ABSTRACTPermafrost microbial research has flourished in the past decades, due in part to improvements in sampling and molecular techniques, but also the increased focus on the permafrost greenhouse gas feedback to climate change and other ecological processes in high latitude and alpine permafrost soils. Permafrost microorganisms are adapted to these extreme environments and remain active at low temperatures and when resources are limited. They are also an important component of global elemental cycles as they regulate organic matter turnover and greenhouse gas production, particularly as permafrost thaws. Here we review the permafrost microbiology literature coupled with an exploration of its historical aspects, with a particular focus on a new understanding advanced by molecular biology techniques. We further identify knowledge gaps and ways forward to improve our understanding of microbial contributions to ecosystem biogeochemistry of permafrost‐affected systems.

Climate change is inducing wide-scale permafrost thaw in the Arctic and subarctic, triggering concerns that long-dormant pathogens could reemerge from the thawing ground and initiate epidemics or pandemics. Viruses, as opposed to bacterial pathogens, garner particular interest because outbreaks cannot be controlled with antibiotics, though the effects can be mitigated by vaccines and newer antiviral drugs. To evaluate the potential hazards posed by viral pathogens emerging from thawing permafrost, we review information from a diverse range of disciplines. This includes efforts to recover infectious virus from human remains, studies on disease occurrence in polar animal populations, investigations into viral persistence and infectivity in permafrost, and assessments of human exposure to the enormous viral diversity present in the environment. Based on currently available knowledge, we conclude that the risk posed by viruses from thawing permafrost is no greater than viruses in other environments such as temperate soils and aquatic systems.

Neodothiora populina is a black yeast-like fungus in the family of Dothioraceae. It causes an aggressive canker disease of trembling aspen that results in widespread mortality of aspen across the boreal forest of Interior Alaska. Here we report a high-quality draft genome including functional annotation of this emerging fungal pathogen based on Nanopore Technology longread sequences. Our initial genome assembly totaled 23,960,169 bp and contained 18 contigs and we identified 7,343 genes. This resource announcement provides new genomic data useful long-term to improve our understanding of forest health in Alaska.

ABSTRACTArbuscular mycorrhizal fungi (AMF, phylum Glomeromycota) are essential to plant community diversity and ecosystem functioning. However, increasing human land use represents a major threat to native AMF globally. Characterizing the loss of AMF diversity remains challenging because many taxa are undescribed, resulting in poor documentation of their biogeography and family‐level disturbance sensitivity. We survey sites representing native and human‐altered ecosystems across the American continents—in Alaska, Kansas, and Brazil—to shed light on these gaps. Using a recently developed pipeline for phylogenetic placement of eDNA, we find evidence for three putative novel clades within the Glomeromycota, sister to Entrophosporaceae, Glomeraceae, and Archaeosporaceae, with evidence for geographic structuring. We further find that taxa in the Diversisporaceae, Glomeraceae, and Entrophosporaceae relatively high families are overrepresented and more diverse in temperate samples. By contrast, the diversity of taxa that cannot be placed into a family is higher in tropical samples, suggesting that tropical sites harbor relatively high undescribed AMF diversity. Moreover, we find evidence that Entrophosporaceae is more tolerant, while Glomeraceae is more sensitive to disturbance. These results underscore the vast undescribed diversity of AMF while highlighting a way forward to systematically improve our understanding of AMF biogeography and response to human disturbance.

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 and nitrogen 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 or nitrogen gas, 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 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.

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.

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.

Lakes are currently responsible for a significant amount of total natural methane emission. Microbial oxidation of methane plays a central role in Arctic carbon cycling, potentially reducing methane emissions from lakes, though little is known about methane cycling in the water column of Arctic lakes. We previously detected surprisingly large enrichments of heavy carbon and hydrogen isotopes of methane in three small lakes in Greenland suggesting unusually efficient methanotrophic communities in these Arctic lakes. Using stable isotope and 16S rRNA gene sequencing we determined carbon and hydrogen isotopes and microbial community composition down the water column of Teardrop lake, under open-water conditions. We found that isotopic values of methane in Teardrop lake were again highly enriched 13C and 2H at 4 m depth with −13.2‰ and −27.1‰ values for carbon and hydrogen isotopes, respectively. Methane concentrations slightly increased at the depth interval with isotope enrichment, not typical of classic methanotrophy. Consistent with isotopic enrichment of the heavy isotopes we detected the highest relative abundance of putative methanotrophs, in particular Methylovulum at 4 m. The highest relative abundance of putative methanogens was detected at 3 m as well as at 5 m. At the same depth interval, temperature and oxidation reduction potential also increase, supporting increased microbial activity within the water column. Based on geochemical and microbial observations, we suggest that the methane cycling in Teardrop lake is decoupled from a traditional depth dependent model where the dominant source of methane is in the anoxic sediments. Instead, methane in the water column is likely from a combination of anoxic sediment, littoral transport and oxic methanogenesis in the mid-water column, and recycling of carbon within the water column is leading to extreme isotope enrichments. Thus, understanding linkages between depth-dependent microbial dynamics and methane biogeochemistry are necessary to constrain the sensitivity of the methane cycle to future climate change.

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.

Arbuscular mycorrhizal fungi (AMF – phylum Glomeromycota) form symbioses with most plant species worldwide and play critical roles in plant nutrient and water uptake, pathogen resistance and soil aggregation (Smith & Read, 2008; Delavaux et al., 2017; Brundrett & Tedersoo, 2018). Because AMF community composition influences ecological function (van der Heijden et al., 1998; Vogelsang et al., 2006; Koziol et al., 2018), understanding patterns of AMF composition is a research priority. Hyphae of AMF species are not morphologically distinguishable, and therefore quantification of AMF species diversity and community composition has increasingly relied on metabarcoding of ribosomal RNA (rRNA) gene sequences from field samples (Öpik et al., 2014). However, to date, no single region of the rRNA gene has been universally accepted as optimal for AMF environmental sequencing. The internal transcribed spacer (ITS) region of the rRNA gene has been suggested as the universal fungal marker (Schoch et al., 2012; Lindahl et al., 2013) and has been used for AMF biogeographical studies (Tedersoo et al., 2014) and environmental sequencing (Öpik et al., 2014). However, this region is suboptimal as a marker gene for AMF (Stockinger et al., 2010; Schoch et al., 2012). The sequence matching approach used for ITS sequences with other fungi is of limited utility for AMF because of the poor representation and poor curation of AMF sequences in ITS sequence databases (Bidartondo, 2008; Stockinger et al., 2010). This database problem cannot be easily rectified because a high proportion of AMF encountered in environmental samples are undescribed. While phylogenetic approaches can be used to identify new sequences as AMF, this approach cannot be used for ITS amplicons because its rapid sequence evolution (Nilsson et al., 2008) does not generate reliable trees. The most commonly used region of the rRNA gene for environmental sequencing of AMF is the small subunit, or SSU (Öpik et al., 2014). The utility of this region is enhanced by a well-developed and curated database for AMF (Öpik et al., 2010; Davison et al., 2015). However, the SSU region has the disadvantage of being slow-evolving and therefore not sufficiently variable to adequately resolve AMF species (Krüger et al., 2009; Bruns & Taylor, 2016; Schlaeppi et al., 2016). By contrast, the large subunit (LSU) region consistently shows greater utility for taxonomic resolution for AMF (Krüger et al., 2012; Hart et al., 2015; House et al., 2016), making it potentially more useful in environmental AMF sequencing. Thus far, the LSU region has rarely been used in environmental sequencing of AMF (Gollotte et al., 2004; Lekberg et al., 2013; House & Bever, 2018; Vieira et al., 2018; Schütte et al., 2019), perhaps because of bioinformatical challenges in implementation. Here, we aim to expand the utility and ease the adoption of the LSU for amplicon sequencing of AMF by providing a well-curated LSU reference database, a reference backbone tree for phylogenetic placement and a computational pipeline easily implemented using current bioinformatical tools. We present a current curated database and reference tree using AMF sequences from several sources: a subset of sequences published by Krüger et al. (2012) available on the National Center for Biotechnology Information (NCBI), a database of unpublished spore-derived sequences from the International Culture Collection of (Vesicular) and Arbuscular Mycorrhizal Fungi (INVAM, Morgantown, WV, USA) and additional recently described sequences from NCBI. Sequences amplified by Krüger et al. (2012) generally used primers SSUmAf and LSUmAr, with a second amplification round with SSUmCf and LSUmBr or SSU-Glom1-NDL22, while sequences amplified by INVAM used primers 1TS1 and NDL22, followed by a second amplification round using primers LR1 and NDL22 (Morton & Msiska, 2010); additional primers used in recently described sequences from NCBI can be found in each respective publication associated with the accession numbers detailed in Fig. 1. To build the reference backbone tree, representative sequences were chosen to maintain the tree structure, conserving clear clades within the tree (using the Interactive Tree of Life to view the tree; Letunic & Bork, 2019), but were kept at a minimum, to make the use of the tree as a reference computationally feasible. These sequences were aligned using Mafft (Katoh & Standley, 2013) and a tree constructed using Raxml v.8 (Stamatakis, 2014) with 1000 bootstrap replicates and the evolutionary model Gtrgamma in Qiime2 (https://qiime2.org). Outgroups were Mortierella elongata (MH047197, Mucoromycota), Exophiala spinifera (MH876260; Basidiomycota) and Rhodotorula hordea (AY631901; Ascomycota). In addition, we included LSU sequences of a plant, Citrus limon (X05910, Rutaceae), and an animal, Rutilus rutilus (EF417167, Cyprinidae). We use the reference database as the reference for open (closed and then de novo) operational taxonomic unit (OTU) clustering and use a phylogenetic tree generated from these same sequences (Fig. 1; Supporting Information Fig. S1) as a backbone constraint in constructing phylogenies to place study sequences. All previously unreported reference sequences have been uploaded to Genbank (MT832155–MT832238). We present a pipeline starting from raw Illumina LSU sequences (MiSeq V3, 2x300 bp) and ending with an OTU table of phylogenetically defined putative AMF OTUs for downstream analyses (Fig. 2). This pipeline is built for use with a high-performance computing (HPC) cluster using a Simple Linux Utility for Resource Management (SLURM) workload manager. Our pipeline covers key bioinformatical steps, including primer removal, quality control, and OTU clustering (Vsearch algorithm (Rognes et al., 2016); for a discussion of why OTUs may be preferable over amplicon sequence variants (ASVs) for this particular application, see Methods S1. Importantly, our pipeline maintains nonoverlapping forward and reverse reads for each sequence, retaining all possible data in long, non-paired reads (700–900 bp; see Table S1 for a forward and reverse read concatenation test). In addition, the pipeline places representative OTU sequences within a tree using our backbone phylogeny and subsequently extracts those that fall within the AMF clade. Operationally, our software handles many batches of OTUs in parallel, thereby greatly improving processing speed. Finally, the output of all batches is joined into a single OTU table containing counts of putative AMF OTUs in each sample, along with a Fasta file containing the representative sequences of all phylogenetically determined putative AMF OTUs. The analogous data files for the OTUs that fall outside of the AMF clade are also provided by this final step. The full bioinformatical pipeline and description can be found in Methods S1; all required files are also supplied (Methods S2; https://github.com/c383d893/AMF-LSU-Database-and-Pipeline). In the age of metagenomics, it is attractive to declare one marker suitable for all fungi (Lekberg et al., 2018). Universal fungal primers would facilitate efforts to compare relative abundances between taxonomic and functional groups and to identify global scale biogeographic patterns. Nonetheless, here we confirm that the general primers targeting the ITS region are not adequate for detecting a majority of undescribed AMF species. We estimate that nearly 90% of phylogenetically defined putative AMF OTUs in our test dataset derived from a Midwestern US grassland are undescribed (i.e. have no Glomeromycota Blast match in NCBI) and c. 30% do not group with described families in our phylogenetic tree (Table S2). Given this severe limitation in building a database and current database constraints (Stockinger et al., 2010; Schoch et al., 2012; Hart et al., 2015), the sequence matching algorithms are not adequate for environmental sequencing of AMF regardless of rRNA gene region. The phylogenetic approach we use here can accommodate undescribed AMF taxa in environmental samples. While the ITS region evolves too quickly to allow reliable tree construction, the LSU region can be used to build a phylogenetic tree and place all study sequences inside or outside of the conserved AMF clade. This allows identification of undescribed putative AMF through clade placement of any environmental sequence. We illustrate this benefit in two studies in which analyses of LSU amplicons reveals significant environmental patterns in AMF composition in US grasslands and boreal forests that were not evident in analyses of ITS amplicons (Methods S3; Table S3). The SSU region, like the LSU region, can be used to build phylogenetic trees and place environmental sequences in the AMF clade (Öpik et al., 2014; Stefani et al., 2020). In addition, the SSU amplicons from commonly used primers have been easier to handle bioinformatically because the small length of c. 500 bp allows for merging of short, paired-end Illumina reads (Lee et al., 2008; Dumbrell et al., 2011). Second, environmental sequences can be directly assigned to virtual taxa in a large publicly available database (Öpik et al., 2010, 2014). However, the SSU region has the disadvantage of being slowly evolving, thereby limiting inferences to taxonomically coarse designations (Krüger et al., 2009; Stockinger et al., 2010; Schoch et al., 2012). Individual virtual taxa assigned using the MaarjAM database include many distinct species and have been suggested to be analogous to genera (Bruns & Taylor, 2016). Analogous problems are present in the LSU in that sequence variation within isolates can be attributed to different OTUs, and individual OTUs can include sequences of different species, particularly for the Claroideoglomeraceae (Stockinger et al., 2010; House et al., 2016). Nonetheless, the LSU does a much better job of capturing other AMF families (Krüger et al., 2012; House et al., 2016) and has been suggested as the most suitable region within short read length restrictions (Stockinger et al., 2010). How these differences translate into inferences on issues such as environmental dependence of AMF distributions or frequency of endemism remains to be evaluated. Here we provide a well-curated LSU reference database, a backbone phylogeny and a computational pipeline that uses these resources to process environmentally derived amplicon sequences. We are optimistic that this set of tools will facilitate molecular work with AMF within the LSU region, leading to finer scale assessments of ecological inferences from AMF community structure. The authors thank the Center for Resource Computing at the University of Kansas as well as RAxML and QIIME2 communities for their support. The authors acknowledge fruitful conversations with Benjamin Sikes about the manuscript pipeline, Bill Wheeler for assistance with INVAM culture meta-data, Rob Ramos for help with initial code development, friendly reviews by Ylva Lekberg and Maarja Öpik, and financial support from the National Science Foundation (DEB-1556664, DEB-1738041, OIA-1656006, IOS-2016351, DEB-0076066). SLS would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a Research Assistantship (Process 307.995/2019-4) and Universidade Regional de Blumenau (FURB) for supporting a sabbatical leave at the University of Kansas. CSD and JDB designed the study. JBM generated the LSU sequences from INVAM isolates. CSD, JDB and SLS led the phylogenetic tree analysis. MRW and CSD developed the bioinformatical pipeline. CSD and JDB led interpretation and manuscript writing, with substantial contributions from all authors (CSD, SLS, MRW, US, JBM, JDB). The unpublished sequences that support the findings of this study are openly available at NCBI at https://www.ncbi.nlm.nih.gov/, accession numbers MT832155–MT832238. Data used to test the pipeline are uploaded under NCBI Project no. PRJNA648993. All other code is presented in Methods S1 and S2 and can be found at https://github.com/c383d893/AMF-LSU-Database-and-Pipeline. Fig. S1 Backbone tree with bootstrap support values. Methods S1 Bioinformatical pipeline. Methods S2 Pipeline scripts and instructions. Methods S3 Comparing ecological inference between ITS database and LSU phylogenetic results. Table S1 Forward and reverse read concatenation test. Table S2 Family classification of OTUs generated from our pipeline with test dataset. Table S3 Comparing ecological inference between ITS database and LSU phylogenetic results. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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 growth 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 growth was significantly affected by the microbial soil inoculants. Plants inoculated with communities from above thawing permafrost showed decreased growth compared to plants inoculated with microbes from undisturbed soils. We used metagenomic sequencing to determine that microbial communities from disturbed soils above thawing permafrost have differences in taxonomy from microbial communities in undisturbed soils above intact permafrost. The combination of these results indicates that a decrease in plant growth 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 growth in Boreal forests and more broadly, ecosystem health.

Permafrost thaw is leading to rapid shifts in boreal ecosystem function. Permafrost thaw affects soil carbon turnover through changes in soil hydrology; however, the biotic mechanisms regulating plant community response remain elusive. Here, we measured the response of fungal community composition and soil nutrient content in an intact permafrost plateau forest soil and an adjacent thermokarst bog and evaluated their potential to mediate shifts in plant composition. We used barcoded amplicon targeting ITS2 and 28S rRNA genes to determine fungal community composition. Next, we used the soils from the permafrost plateau and the thermokarst bog as soil inoculum in a greenhouse experiment to measure whether shifts in fungal community and soil water level regulate plant productivity and composition. Overall, we found that fungal community composition differed significantly between the thawed and intact permafrost sites, but soil nutrient content did not. Relative abundance of mycorrhizal fungal taxa decreased while relative abundance of putative fungal pathogens increased with permafrost thaw. In the greenhouse, we found that ecto‐ and arbuscular‐associated host plants had higher productivity in permafrost‐intact soils relative to thawed soils. However, productivity of non‐mycorrhizal tussock grass was more affected by soil water levels than soil communities. Synthesis. Our results suggest that fungal communities are crucial in mediating plant community response to permafrost thaws inducing hydrology changes.

ABSTRACT 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.

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.

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.

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.

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

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.

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.

Data derived from molecular microbiological investigations of the human vagina have led to the discovery of resident bacterial communities that exhibit marked differences in terms of species composition. All undergo dynamic changes that are likely due to intrinsic host and behavioral factors. Similar types of bacteria have been found in both amniotic fluid and the vagina, suggesting a potential route of colonization. Given that not all of the species involved in intrauterine infections are readily cultivated, it is important that culture-independent methods of analysis must be used to understand the etiology of these infections. Further research is needed to establish whether an ascending pathway from the vagina to the amniotic cavity enables the development of intrauterine infections.