Rare Operational Taxonomic Units Research Articles

Biogas production and microbial community structure in a stable‐stage of a two‐stage anaerobic digester

AbstractIn a pilot plant under the stable‐stage of the two‐stage anaerobic digester, the dynamic of the archaeal, bacterial, and eukaryal community structure was studied as well as their link to performance and biogas production. An effective synergic action between microorganisms was reached since biogas production and particularly methane (CH4) generation, were more than two‐times higher in the methanogenic respect to the acidogenic bioreactor. The α‐ and β‐diversity analyses showed that dominant and rare operational taxonomic units of prokaryotic and eukaryotic microorganisms were critical in developing a functional successful anaerobic digestion process. A canonical correspondence analysis showed that acid/alkalinity ratio, volatile fatty acid, and pH parameters mainly affected prokaryotic and eukaryotic phylotypes and a clear positive relationship was found between several phylotypes, such as Methanobacteriaceae, Unknown Thaumarchaeota, Intestinibacter, Coprothermobacter, and Magnoliophyta, with biogas and CH4 production and performance parameters.

Importance of Species Sorting and Immigration on the Bacterial Assembly of Different-Sized Aggregates in a Full-Scale Aerobic Granular Sludge Plant.

In aerobic granular sludge (AGS) systems, different-sized microbial aggregates having different solids retention time (SRT) coexist in the same reactor compartment and are subjected to the same influent wastewater. Thus, the AGS system provides a unique ecosystem to study the importance of local (species sorting) and regional (immigration) processes in bacterial community assembly. The microbial communities of different-sized aggregates (flocs <0.2 mm, small granules (0.2-1.0 mm) and large granules >1.0 mm), influent wastewater, excess sludge and effluent of a full-scale AGS plant were characterized over a steady-state operation period of 6 months. Amplicon sequencing was integrated with mass balance to determine the SRT and net growth rate of operational taxonomic units (OTUs). We found strong evidence of species sorting as opposed to immigration, which was significantly higher at short SRT (i.e., flocs and small granules) than that at long SRT (large granules). Rare OTUs in wastewater belonging to putative functional groups responsible for nitrogen and phosphorus removal were progressively enriched with an increase in microbial aggregates size. In contrast, fecal- and sewage infrastructure-derived microbes progressively decreased in relative abundance with increase in microbial aggregate size. These findings highlight the importance of AGS as a unique model ecosystem to study fundamental microbial ecology concepts.

Outpatient-to-Inpatient Transition Causes Marked Dysbiosis in Allogeneic Hematopoietic Cell Transplantation Recipients

Features of gut dysbiosis such as low diversity and intestinal domination have been associated with worse outcomes after allogeneic hematopoietic cell transplantation (allo-HCT). Potential sources of insult to the microbiome vary with time. We hypothesized that outpatient-to-inpatient transition has a different impact on the microbiome than later events such as antibiotic pressure and cumulative conditioning toxicity. We performed 16S rRNA gene profiling of 167 stool samples collected thrice weekly from 20 allograft recipients (12 men, 8 women; median [range] age: 52 [22-68] years) between admission (day 1) and 14 days post-HCT. No exclusion criteria were used. Patients were admitted 7-8 days before HCT. None were receiving antibacterial antibiotics at admission. Patients received levofloxacin starting 1 day pre-HCT and cefepime upon neutropenic fever. Conditioning included TBI in all patients (50% myeloablative). The most common underlying diagnosis was acute leukemia (75%). Donors were: HLA-haploidentical relative (40%), HLA-matched sibling (35%), cord blood (20%), and matched unrelated donor (5%). Non-cord graft sources were bone marrow (50%) and peripheral blood (30%). With an estimated Good's coverage of 98.4±0.2%, the 3 most abundant genera (aggregate over time) were Bacteroides (mean relative abundance 27%), Clostridium XIVa (10%), and Parabacteroides (5%). Consistent with previous studies, the remaining 48% of taxa were rare operational taxonomic units (OTUs; each A parsimonious explanation of these findings from a dense sampling protocol and using 3 community-level indices suggests two phases of microbiota injury during the first 3 weeks of hospitalization. In phase 1 (admission to HCT), characterized by outpatient-to-inpatient transition, a large fraction of the microbiome is replaced with new communities. In phase 2 (HCT to engraftment), characterized by antibiotic pressure and conditioning toxicity, microbiome turnover decelerates and the more successful taxa expand. These results identify week 1 of hospitalization as a potential window for microbiota therapeutics to prevent dysbiosis-related adverse outcomes.

