification in boreal forests could constrain the capacity for N-limited primary producers to maintain C stocks in soils following permafrost thaw.

Unearthing Shifts in Microbial Communities Across a Soil Disturbance Gradient.

Permafrost, an important source of soil disturbance, is particularly vulnerable to climate change in Alaska where 85% of the land is underlained with discontinuous permafrost. Boreal forests, home to plants integral to subsistence diets of many Alaska Native communities, are not immune to the effects of climate change. Soil disturbance events, such as permafrost thaw, wildfires, and land use change can influence abiotic conditions, which can then affect active layer soil microbial communities. In a previous study, we found negative effects on boreal plants inoculated with microbes impacted by soil disturbance compared to plants inoculated with microbes from undisturbed soils. Here, we identify key shifts in microbial communities altered by soil disturbance using 16S rRNA gene sequencing and make connections between microbial community changes and previously observed plant growth. Additionally, we identify further community shifts in potential functional mechanisms using long read metagenomics. Across a soil disturbance gradient, microbial communities differ significantly based on the level of soil disturbance. Consistent with the earlier study, the family Acidobacteriaceae, which consists of known plant growth promoters, was abundant in undisturbed soil, but practically absent in most disturbed soil. In contrast, Comamonadaceae, a family with known agricultural pathogens, was overrepresented in most disturbed soil communities compared to undisturbed. Within our metagenomic data, we found that soil disturbance level is associated with differences in microbial community function, including mechanisms potentially involved in plant pathogenicity. These results indicate that a decrease in plant growth can be linked to changes in the microbial community and functional composition driven by soil disturbance and climate change. Together, these results build a genomic understanding of how shifting soil microbiomes may affect plant productivity and ecosystem health as the Arctic warms.

Microbiome assembly in thawing permafrost and its feedbacks to climate.

The physical and chemical changes that accompany permafrost thaw directly influence the microbial communities that mediate the decomposition of formerly frozen organic matter, leading to uncertainty in permafrost-climate feedbacks. Although changes to microbial metabolism and community structure are documented following thaw, the generality of post-thaw assembly patterns across permafrost soils of the world remains uncertain, limiting our ability to predict biogeochemistry and microbial community responses to climate change. Based on our review of the Arctic microbiome, permafrost microbiology, and community ecology, we propose that Assembly Theory provides a framework to better understand thaw-mediated microbiome changes and the implications for community function and climate feedbacks. This framework posits that the prevalence of deterministic or stochastic processes indicates whether the community is well-suited to thrive in changing environmental conditions. We predict that on a short timescale and following high-disturbance thaw (e.g., thermokarst), stochasticity dominates post-thaw microbiome assembly, suggesting that functional predictions will be aided by detailed information about the microbiome. At a longer timescale and lower-intensity disturbance (e.g., active layer deepening), deterministic processes likely dominate, making environmental parameters sufficient for predicting function. We propose that the contribution of stochastic and deterministic processes to post-thaw microbiome assembly depends on the characteristics of the thaw disturbance, as well as characteristics of the microbial community, such as the ecological and phylogenetic breadth of functional guilds, their functional redundancy, and biotic interactions. These propagate across space and time, potentially providing a means for predicting the microbial forcing of greenhouse gas feedbacks to global climate change.

Soil Disturbance Affects Plant Productivity via Soil Microbial Community Shifts.

Recent advances in climate research have discovered that permafrost is particularly vulnerable to the changes occurring in the atmosphere and climate, especially in Alaska where 85% of the land is underlain by mostly discontinuous permafrost. As permafrost thaws, research has shown that natural and anthropogenic soil disturbance causes microbial communities to undergo shifts in membership composition and biomass, as well as in functional diversity. Boreal forests are home to many plants that are integral to the subsistence diets of many Alaska Native communities. Yet, it is unclear how the observed shifts in soil microbes can affect above ground plant communities that are relied on as a major source of food. In this study, we tested the hypothesis that microbial communities associated with permafrost thaw affect plant productivity by growing five plant species found in Boreal forests and Tundra ecosystems, including low-bush cranberry and bog blueberry, with microbial communities from the active layer soils of a permafrost thaw gradient. We found that plant productivity was significantly affected by the microbial soil inoculants. Plants inoculated with communities from above thawing permafrost showed decreased productivity compared to plants inoculated with microbes from undisturbed soils. We used metagenomic sequencing to determine that microbial communities from disturbed soils above thawing permafrost differ in taxonomy from microbial communities in undisturbed soils above intact permafrost. The combination of these results indicates that a decrease in plant productivity can be linked to soil disturbance driven changes in microbial community membership and abundance. These data contribute to an understanding of how microbial communities can be affected by soil disturbance and climate change, and how those community shifts can further influence plant productivity in Boreal forests and more broadly, ecosystem health.

