Incorporating functional dissimilarities into sample‐based rarefaction curves: from taxon resampling to functional resampling

Question: Indices of functional diversity have been seen as the key for integrating information on species richness with measures that focus on those components of community composition related to ecosystem functioning. For comparing species richness among habitats on an equal‐effort basis, so‐called sample‐based rarefaction curves may be used. Given a study area that is sampled for species presence and absence in N plots, sample‐based rarefaction generates the expected number of accumulated species as the number of sampled plots increases from 1 to N. Accordingly, the question for this study is: can we construct a ‘functional rarefaction curve’ that summarizes the expected functional dissimilarity between species when n plots are drawn at random from a larger pool of N plots? Methods: In this paper, we propose a parametric measure of functional diversity that is obtained by combining sample‐based rarefaction techniques that are usually applied to species richness with Rao's quadratic diversity. For a given set of N presence/absence plots, the resulting measure summarizes the expected functional dissimilarity at an increasingly larger cumulative number of plots n (n≤N). Results and Conclusions: Due to its parametric nature, the proposed measure is progressively more sensitive to rare species with increasing plot number, thus rendering this measure adequate for comparing the functional diversity of species assemblages that have been sampled with variable effort.     

Comparison of Bacterial and Fungal Communities Between Natural and Planted Pine Forests in Subtropical China

To improve our understanding of the changes in bacterial and fungal diversity in natural pine and planted forests in subtropical region of China, we examined bacterial and fungal communities from a native and a nearby planted pine forest of the Mt. Lushan by constructing clone libraries of 16S and 18S rRNA genes. For bacterial communities, Proteobacteria and Acidobacteria were dominant bacterial taxa in both two types of forest soils. The Shannon–Wiener diversity index, rarefaction curve analysis, and LibShuff analysis suggest that these two forests contained similar diversity of bacterial communities. Low soil acidity (pH ≈ 4) of our study forests might be one of the most important selection factors determining growth of acidophilic Acidobacteria and Proteobacteria. However, the natural forest harbored greater level of fungal diversity than the planted forest according to the Shannon–Wiener diversity index and rarefaction curve analysis. Basidiomycota and Ascomycota were dominant fungal taxa in the soils of natural and planted forests, respectively. Our results suggest that fungal community was more sensitive than the bacterial community in characterizing the differences in plant cover impacts on the microbial flora in the natural and planted forests. The natural and planted forests may function differently due to the differences in soil fungal diversity and relative abundance.     

It's all relative: ranking the diversity of aquatic bacterial communities

The study of microbial diversity patterns is hampered by the enormous diversity of microbial communities and the lack of resources to sample them exhaustively. For many questions about richness and evenness, however, one only needs to know the relative order of diversity among samples rather than total diversity. We used 16S libraries from the Global Ocean Survey to investigate the ability of 10 diversity statistics (including rarefaction, non-parametric, parametric, curve extrapolation and diversity indices) to assess the relative diversity of six aquatic bacterial communities. Overall, we found that the statistics yielded remarkably similar rankings of the samples for a given sequence similarity cut-off. This correspondence, despite the different underlying assumptions of the statistics, suggests that diversity statistics are a useful tool for ranking samples of microbial diversity. In addition, sequence similarity cut-off influenced the diversity ranking of the samples, demonstrating that diversity statistics can also be used to detect differences in phylogenetic structure among microbial communities. Finally, a subsampling analysis suggests that further sequencing from these particular clone libraries would not have substantially changed the richness rankings of the samples.     

A unifying quantitative framework for exploring the multiple facets of microbial biodiversity across diverse scales

Recent developments of molecular tools have revolutionized our knowledge of microbial biodiversity by allowing detailed exploration of its different facets and generating unprecedented amount of data. One key issue with such large datasets is the development of diversity measures that cope with different data outputs and allow comparison of biodiversity across different scales. Diversity has indeed three components: local (α), regional (γ) and the overall difference between local communities (β). Current measures of microbial diversity, derived from several approaches, provide complementary but different views. They only capture the β component of diversity, compare communities in a pairwise way, consider all species as equivalent or lack a mathematically explicit relationship among the α, β and γ components. We propose a unified quantitative framework based on the Rao quadratic entropy, to obtain an additive decomposition of diversity (γ = α + β), so the three components can be compared, and that integrate the relationship (phylogenetic or functional) among Microbial Diversity Units that compose a microbial community. We show how this framework is adapted to all types of molecular data, and we highlight crucial issues in microbial ecology that would benefit from this framework and propose ready‐to‐use R‐functions to easily set up our approach.     