Rare taxa of alkaline phosphomonoesterase-harboring microorganisms mediate soil phosphorus mineralization

As a homologous gene encoding microbial alkaline phosphomonoesterase, the expression of phoD is critically controlled by P availability and thus contributes to the mineralization of soil organic P under P-depleted condition. However, its role in the regulation of soil P turnover is largely unknown due to the complex coupling of physiochemical and biological processes in the P cycle, especially in paddy field. We hypothesized that 1) P fertilization would decrease the abundance of phoD gene and change the composition of phoD-harboring microbial community and 2) the high abundance of phoD-harboring microorganisms in P-poor soil would stimulate the synthesis of alkaline phosphomonoesterase, thus mitigating P limitation via the mineralization of organic P. After 42 days of rice growth, the phoD abundance negatively correlated with soil P availability, and it was significantly higher in non-fertilized treatments than in P-fertilized treatments for both rhizosphere and bulk soils. A stronger competition among phoD-harboring microorganisms was detected in non-fertilized soil than in P-fertilized soil, with Bradyrhizobium, Methylobacterium, and Methylomonas being the dominant taxa in all samples. However, the high phoD gene abundance under P-poor condition was mainly due to the growth of rare operational taxonomic units (OTUs) affiliated to Actinobacteria and Cyanobacteria (relative abundance < 3%). Consistent with our hypothesis, the growth of phoD-harboring microorganisms stimulated the hydrolysis of organic P in non-fertilized soil. However, in the P-fertilized treatments, the increase in OTU abundance was accompanied by the depletion of exchangeable P and accumulation of microbial biomass P. Our findings suggest that phoD-harboring microorganisms have the potential to immobilize P in biomass when the supply is sufficient while mineralize organic P under P-poor condition, during which the rare taxa play an important role.

Evaluation of plant contamination in metabarcoding diet analysis of a herbivore

Fecal DNA metabarcoding is currently used in various fields of ecology to determine animal diets. Contamination of non-food DNA from complex field environments is a considerable challenge to the reliability of this method but has rarely been quantified. We evaluated plant DNA contamination by sequencing the chloroplast trnL P6 loop region from food-controlled geese feces. The average percentage of contaminant sequences per sample was 1.86%. According to the results of generalized linear models, the probability of contamination was highest in samples placed in wet soil. The proportion of contaminant sequences was lowest at the earliest sampling point and was slightly higher in samples placed in open conditions. Exclusion of rare OTUs (operational taxonomic units) was effective for obtaining reliable dietary data from the obtained sequences, and a 1% cutoff reduced the percentage of contaminated samples to less than 30%. However, appropriate interpretation of the barcoding results considering inevitable contamination is an important issue to address. We suggest the following procedures for fecal sampling and sequence data treatment to increase the reliability of DNA metabarcoding diet analyses: (i) Collect samples as soon as possible after deposition, (ii) avoid samples from deposits on wet soil, and (iii) exclude rare OTUs from diet composition estimations.

Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event.

The Pacific oyster, Crassostrea gigas, is a key commercial species that is cultivated globally. In recent years, disease outbreaks have heavily impacted C. gigas stocks worldwide, with many losses incurred during summer. A number of infectious agents have been associated with these summer mortality events, including viruses (particularly Ostreid herpesvirus 1, OsHV-1) and bacteria; however, cases where no known aetiological agent can be identified are common. In this study, we examined the microbiome of disease-affected and disease-unaffected C. gigas during a 2013-2014 summer mortality event in Port Stephens (Australia) where known oyster pathogens including OsHV-1 were not detected. The adductor muscle microbiomes of 70 C. gigas samples across 12 study sites in the Port Stephens estuary were characterised using 16S rRNA (V1-V3 region) amplicon sequencing, with the aim of comparing the influence of spatial location and disease state on the oyster microbiome. Spatial location was found to be a significant determinant of the disease-affected oyster microbiome. Furthermore, microbiome comparisons between disease states identified a significant increase in rare operational taxonomic units (OTUs) belonging to Vibrio harveyi and an unidentified member of the Vibrio genus in the disease-affected microbiome. This is indicative of a potential role of Vibrio species in oyster disease and supportive of previous culture-based examination of this mortality event.