Unanticipated Geochemical and Microbial Community Structure under Seasonal Ice Cover in a Dilute, Dimictic Arctic Lake.

Despite most lakes in the Arctic being perennially or seasonally frozen for at least 40% of the year, little is known about microbial communities and nutrient cycling under ice cover. We assessed the vertical microbial community distribution and geochemical composition in early spring under ice in a seasonally ice-covered lake in southwest Greenland using amplicon-based sequencing that targeted 16S rRNA genes and using a combination of field and laboratory aqueous geochemical methods. Microbial communities changed consistently with changes in geochemistry. Composition of the abundant members responded strongly to redox conditions, shifting downward from a predominantly heterotrophic aerobic community in the suboxic waters to a heterotrophic anaerobic community in the anoxic waters. Operational taxonomic units (OTUs) of Sporichthyaceae, Comamonadaceae, and the SAR11 Clade had higher relative abundances above the oxycline and OTUs within the genus Methylobacter, the phylum Lentisphaerae, and purple sulfur bacteria (PSB) below the oxycline. Notably, a 13-fold increase in sulfide at the oxycline was reflected in an increase and change in community composition of potential sulfur oxidizers. Purple non-sulfur bacteria were present above the oxycline and green sulfur bacteria and PSB coexisted below the oxycline, however, PSB were most abundant. For the first time we show the importance of PSB as potential sulfur oxidizers in an Arctic dimictic lake.

Phylogenetically Structured Differences in rRNA Gene Sequence Variation among Species of Arbuscular Mycorrhizal Fungi and Their Implications for Sequence Clustering.

UNLABELLED: Arbuscular mycorrhizal (AM) fungi form mutualisms with plant roots that increase plant growth and shape plant communities. Each AM fungal cell contains a large amount of genetic diversity, but it is unclear if this diversity varies across evolutionary lineages. We found that sequence variation in the nuclear large-subunit (LSU) rRNA gene from 29 isolates representing 21 AM fungal species generally assorted into genus- and species-level clades, with the exception of species of the genera Claroideoglomus and Entrophospora However, there were significant differences in the levels of sequence variation across the phylogeny and between genera, indicating that it is an evolutionarily constrained trait in AM fungi. These consistent patterns of sequence variation across both phylogenetic and taxonomic groups pose challenges to interpreting operational taxonomic units (OTUs) as approximations of species-level groups of AM fungi. We demonstrate that the OTUs produced by five sequence clustering methods using 97% or equivalent sequence similarity thresholds failed to match the expected species of AM fungi, although OTUs from AbundantOTU, CD-HIT-OTU, and CROP corresponded better to species than did OTUs from mothur or UPARSE. This lack of OTU-to-species correspondence resulted both from sequences of one species being split into multiple OTUs and from sequences of multiple species being lumped into the same OTU. The OTU richness therefore will not reliably correspond to the AM fungal species richness in environmental samples. Conservatively, this error can overestimate species richness by 4-fold or underestimate richness by one-half, and the direction of this error will depend on the genera represented in the sample. IMPORTANCE: Arbuscular mycorrhizal (AM) fungi form important mutualisms with the roots of most plant species. Individual AM fungi are genetically diverse, but it is unclear whether the level of this diversity differs among evolutionary lineages. We found that the amount of sequence variation in an rRNA gene that is commonly used to identify AM fungal species varied significantly between evolutionary groups that correspond to different genera, with the exception of two genera that are genetically indistinguishable from each other. When we clustered groups of similar sequences into operational taxonomic units (OTUs) using five different clustering methods, these patterns of sequence variation caused the number of OTUs to either over- or underestimate the actual number of AM fungal species, depending on the genus. Our results indicate that OTU-based inferences about AM fungal species composition from environmental sequences can be improved if they take these taxonomically structured patterns of sequence variation into account.