Microbial diversity and biogeochemistry of the Guaymas Basin deep‐sea hydrothermal plume

Hydrothermal plumes are hot spots of microbial biogeochemistry in the deep ocean, yet little is known about the diversity or ecology of microorganisms inhabiting plumes. Recent biogeochemical evidence shows that Mn(II) oxidation in the Guaymas Basin (GB) hydrothermal plume is microbially mediated and suggests that the plume microbial community is distinct from deep‐sea communities. Here we use a molecular approach to compare microbial diversity in the GB plume and in background deep seawater communities, and cultivation to identify Mn(II)‐oxidizing bacteria from plumes and sediments. Despite dramatic differences in Mn(II) oxidation rates between plumes and background seawater, microbial diversity and membership were remarkably similar. All bacterial clone libraries were dominated by Gammaproteobacteria and archaeal clone libraries were dominated by Crenarchaeota. Two lineages, both phylogenetically related to methanotrophs and/or methylotrophs, were consistently over‐represented in the plume. Eight Mn(II)‐oxidizing bacteria were isolated, but none of these or previously identified Mn(II) oxidizers were abundant in clone libraries. Taken together with Mn(II) oxidation rates measured in laboratory cultures and in the field, these results suggest that Mn(II) oxidation in the GB hydrothermal plume is mediated by genome‐level dynamics (gene content and/or expression) of microorganisms that are indigenous and abundant in the deep sea but have yet to be unidentified as Mn(II) oxidizers.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Vector‐borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito‐associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next‐generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human‐impacted landscapes that may ultimately affect vector‐borne disease risk.     

Long‐term nitrogen fertilization decreases bacterial diversity and favors the growth of Actinobacteria and Proteobacteria in agro‐ecosystems across the globe

Long‐term elevated nitrogen (N) input from anthropogenic sources may cause soil acidification and decrease crop yield, yet the response of the belowground microbial community to long‐term N input alone or in combination with phosphorus (P) and potassium (K) is poorly understood. We explored the effect of long‐term N and NPK fertilization on soil bacterial diversity and community composition using meta‐analysis of a global dataset. Nitrogen fertilization decreased soil pH, and increased soil organic carbon (C) and available N contents. Bacterial taxonomic diversity was decreased by N fertilization alone, but was increased by NPK fertilization. The effect of N fertilization on bacterial diversity varied with soil texture and water management, but was independent of crop type or N application rate. Changes in bacterial diversity were positively related to both soil pH and organic C content under N fertilization alone, but only to soil organic C under NPK fertilization. Microbial biomass C decreased with decreasing bacterial diversity under long‐term N fertilization. Nitrogen fertilization increased the relative abundance of Proteobacteria and Actinobacteria, but reduced the abundance of Acidobacteria, consistent with the general life history strategy theory for bacteria. The positive correlation between N application rate and the relative abundance of Actinobacteria indicates that increased N availability favored the growth of Actinobacteria. This first global analysis of long‐term N and NPK fertilization that differentially affects bacterial diversity and community composition provides a reference for nutrient management strategies for maintaining belowground microbial diversity in agro‐ecosystems worldwide.     

Abrupt burial imparts persistent changes to the bacterial diversity of turbidite‐associated sediment profiles