Biphenyl-Metabolizing Microbial Community and a Functional Operon Revealed in E-Waste-Contaminated Soil.

Primitive electronic waste (e-waste) recycling activities release massive amounts of persistent organic pollutants (POPs) and heavy metals into surrounding soils, posing a major threat to the ecosystem and human health. Microbes capable of metabolizing POPs play important roles in POPs remediation in soils, but their phylotypes and functions remain unclear. Polychlorinated biphenyls (PCBs), one of the main pollutants in e-waste contaminated soils, have drawn increasing attention due to their high persistence, toxicity, and bioaccumulation. In the present study, we employed the culture-independent method of DNA stable-isotope probing to identify active biphenyl and PCB degraders in e-waste-contaminated soil. A total of 19 rare operational taxonomic units and three dominant bacterial genera ( Ralstonia, Cupriavidus, and uncultured bacterium DA101) were enriched in the 13C heavy DNA fraction, confirming their functions in PCBs metabolism. Additionally, a 13.8 kb bph operon was amplified, containing a bphA gene labeled by 13C that was concentrated in the heavy DNA fraction. The tetranucleotide signature characteristics of the bph operon suggest that it originated from Ralstonia. The bph operon may be shared by horizontal gene transfer because it contains a transposon gene and is found in various bacterial species. This study gives us a deeper understanding of PCB-degrading mechanisms and provides a potential resource for the bioremediation of PCBs-contaminated soils.

Sequence clustering threshold has little effect on the recovery of microbial community structure.

Analysis of microbial community structure by multivariate ordination methods, using data obtained by high-throughput sequencing of amplified markers (i.e., DNA metabarcoding), often requires clustering of DNA sequences into operational taxonomic units (OTUs). Parameters for the clustering procedure tend not to be justified but are set by tradition rather than being based on explicit knowledge. In this study, we explore the extent to which ordination results are affected by variation in parameter settings for the clustering procedure. Amplicon sequence data from nine microbial community studies, representing different sampling designs, spatial scales and ecosystems, were subjected to clustering into OTUs at seven different similarity thresholds (clustering thresholds) ranging from 87% to 99% sequence similarity. The 63 data sets thus obtained were subjected to parallel DCA and GNMDS ordinations. The resulting community structures were highly similar across all clustering thresholds. We explain this pattern by the existence of strong ecological structuring gradients and phylogenetically diverse sets of abundant OTUs that are highly stable across clustering thresholds. Removing low-abundance, rare OTUs had negligible effects on community patterns. Our results indicate that microbial data sets with a clear gradient structure are highly robust to choice of sequence clustering threshold.

Bacterioplankton composition in tropical high-elevation lakes of the Andean plateau.

High-elevation lakes in the tropics are subject to extreme environmental fluctuations and microbes may harbor a unique genomic repertoire, but their composition and diversity are largely unknown. Here, we compared the planktonic bacterial community composition (BCC) and diversity of three tropical lakes located in the high Andean plateau (≥4400 m above sea level) during the dry and wet season. Diversity in these lakes was higher in the cool and wet season than in the warm and dry one. Operational taxonomic units (OTUs) composition was significantly different among lakes and between seasons. Members of the class Opitutae, Spartobacteria, Burkholderiales and Actinobacteria were dominant, but only the hgcI clade (Actinobacteria) and the Comamonadaceae family (Burkholderiales) were shared between seasons among the three lakes. In general, a large percentage (up to 42%) of the rare OTUs was unclassified even at the family level. In one lake, a pycnocline and an anoxic water layer with high abundance of Thiocapsa sp. was found in the wet season indicating that the known polymictic thermal condition is not always given. Our study highlights the particular BCC of tropical high-elevation lakes and also how little is known about the variability in physico-chemical conditions of these ecosystems.