Combined Flux Chamber and Genomics Approach Links Nitrous Acid Emissions to Ammonia Oxidizing Bacteria and Archaea in Urban and Agricultural Soil.

Nitrous acid (HONO) is a photochemical source of hydroxyl radical and nitric oxide in the atmosphere that stems from abiotic and biogenic processes, including the activity of ammonia-oxidizing soil microbes. HONO fluxes were measured from agricultural and urban soil in mesocosm studies aimed at characterizing biogenic sources and linking them to indigenous microbial consortia. Fluxes of HONO from agricultural and urban soil were suppressed by addition of a nitrification inhibitor and enhanced by amendment with ammonium (NH4(+)), with peaks at 19 and 8 ng m(-2) s(-1), respectively. In addition, both agricultural and urban soils were observed to convert (15)NH4(+) to HO(15)NO. Genomic surveys of soil samples revealed that 1.5-6% of total expressed 16S rRNA sequences detected belonged to known ammonia oxidizing bacteria and archaea. Peak fluxes of HONO were directly related to the abundance of ammonia-oxidizer sequences, which in turn depended on soil pH. Peak HONO fluxes under fertilized conditions are comparable in magnitude to fluxes reported during field campaigns. The results suggest that biogenic HONO emissions will be important in soil environments that exhibit high nitrification rates (e.g., agricultural soil) although the widespread occurrence of ammonia oxidizers implies that biogenic HONO emissions are also possible in the urban and remote environment.

Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes.

Maize, genetically modified with the insect toxin genes of Bacillus thuringiensis (Bt), is widely cultivated, yet its impacts on soil organisms are poorly understood. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and may be uniquely sensitive to genetic changes within a plant host. In this field study, the effects of nine different lines of Bt maize and their corresponding non-Bt parental isolines were evaluated on AMF colonization and community diversity in plant roots. Plants were harvested 60 days after sowing, and data were collected on plant growth and per cent AMF colonization of roots. AMF community composition in roots was assessed using 454 pyrosequencing of the 28S rRNA genes, and spatial variation in mycorrhizal communities within replicated experimental field plots was examined. Growth responses, per cent AMF colonization of roots and AMF community diversity in roots did not differ between Bt and non-Bt maize, but root and shoot biomass and per cent colonization by arbuscules varied by maize cultivar. Plot identity had the most significant effect on plant growth, AMF colonization and AMF community composition in roots, indicating spatial heterogeneity in the field. Mycorrhizal fungal communities in maize roots were autocorrelated within approximately 1 m, but at greater distances, AMF community composition of roots differed between plants. Our findings indicate that spatial variation and heterogeneity in the field has a greater effect on the structure of AMF communities than host plant cultivar or modification by Bt toxin genes.

mcaGUI: microbial community analysis R-Graphical User Interface (GUI).

UNLABELLED: Microbial communities have an important role in natural ecosystems and have an impact on animal and human health. Intuitive graphic and analytical tools that can facilitate the study of these communities are in short supply. This article introduces Microbial Community Analysis GUI, a graphical user interface (GUI) for the R-programming language (R Development Core Team, 2010). With this application, researchers can input aligned and clustered sequence data to create custom abundance tables and perform analyses specific to their needs. This GUI provides a flexible modular platform, expandable to include other statistical tools for microbial community analysis in the future. AVAILABILITY: The mcaGUI package and source are freely available as part of Bionconductor at http://www.bioconductor.org/packages/release/bioc/html/mcaGUI.html

Temporal dynamics of the human vaginal microbiota.

Elucidating the factors that impinge on the stability of bacterial communities in the vagina may help in predicting the risk of diseases that affect women's health. Here, we describe the temporal dynamics of the composition of vaginal bacterial communities in 32 reproductive-age women over a 16-week period. The analysis revealed the dynamics of five major classes of bacterial communities and showed that some communities change markedly over short time periods, whereas others are relatively stable. Modeling community stability using new quantitative measures indicates that deviation from stability correlates with time in the menstrual cycle, bacterial community composition, and sexual activity. The women studied are healthy; thus, it appears that neither variation in community composition per se nor higher levels of observed diversity (co-dominance) are necessarily indicative of dysbiosis.

Characterization of the diversity and temporal stability of bacterial communities in human milk.

Recent investigations have demonstrated that human milk contains a variety of bacterial genera; however, as of yet very little work has been done to characterize the full diversity of these milk bacterial communities and their relative stability over time. To more thoroughly investigate the human milk microbiome, we utilized microbial identification techniques based on pyrosequencing of the 16S ribosomal RNA gene. Specifically, we characterized the bacterial communities present in milk samples collected from 16 women at three time-points over four weeks. Results indicated that milk bacterial communities were generally complex; several genera represented greater than 5% of the relative community abundance, and the community was often, yet not always, stable over time within an individual. These results support the conclusion that human milk, which is recommended as the optimal nutrition source for almost all healthy infants, contains a collection of bacteria more diverse than previously reported. This finding begs the question as to what role this community plays in colonization of the infant gastrointestinal tract and maintaining mammary health.

Bacterial diversity in a glacier foreland of the high Arctic.

Over the past 100 years, Arctic temperatures have increased at almost twice the global average rate. One consequence is the acceleration of glacier retreat, exposing new habitats that are colonized by microorganisms whose diversity and function are unknown. Here, we characterized bacterial diversity along two approximately parallel chronosequences in an Arctic glacier forefield that span six time points following glacier retreat. We assessed changes in phylotype richness, evenness and turnover rate through the analysis of 16S rRNA gene sequences recovered from 52 samples taken from surface layers along the chronosequences. An average of 4500 sequences was obtained from each sample by 454 pyrosequencing. Using parametric methods, it was estimated that bacterial phylotype richness was high, and that it increased significantly from an average of 4000 (at a threshold of 97% sequence similarity) at locations exposed for 5 years to an average of 7050 phylotypes per 0.5 g of soil at sites that had been exposed for 150 years. Phylotype evenness also increased over time, with an evenness of 0.74 for 150 years since glacier retreat reflecting large proportions of rare phylotypes. The bacterial species turnover rate was especially high between sites exposed for 5 and 19 years. The level of bacterial diversity present in this High Arctic glacier foreland was comparable with that found in temperate and tropical soils, raising the question whether global patterns of bacterial species diversity parallel that of plants and animals, which have been found to form a latitudinal gradient and be lower in polar regions compared with the tropics.

The vaginal bacterial communities of Japanese women resemble those of women in other racial groups.

To determine whether different racial groups shared common types of vaginal microbiota, we characterized the composition and structure of vaginal bacterial communities in asymptomatic and apparently healthy Japanese women in Tokyo, Japan, and compared them with those of White and Black women from North America. The composition of vaginal communities was compared based on community profiles of terminal restriction fragments of 16S rRNA genes and phylogenetic analysis of cloned 16S rRNA gene sequences of the numerically dominant bacterial populations. The types of vaginal communities found in Japanese women were similar to those of Black and White women. As with White and Black women, most vaginal communities were dominated by lactobacilli, and only four species of Lactobacillus (Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri) were commonly found. Communities dominated by multiple species of lactobacilli were common in Japanese and White women, but rare in Black women. The incidence, in Japanese women, of vaginal communities with several non-Lactobacillus species at moderately high frequencies was intermediate between Black women and White women. The limited number of community types found among women in different ethnic groups suggests that host genetic factors, including the innate and adaptive immune systems, may be more important in determining the species composition of vaginal bacterial communities than are cultural and behavioral differences.

Bacterial succession in a glacier foreland of the High Arctic.

Succession is defined as changes in biological communities over time. It has been extensively studied in plant communities, but little is known about bacterial succession, in particular in environments such as High Arctic glacier forelands. Bacteria carry out key processes in the development of soil, biogeochemical cycling and facilitating plant colonization. In this study we sampled two roughly parallel chronosequences in the foreland of Midre Lovén glacier on Svalbard, Norway and tested whether any of several factors were associated with changes in the structure of bacterial communities, including time after glacier retreat, horizontal variation caused by the distance between chronosequences and vertical variation at two soil depths. The structures of soil bacterial communities at different locations were compared using terminal restriction fragment length polymorphisms of 16S rRNA genes, and the data were analyzed by sequential analysis of log-linear statistical models. Although no significant differences in community structure were detected between the two chronosequences, statistically significant differences between sampling locations in the surface and mineral soils could be demonstrated even though glacier forelands are patchy and dynamic environments. These findings suggest that bacterial succession occurs in High Arctic glacier forelands but may differ in different soil depths.

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities.

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.