The emplacement of subaqueous gravity‐driven sediment flows imposes a significant physical and geochemical impact on underlying sediment and microbial communities. Although previous studies have established lasting mineralogical and biological signatures of turbidite deposition, the response of bacteria and archaea within and beneath debris flows remains poorly constrained. Both bacterial cells associated with the underlying sediment and those attached to allochthonous material must respond to substantially altered environmental conditions and selective pressures. As a consequence, turbidites and underlying sediments provide an exceptional opportunity to examine (i) the microbial community response to rapid sedimentation and (ii) the preservation and identification of displaced micro‐organisms. We collected Illumina MiSeq sequence libraries across turbidite boundaries at ~26 cm sediment depth in La Jolla Canyon off the coast of California, and at ~50 cm depth in meromictic Twin Lake, Hennepin County, MN. 16S rRNA gene signatures of relict and active bacterial populations exhibit persistent differences attributable to turbidite deposition. In particular, both the marine and lacustrine turbidite boundaries are sharply demarcated by the abundance and diversity of Chloroflexi, suggesting a characteristic sensitivity to sediment disturbance history or to differences in organic substrates across turbidite profiles. Variations in the abundance of putative dissimilatory sulfate‐reducing Deltaproteobacteria across the buried La Jolla Canyon sediment–water interface reflect turbidite‐induced changes to the geochemical environment. Species‐level distinctions within the Deltaproteobacteria clearly conform to the sedimentological boundary, suggesting a continuing impact of genetic inheritance distinguishable from broader trends attributable to selective pressure. Abrupt, <1‐cm scale changes in bacterial diversity across the Twin Lake turbidite contact are consistent with previous studies showing that relict DNA signatures attributable to sediment transport may be more easily preserved in low‐energy, anoxic environments. This work raises the possibility that deep subsurface microbial communities may inherit variations in microbial diversity from sediment flow and deformation events.     

Comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater‐fed rapid sand filter communities

The recent discovery of completely nitrifying Nitrospira demands a re‐examination of nitrifying environments to evaluate their contribution to nitrogen cycling. To approach this challenge, tools are needed to detect and quantify comammox Nitrospira. We present primers for the simultaneous quantification and diversity assessement of both comammox Nitrospira clades. The primers cover a wide range of comammox diversity, spanning all available high quality sequences. We applied these primers to 12 groundwater‐fed rapid sand filters, and found comammox Nitrospira to be abundant in all filters. Clade B comammox comprise the majority (～75%) of comammox abundance in all filters. Nitrosomonadaceae were present in all filters, although at low abundance (mean = 1.8%). Ordination suggests that temperature impacts the structure of nitrifying communities, and in particular that increasing temperature favours Nitrospira. The nitrogen content of the filter material, sulfate concentration and surface ammonium loading rates shape the structure of the comammox guild in the filters. This work provides an assay for simultaneous detection and diversity assessment of clades A and B comammox Nitrospira, expands our current knowledge of comammox Nitrospira diversity and demonstrates a key role for comammox Nitrospira in nitrification in groundwater‐fed biofilters.     

On‐farm habitat restoration counters biotic homogenization in intensively managed agriculture

To slow the rate of global species loss, it is imperative to understand how to restore and maintain native biodiversity in agricultural landscapes. Currently, agriculture is associated with lower spatial heterogeneity and turnover in community composition (β‐diversity). While some techniques are known to enhance α‐diversity, it is unclear whether habitat restoration can re‐establish β‐diversity. Using a long‐term pollinator dataset, comprising ～9,800 specimens collected from the intensively managed agricultural landscape of the Central Valley of California, we show that on‐farm habitat restoration in the form of native plant ‘hedgerows’, when replicated across a landscape, can boost β‐diversity by approximately 14% relative to unrestored field margins, to levels similar to some natural communities. Hedgerows restore β‐diversity by promoting the assembly of phenotypically diverse communities. Intensively managed agriculture imposes a strong ecological filter that negatively affects several important dimensions of community trait diversity, distribution, and uniqueness. However, by helping to restore phenotypically diverse pollinator communities, small‐scale restorations such as hedgerows provide a valuable tool for conserving biodiversity and promoting ecosystem services.     

Elevated carbon dioxide accelerates the spatial turnover of soil microbial communities

Although elevated CO₂ (eCO₂) significantly affects the α‐diversity, composition, function, interaction and dynamics of soil microbial communities at the local scale, little is known about eCO₂ impacts on the geographic distribution of micro‐organisms regionally or globally. Here, we examined the β‐diversity of 110 soil microbial communities across six free air CO₂ enrichment (FACE) experimental sites using a high‐throughput functional gene array. The β‐diversity of soil microbial communities was significantly (P < 0.05) correlated with geographic distance under both CO₂ conditions, but declined significantly (P < 0.05) faster at eCO₂ with a slope of ?0.0250 than at ambient CO₂ (aCO₂) with a slope of ?0.0231 although it varied within each individual site, indicating that the spatial turnover rate of soil microbial communities was accelerated under eCO₂ at a larger geographic scale (e.g. regionally). Both distance and soil properties significantly (P < 0.05) contributed to the observed microbial β‐diversity. This study provides new hypotheses for further understanding their assembly mechanisms that may be especially important as global CO₂ continues to increase.     