DNA Template Dilution Impacts Amplicon Sequencing-Based Estimates of Soil Fungal Diversity

Next generation sequencing of taxonomically relevant marker genes has enabled researchers to sample the richness, diversity, and composition of environmental microbiomes at previously unattainable depths. However, molecular methods may have unintended downstream consequences and the inadvertent undersampling of microbial communities may be a significant pitfall in microbiome profiling. One such procedure, dilution of the DNA template prior to polymerase chain reaction (PCR), may improve marker gene amplification by reducing chimeric read formation and decreasing PCR inhibitor concentrations. However, dilution unavoidably reduces target DNA template number per sample. We evaluated the effects of pre-PCR DNA template dilution on estimates of soil fungal microbiome diversity, composition, and species abundance distributions across a collection of 144 agricultural soil samples. Fungal DNA templates were serially diluted at 0-, 10-, 100-, and 1,000-fold and sequence data of diluted templates were compared with those of an identical set of undiluted templates. For three prairie soil samples, in addition to evaluating variation among replicates of individual samples, we serially diluted fungal DNA extracts from soil samples in triplicate and sequenced undiluted and diluted samples. DNA template dilution significantly reduced estimates of fungal richness and diversity, as compared with undiluted samples. Dilution of DNA template also resulted in reduced relative abundances of rare operational taxonomic units (OTUs) and increased relative abundances of common OTUs. Collectively, changes in OTU abundance distributions following dilution produced substantial shifts in overall fungal community composition. Our results highlight risks associated with sample dilution and point to the potential utility of quantifying pre-PCR template concentration in the estimation of microbiomes. We urge researchers to thoroughly document methods and to reconsider routine dilution of pre-PCR DNA templates particularly for low abundance microbiome samples. As efforts to profile environmental microbiomes using molecular sequencing approaches accelerate, developing an adequate understanding of potential methodological bottlenecks will increase our ability to accurately characterize and compare datasets.

Deep ocean prokaryotic communities are remarkably malleable when facing long-term starvation.

The bathypelagic ocean is one of the largest ecosystems on Earth and sustains half of the ocean's microbial activity. This microbial activity strongly relies on surface-derived particles, but there is growing evidence that the carbon released through solubilization of these particles may not be sufficient to meet the energy demands of deep ocean prokaryotes. To explore how bathypelagic prokaryotes respond to the absence of external inputs of carbon, we followed the long-term (1year) dynamics of an enclosed community. Despite the lack of external energy supply, we observed a continuous succession of active prokaryotic phylotypes, which was driven by recruitment of taxa from the seed bank (i.e., initially rare operational taxonomic units [OTUs]). A single OTU belonging to Marine Group I of Thaumarchaeota, which was originally rare, dominated the microbial community for ∼4months and played a fundamental role in this succession likely by introducing new organic carbon through chemolithoautotrophy. This carbon presumably produced a priming effect, because after the decline of Thaumarchaeota, the diversity and metabolic potential of the community increased back to the levels present at the start of the experiment. Our study demonstrates the profound versatility of deep microbial communities when facing organic carbon deprivation.

Microbial communities in peatlands along a chronosequence on the Sanjiang Plain, China

Microbial communities play crucial roles in the global carbon cycle, particularly in peatland ecosystems under climate change. The peatlands of the Sanjiang Plain could be highly vulnerable to global warming because they are mainly located at the southern limit of northern peatlands. In this study, the alpha diversity and composition of bacterial communities in three different minerotrophic fens along a chronosequence were investigated. We captured a rich microbial community that included many rare operational taxonomic units (OTUs) but was dominated by a few bacterial classes that have frequently been detected in other peatland ecosystems. Notably, a large diversity of methanotrophs affiliated with Alpha- and Gammaproteobacteria was also detected. Bacterial alpha diversity and composition varied as a function of peat depth and its associated physical-chemical properties, such as total carbon, total nitrogen, pH and bulk density. We also found that bacterial community turnover (beta diversity) to be significantly correlated with soil age, whereas bacterial alpha diversity was not.

Membrane biofilm communities in full-scale membrane bioreactors are not randomly assembled and consist of a core microbiome

Finding efficient biofouling control strategies requires a better understanding of the microbial ecology of membrane biofilm communities in membrane bioreactors (MBRs). Studies that characterized the membrane biofilm communities in lab-and pilot-scale MBRs are numerous, yet similar studies in full-scale MBRs are limited. Also, most of these studies have characterized the mature biofilm communities with very few studies addressing early biofilm communities. In this study, five full-scale MBRs located in Seattle (Washington, U.S.A.) were selected to address two questions concerning membrane biofilm communities (early and mature): (i) Is the assembly of biofilm communities (early and mature) the result of random immigration of species from the source community (i.e. activated sludge)? and (ii) Is there a core membrane biofilm community in full-scale MBRs? Membrane biofilm (early and mature) and activated sludge (AS) samples were collected from the five MBRs, and 16S rRNA gene sequencing was applied to investigate the bacterial communities of AS and membrane biofilms (early and mature). Alpha and beta diversity measures revealed clear differences in the bacterial community structure between the AS and biofilm (early and mature) samples in the five full-scale MBRs. These differences were mainly due to the presence of large number of unique but rare operational taxonomic units (∼13% of total reads in each MBR) in each sample. In contrast, a high percentage (∼87% of total reads in each MBR) of sequence reads was shared between AS and biofilm samples in each MBR, and these shared sequence reads mainly belong to the dominant taxa in these samples. Despite the large fraction of shared sequence reads between AS and biofilm samples, simulated biofilm communities from random sampling of the respective AS community revealed that biofilm communities differed significantly from the random assemblages (P < 0.001 for each MBR), indicating that the biofilm communities (early and mature) are unlikely to represent a random sample of the AS community. In addition to the presence of unique operational taxonomic units in each biofilm sample (early or mature), comparative analysis of operational taxonomic units and genera revealed the presence of a core biofilm community in the five full-scale MBRs. These findings provided insight into the membrane biofilm communities in full-scale MBRs. More comparative studies are needed in the future to elucidate the factors shaping the core and unique biofilm communities in full-scale MBRs.

Exploring the Ecological Coherence between the Spatial and Temporal Patterns of Bacterioplankton in Boreal Lakes.

One of the major contemporary challenges in microbial ecology has been to discriminate the reactive core from the random, unreactive components of bacterial communities. In previous work we used the spatial abundance distributions of bacterioplankton across boreal lakes of Québec to group taxa into four distinct categories that reflect either hydrology-mediated dispersal along the aquatic network or environmental selection mechanisms within lakes. Here, we test whether this categorization derived from the spatial distribution of taxa is maintained over time, by analyzing the temporal dynamics of the operational taxonomic units (OTUs) within those spatially derived categories along an annual cycle in the oligotrophic lake Croche (Québec, Canada), and assessing the coherence in the patterns of abundance, occurrence, and environmental range of these OTUs over space and time. We report that the temporal dynamics of most taxa within a single lake are largely coherent with those derived from their spatial distribution over large spatial scales, suggesting that these properties must be intrinsic of particular taxa. We also identified a set of rare taxa cataloged as having a random occupancy based on their spatial distribution, but which showed clear seasonality and abundance peaks along the year, yet these comprised a very small fraction of the total rare OTUs. We conclude that the presence of most rare bacterioplankton taxa in boreal lakes is random, since both their temporal and spatial dynamics suggest links to passive downstream transport and persistence in freshwater networks, rather than environmental selection.

Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding

DNA metabarcoding, the PCR-based profiling of natural communities, is becoming the method of choice for biodiversity monitoring because it circumvents some of the limitations inherent to traditional ecological surveys. However, potential sources of bias that can affect the reproducibility of this method remain to be quantified. The interpretation of differences in patterns of sequence abundance and the ecological relevance of rare sequences remain particularly uncertain. Here we used one artificial mock community to explore the significance of abundance patterns and disentangle the effects of two potential biases on data reproducibility: indexed PCR primers and random sampling during Illumina MiSeq sequencing. We amplified a short fragment of the mitochondrial Cytochrome c Oxidase Subunit I (COI) for a single mock sample containing equimolar amounts of total genomic DNA from 34 marine invertebrates belonging to six phyla. We used seven indexed broad-range primers and sequenced the resulting library on two consecutive Illumina MiSeq runs. The total number of Operational Taxonomic Units (OTUs) was ∼4 times higher than expected based on the composition of the mock sample. Moreover, the total number of reads for the 34 components of the mock sample differed by up to three orders of magnitude. However, 79 out of 86 of the unexpected OTUs were represented by <10 sequences that did not appear consistently across replicates. Our data suggest that random sampling of rare OTUs (e.g., small associated fauna such as parasites) accounted for most of variation in OTU presence–absence, whereas biases associated with indexed PCRs accounted for a larger amount of variation in relative abundance patterns. These results suggest that random sampling during sequencing leads to the low reproducibility of rare OTUs. We suggest that the strategy for handling rare OTUs should depend on the objectives of the study. Systematic removal of rare OTUs may avoid inflating diversity based on common β descriptors but will exclude positive records of taxa that are functionally important. Our results further reinforce the need for technical replicates (parallel PCR and sequencing from the same sample) in metabarcoding experimental designs. Data reproducibility should be determined empirically as it will depend upon the sequencing depth, the type of sample, the sequence analysis pipeline, and the number of replicates. Moreover, estimating relative biomasses or abundances based on read counts remains elusive at the OTU level.

Geographical variation in soil bacterial community structure in tropical forests in Southeast Asia and temperate forests in Japan based on pyrosequencing analysis of 16S rRNA.

Geographical variation in soil bacterial community structure in 26 tropical forests in Southeast Asia (Malaysia, Indonesia and Singapore) and two temperate forests in Japan was investigated to elucidate the environmental factors and mechanisms that influence biogeography of soil bacterial diversity and composition. Despite substantial environmental differences, bacterial phyla were represented in similar proportions, with Acidobacteria and Proteobacteria the dominant phyla in all forests except one mangrove forest in Sarawak, although highly significant heterogeneity in frequency of individual phyla was detected among forests. In contrast, species diversity (α-diversity) differed to a much greater extent, being nearly six-fold higher in the mangrove forest (Chao1 index = 6,862) than in forests in Singapore and Sarawak (~1,250). In addition, natural mixed dipterocarp forests had lower species diversity than acacia and oil palm plantations, indicating that aboveground tree composition does not influence soil bacterial diversity. Shannon and Chao1 indices were correlated positively, implying that skewed operational taxonomic unit (OTU) distribution was associated with the abundance of overall and rare (singleton) OTUs. No OTUs were represented in all 28 forests, and forest-specific OTUs accounted for over 70% of all detected OTUs. Forests that were geographically adjacent and/or of the same forest type had similar bacterial species composition, and a positive correlation was detected between species divergence (β-diversity) and direct distance between forests. Both α- and β-diversities were correlated with soil pH. These results suggest that soil bacterial communities in different forests evolve largely independently of each other and that soil bacterial communities adapt to their local environment, modulated by bacterial dispersal (distance effect) and forest type. Therefore, we conclude that the biogeography of soil bacteria communities described here is non-random, reflecting the influences of contemporary environmental factors and evolutionary history.

Microbial eukaryote communities exhibit robust biogeographical patterns along a gradient of Patagonian and Antarctic lakes.

Microbial eukaryotes play important roles in aquatic ecosystem functioning. Unravelling their distribution patterns and biogeography provides important baseline information to infer the underlying mechanisms that regulate the biodiversity and complexity of ecosystems. We studied the distribution patterns and factors driving diversity gradients in microeukaryote communities (total, abundant, uncommon and rare community composition) along a latitudinal gradient of lakes distributed from Argentinean Patagonia to Maritime Antarctica using both denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (Illumina HiSeq). DGGE and abundant Illumina operational taxonomic units (OTUs) showed both decreasing richness with latitude and significant differences between Patagonian and Antarctic lakes communities. In contrast, total richness did not change significantly across the latitudinal gradient, although evenness and diversity indices were significantly higher in Patagonian lakes. Beta-diversity was characterized by a high species turnover, influenced by both environmental and geographical descriptors, although this pattern faded in the rare community. Our results suggest the co-existence of a 'core biosphere' containing reduced number of abundant/dominant OTUs on which classical ecological rules apply, together with a much larger seedbank of rare OTUs driven by stochastic and reduced dispersal processes. These findings shed new light on the biogeographical patterns and forces structuring inland microeukaryote composition across broad spatial scales.

Bacterial community structure and function shift across a northern boreal forest fire chronosequence.

Soil microbial responses to fire are likely to change over the course of forest recovery. Investigations on long-term changes in bacterial dynamics following fire are rare. We characterized the soil bacterial communities across three different times post fire in a 2 to 152-year fire chronosequence by Illumina MiSeq sequencing, coupled with a functional gene array (GeoChip). The results showed that the bacterial diversity did not differ between the recently and older burned areas, suggesting a concomitant recovery in the bacterial diversity after fire. The differences in bacterial communities over time were mainly driven by the rare operational taxonomic units (OTUs < 0.1%). Proteobacteria (39%), Acidobacteria (34%) and Actinobacteria (17%) were the most abundant phyla across all sites. Genes involved in C and N cycling pathways were present in all sites showing high redundancy in the gene profiles. However, hierarchical cluster analysis using gene signal intensity revealed that the sites with different fire histories formed separate clusters, suggesting potential differences in maintaining essential biogeochemical soil processes. Soil temperature, pH and water contents were the most important factors in shaping the bacterial community structures and function. This study provides functional insight on the impact of fire disturbance on soil bacterial community.

Status of the Archaeal and Bacterial Census: an Update.

ABSTRACTA census is typically carried out for people across a range of geographical levels; however, microbial ecologists have implemented a molecular census of bacteria and archaea by sequencing their 16S rRNA genes. We assessed how well the census of full-length 16S rRNA gene sequences is proceeding in the context of recent advances in high-throughput sequencing technologies because full-length sequences are typically used as references for classification of the short sequences generated by newer technologies. Among the 1,411,234 and 53,546 full-length bacterial and archaeal sequences, 94.5% and 95.1% of the bacterial and archaeal sequences, respectively, belonged to operational taxonomic units (OTUs) that have been observed more than once. Although these metrics suggest that the census is approaching completion, 29.2% of the bacterial and 38.5% of the archaeal OTUs have been observed more than once. Thus, there is still considerable diversity to be explored. Unfortunately, the rate of new full-length sequences has been declining, and new sequences are primarily being deposited by a small number of studies. Furthermore, sequences from soil and aquatic environments, which are known to be rich in bacterial diversity, represent only 7.8 and 16.5% of the census, while sequences associated with host-associated environments represent 55.0% of the census. Continued use of traditional approaches and new technologies such as single-cell genomics and short-read assembly are likely to improve our ability to sample rare OTUs if it is possible to overcome this sampling bias. The success of ongoing efforts to use short-read sequencing to characterize archaeal and bacterial communities requires that researchers strive to expand the depth and breadth of this census.

Structure of phyllosphere fungal communities in a tropical dipterocarp plantation: A massively parallel next-generation sequencing analysis

Phyllosphere fungi show high species diversity and fulfill important ecological functions not only in natural forests but also in plantations. We sought to estimate the species diversity and community structure of phyllosphere fungi from tropical tree plantation plots in Central Kalimantan, Indonesia. We conducted a massively parallel amplicon sequencing analysis of fungi collected from the leaves of Shorea leprosula (Dipterocarpaceae), an ecologically and commercially important tree species. Phyllosphere fungal compositions and spatial variability were investigated for 31 S. leprosula trees across four plots within a plantation stand. In total, 488 fungal operational taxonomic units (OTUs) were recognized in 153,194 ribosomal internal transcribed spacer reads at 95% OTU identity level. Rare OTUs accounted for the majority of fungal diversity detected in the study site; 200 OTUs (41%) comprised fewer than 10 reads and 465 OTUs (95%) were found in fewer than half of the samples. Fungal OTU compositions of S. leprosula trees were differentiated within a narrow area of the plantation and even between plots that were separated by 15 m. These findings indicate that highly diverse fungal OTUs form spatially structured communities even within a tropical plantation stand of single tree species.