Internal Porosity of Mineral Coating Supports Microbial Activity in Rapid Sand Filters for Groundwater Treatment

A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH₄⁺ removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 μm) of the mineral coating, which had a thickness of up to 600 ± 51 μm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

A meta‐analysis of temperature sensitivity as a microbial trait

Traits‐based approaches in microbial ecology provide a valuable way to abstract organismal interaction with the environment and to generate hypotheses about community function. Using macromolecular rate theory (MMRT), we recently identified that temperature sensitivity can be characterized as a distinct microbial trait. As temperature is fundamental in controlling biological reactions, variation in temperature sensitivity across communities, organisms, and processes has the potential to vastly improve understanding of microbial response to climate change. These microbial temperature sensitivity traits include the heat capacity (), temperature optimum (T_opt), and point of maximum temperature sensitivity (TS_max), each of which provide unique insights about organismal response to changes in temperature. In this meta‐analysis, we analyzed the distribution of these temperature sensitivity traits from bacteria, fungi, and mixed communities across a variety of biological systems (e.g., soils, oceans, foods, wastewater treatment plants) in order to identify commonalities in temperature responses across these diverse organisms and reaction rates. Our analysis of temperature sensitivity traits from over 350 temperature response curves reveals a wide distribution of temperature sensitivity traits, with T_opt and TS_max well within biological relevant temperatures. We find that traits vary significantly depending on organism type, microbial diversity, source environment, and biological process, with higher temperature sensitivity found in fungi than bacteria and in less diverse systems. Carbon dioxide production was found to be less temperature sensitive than denitrification, suggesting that changes in temperature will have a potentially larger impact on nitrogen‐related processes. As climate changes, these results have important implications for basic understanding of the temperature sensitivity of biological reactions and for ecological understanding of species’ trait distributions, as well as for improved treatment of temperature sensitivity in models.     

Landscape partitioning by nocturnal rodent assemblages in the Llanos de Ojuelos,in Mexico’s Central High Plateau

Aim The Llanos de Ojuelos in Mexico’s Central High Plateau supports unique Opuntia scrublands and the southernmost Chihuahuan grasslands. Although human activities have modified strongly its landscape and impacted its biodiversity at an unknown scale, such impacts are poorly known. We aimed at understanding how nocturnal rodent species distributed across the landscape and formed assemblages and on the role and integration of the different habitats at the landscape level. Location The study was carried out at 43 sites in a study area of approximately 3350 km² in the Llanos de Ojuelos. Methods During the Spring of 2008, we surveyed nocturnal rodents, through live‐trapping. We redefined habitat classes based on log‐linear multinomial regressions of rodent captures. Species rarefaction curves and true alpha, beta and gamma diversities were calculated for the different habitat classes. A map of the different habitats was constructed based on Landsat imagery. Results We captured 458 individuals of 20 rodent species. Multinomial regressions caused the merger of 11 a priori defined habitat classes into 7. Leguminous scrub and mixed nopaleras, both secondary habitats, had the highest alpha and gamma diversity values. Closed arboreal nopaleras and grasslands had the highest within‐habitat variability (¹D_β) and the lowest area coverage. Within‐habitat ¹D_β was larger than landscape ¹D_β, because of the great overlap in rodent assemblage composition between the habitats. Main conclusions There are no ‘typical’ rodent assemblages per habitat class, but they are organized loosely and have fuzzy borders. Rodent community organization was highly species‐centred. At the landscape level, secondary habitats have a profound effect on rodent diversity and should be included in management schemes for biological conservation. The most endangered habitats seem to be the closed arboreal nopaleras and grasslands. Any conservation efforts must consider their conservation and an increase in the size of remaining patches.     

Bacterial community collapse: a meta‐analysis of the sinonasal microbiota in chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is a common, debilitating condition characterized by long‐term inflammation of the nasal cavity and paranasal sinuses. The role of the sinonasal bacteria in CRS is unclear. We conducted a meta‐analysis combining and reanalysing published bacterial 16S rRNA sequence data to explore differences in sinonasal bacterial community composition and predicted function between healthy and CRS affected subjects. The results identify the most abundant bacteria across all subjects as Staphylococcus, Propionibacterium, Corynebacterium, Streptococcus and an unclassified lineage of Actinobacteria. The meta‐analysis results suggest that the bacterial community associated with CRS patients is dysbiotic and ecological networks fostering healthy communities are fragmented. Increased dispersion of bacterial communities, significantly lower bacterial diversity, and increased abundance of members of the genus Corynebacterium are associated with CRS. Increased relative abundance and diversity of other members belonging to the phylum Actinobacteria and members from the genera Propionibacterium differentiated healthy sinuses from those that were chronically inflamed. Removal of Burkholderia and Propionibacterium phylotypes from the healthy community dataset was correlated with a significant increase in network fragmentation. This meta‐analysis highlights the potential importance of the genera Burkholderia and Propionibacterium as gatekeepers, whose presence may be important in maintaining a stable sinonasal bacterial community.     

Probiotic roles of Lactobacillus sp. in swine: insights from gut microbiota

The use of lactobacilli as probiotics in swine has been gaining attention due to their ability to improve growth performance and carcass quality, prevent gastrointestinal infection and most importantly, their ‘generally recognized as safe’ status. Previous studies support the potential of lactobacilli to regulate host immune systems, enhance gut metabolic capacities and maintain balance in the gut microbiota. Research on swine gut microbiota has revealed complex gut microbial community structure and showed the importance of Lactobacillus to the host's health. However, the species‐ and strain‐specific characteristics of lactobacilli that confer probiotic benefits are still not well understood. The diversity of probiotic traits in a complex gut ecosystem makes it challenging to infer the relationships between specific functions of Lactobacillus sp. and host health. In this review, we provide an overview of how lactobacilli play a pivotal role in the swine gut ecosystem and identify key characteristics that influence gut microbial community structure and the health of pigs. In addition, based on recent and ongoing meta‐omics and omics research on the gut microbiota of pigs, we suggest a workflow combining culture‐dependent and culture‐independent approaches for more effective selection of probiotic lactobacilli.     

Change in Bacterial Community Structure in Response to Disturbance of Natural Hardwood and Secondary Coniferous Forest Soils in Central Taiwan

Forest management often results in changes in the soil and its microbial communities. In the present study, differences in the soil bacterial community caused by forest management practices were characterized using small subunit (SSU) ribosomal RNA (rRNA) gene clone libraries. The communities were from a native hardwood forest (HWD) and two adjacent conifer plantations in a low-elevation montane, subtropical experimental forest at the Lienhuachi Experimental Forest (LHCEF) in central Taiwan. At this locality, the elevation ranges from 600 to 950 m, the mean annual precipitation is 2,200 mm, the mean annual temperature is 20.8°C, and the soil pH is 4. The conifer forests included a Cunninghamia konishii Hay (CNH) plantation of 40 years and an old growth Calocedrus formosana (Florin) Florin (CLC) forest of 80 years. A total of 476 clones were sequenced and assigned into 12 phylogenetic groups. Proteobacteria-affiliated clones (53%) predominated in the library from HWD soils. In contrast, Acidobacteria was the most abundant phylum and comprised 39% and 57% in the CLC and CNH libraries, respectively. Similarly, the most abundant OTUs in HWD soils were greatly reduced or absent in the CLC and CNH soils. Based on several diversity indices, the numbers of abundant OTUs and singletons, and rarefaction curves, the diversity of the HWD community (0.95 in evenness and Shannon diversity indices) was somewhat less than that in the CNH soils (0.97 in evenness and Shannon diversity indices). The diversity of the community in CLC soils was intermediate. The differences in diversity among the three communities may also reflect changes in abundances of a few OTUs. The CNH forest soil community may be still in a successional phase that is only partially stabilized after 40 years. Analysis of molecular variance also revealed that the bacterial community composition of HWD soils was significantly different from CLC and CNH soils (p = 0.001). These results suggest that the disturbance of forest conversion and tree species composition are important factors influencing the soil bacterial community among three forest ecosystems in the same climate.     

The effects of variable sample biomass on comparative metagenomics

Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana‐associated microbial community. We modified the Illumina Nextera kit to generate high‐quality large‐insert (680 bp) paired‐end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly‐based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low‐GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case‐by‐case basis. Overall, this study provides recommendations for quality filtering and de‐replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys.