VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing

Background

The 16S rRNA gene-based amplicon sequencing analysis is widely used to determine the taxonomic composition of microbial communities. Once the taxonomic composition of each community is obtained, evolutionary relationships among taxa are inferred by a phylogenetic tree. Thus, the combined representation of taxonomic composition and phylogenetic relationships among taxa is a powerful method for understanding microbial community structure; however, applying phylogenetic tree-based representation with information on the abundance of thousands or more taxa in each community is a difficult task. For this purpose, we previously developed the tool VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community), which is based on the genome-sequenced microbes’ phylogenetic information. Here, we introduce VITCOMIC2, which incorporates substantive improvements over VITCOMIC that were necessary to address several issues associated with 16S rRNA gene-based analysis of microbial communities.

Results

We developed VITCOMIC2 to provide (i) sequence identity searches against broad reference taxa including uncultured taxa; (ii) normalization of 16S rRNA gene copy number differences among taxa; (iii) rapid sequence identity searches by applying the graphics processing unit-based sequence identity search tool CLAST; (iv) accurate taxonomic composition inference and nearly full-length 16S rRNA gene sequence reconstructions for metagenomic shotgun sequencing; and (v) an interactive user interface for simultaneous representation of the taxonomic composition of microbial communities and phylogenetic relationships among taxa. We validated the accuracy of processes (ii) and (iv) by using metagenomic shotgun sequencing data from a mock microbial community.

Conclusions

The improvements incorporated into VITCOMIC2 enable users to acquire an intuitive understanding of microbial community composition based on the 16S rRNA gene sequence data obtained from both metagenomic shotgun and amplicon sequencing.

     

Amplification by PCR artificially reduces the proportion of the rare biosphere in microbial communities

The microbial world has been shown to hold an unimaginable diversity. The use of rRNA genes and PCR amplification to assess microbial community structure and diversity present biases that need to be analyzed in order to understand the risks involved in those estimates. Herein, we show that PCR amplification of specific sequence targets within a community depends on the fractions that those sequences represent to the total DNA template. Using quantitative, real-time, multiplex PCR and specific Taqman probes, the amplification of 16S rRNA genes from four bacterial species within a laboratory community were monitored. Results indicate that the relative amplification efficiency for each bacterial species is a nonlinear function of the fraction that each of those taxa represent within a community or multispecies DNA template. Consequently, the low-proportion taxa in a community are under-represented during PCR-based surveys and a large number of sequences might need to be processed to detect some of the bacterial taxa within the 'rare biosphere'. The structure of microbial communities from PCR-based surveys is clearly biased against low abundant taxa which are required to decipher the complete extent of microbial diversity in nature.     

水热增加下黑土细菌群落共生网络特征

黑土是有机质含量高且肥沃的土壤类型之一,气候变化会显著改变黑土中微生物群落的结构,同时影响群落间的潜在相互作用关系。[目的] 揭示水热增加对黑土中的细菌群落结构及潜在互作关系的影响。[方法] 基于土壤移置试验,采用16S rRNA高通量测序解析农田黑土（原位黑土、水热增加1和水热增加2）中的细菌群落结构对水热增加的响应;使用CoNet构建微生物群落共生网络,识别共生网络中的枢纽微生物;利用结构方程模型、相关性分析探究水热条件变化下土壤性质、微生物交互作用、多样性之间的直接、间接关系。[结果] 黑土中的微生物以疣微菌、变形杆菌、酸性杆菌和放线菌为主。水热增加下土壤微生物共生网络的拓扑性质发生显著变化,网络中表征微生物潜在竞争关系的负连线随着水热增加而显著增加。气候因素通过改变微生物潜在相互作用影响了群落水平分类多样性。物种竞争增强可能直接导致了土壤有机碳含量的降低。[结论] 水热增加会显著改变黑土中微生物之间的潜在交互作用,枢纽微生物的响应更加敏感。     

Microbial Community Profiling to Investigate Transmission of Bacteria Between Life Stages of the Wood-Boring Beetle, Anoplophora glabripennis

Many insects harbor specific bacteria in their digestive tract, and these gut microbiota often play important roles in digestion and nutrient provisioning. While it is common for a given insect species to harbor a representative gut microbial community as a population, how this community is acquired and maintained from generation to generation is not known for most xylophagous insects, except termites. In this study, we examined acquisition of gut microbiota by the wood-feeding beetle, Anoplophora glabripennis, by identifying and comparing microbial community members among different life stages of the insect and with microbes it encounters in the environment. Automated ribosomal intergenic spacer analysis was employed to compare bacterial communities present in the egg and larval stages of A. glabripennis as well as with microbes found in the oviposition site and the surrounding woody tissue. Multivariate analyses were used to identify relationships between sample type and specific bacterial types (operational taxonomic units). From this analysis, bacteria that were derived from the environment, the oviposition site, and/or the egg were identified and compared with taxa found in larvae. Results showed that while some larval microbes were derived from environmental sources, other members of the larval microbial community appear to be vertically transmitted. These findings could lead to a better understanding of which microbial species are critical for the survival of this insect and to development of techniques that could be used to alter this community to disrupt the digestive physiology of the host insect as a biological control measure.     

Butterfly community structure in fragmented habitats

We analysed effects of habitat fragmentation on the diversity, abundance, and life history traits of butterflies on 33 calcareous grasslands. Diversity of butterflies was positively correlated with habitat area (as was plant diversity), but not with habitat isolation. In contrast to expectations, butterfly densities of polyphagous and oligophagous species declined with habitat area whereas densities of monophagous species increased. The z -values, i.e. the slope of species–area relationships, increased with food plant specialization, from 0.07 in polyphagous, 0.11 in oligophagous, 0.16 in strongly oligophagous to 0.22 in monophagous species, and were 0.14 in plant species. Significant z -values were not only found for total species richness, based on a sample size adjusted to fragment area ( z  = 0.12), but also for the local density of butterfly species richness, based on equal sample size across all habitat fragments ( z  = 0.06). To our knowledge, this is the first study to show differential responses of monophagous, oligophagous and polyphagous species to area with respect to species richness and population density.     

What is the extent of prokaryotic diversity?

The extent of microbial diversity is an intrinsically fascinating subject of profound practical importance. The term 'diversity' may allude to the number of taxa or species richness as well as their relative abundance. There is uncertainty about both, primarily because sample sizes are too small. Non-parametric diversity estimators make gross underestimates if used with small sample sizes on unevenly distributed communities. One can make richness estimates over many scales using small samples by assuming a species/taxa-abundance distribution. However, no one knows what the underlying taxa-abundance distributions are for bacterial communities. Latterly, diversity has been estimated by fitting data from gene clone libraries and extrapolating from this to taxa-abundance curves to estimate richness. However, since sample sizes are small, we cannot be sure that such samples are representative of the community from which they were drawn. It is however possible to formulate, and calibrate, models that predict the diversity of local communities and of samples drawn from that local community. The calibration of such models suggests that migration rates are small and decrease as the community gets larger. The preliminary predictions of the model are qualitatively consistent with the patterns seen in clone libraries in 'real life'. The validation of this model is also confounded by small sample sizes. However, if such models were properly validated, they could form invaluable tools for the prediction of microbial diversity and a basis for the systematic exploration of microbial diversity on the planet.     

On the determinants of census area: implications for mammalian macroecological patterns

The aim of this study was to examine the effects of various biological factors such as body mass, trophic level, climate and geography on census area in terrestrial mammals. We also examine the effects of census area on the population density–body mass relationship. The geographic areas covered in this study include most major terrestrial biomes including taïga, desert, savanna, grassland, tropical dry forest, temperate dry forest, tropical rain forest and temperate rain forest. An extensive literature search was conducted and we compiled data on census area and body mass from 377 mammalian populations and 59 communities. Statistical analyses include linear regression, Kruskal–Wallis analysis of variance, LOWESS, and multiple regression. Overall, the regression between log census area (A) and log body mass (M) yielded a slope of 0.710, which did not differ significantly from 0.75, but it was significantly different from 1.0. The analyses also showed that the log A–log M relationship is constrained within a well‐defined space in a similar fashion to the home range–body mass relationship. When mammals were separated into trophic groups, regression lines differed significantly in their intercepts, but not in slopes. At the community level, the census area was particularly affected by the population with the largest body mass within the community. Both the number of species and number of taxa encompassed by the community were found to be correlated positively with log A (r = 0.26, P = 0.0464 and r = 0.27, P = 0.0398, respectively). Sampling of mammalian species is not usually random. Not only is census area significantly associated with the technique used to sample a given species, but it is also influenced by biological factors that have been shown previously to influence population density. Striking similarities were found between the census area–body mass relationship and the home range–body mass relationship, suggesting that investigators may sample mammalian populations over areas that actually reflect the use of space of their focal species.     

Are you what you eat? A highly transient and prey‐influenced gut microbiome in the grey house spider Badumna longinqua

Stable core microbial communities have been described in numerous animal species and are commonly associated with fitness benefits for their hosts. Recent research, however, highlights examples of species whose microbiota are transient and environmentally derived. Here, we test the effect of diet on gut microbial community assembly in the spider Badumna longinqua. Using 16S rRNA gene amplicon sequencing combined with quantitative PCR, we analyzed diversity and abundance of the spider's gut microbes, and simultaneously characterized its prey communities using nuclear rRNA markers. We found a clear correlation between community similarity of the spider's insect prey and gut microbial DNA, suggesting that microbiome assembly is primarily diet‐driven. This assumption is supported by a feeding experiment, in which two types of prey—crickets and fruit flies—both substantially altered microbial diversity and community similarity between spiders, but did so in different ways. After cricket consumption, numerous cricket‐derived microbes appeared in the spider's gut, resulting in a rapid homogenization of microbial communities among spiders. In contrast, few prey‐associated bacteria were detected after consumption of fruit flies; instead, the microbial community was remodelled by environmentally sourced microbes, or abundance shifts of rare taxa in the spider's gut. The reshaping of the microbiota by both prey taxa mimicked a stable core microbiome in the spiders for several weeks post feeding. Our results suggest that the spider's gut microbiome undergoes pronounced temporal fluctuations, that its assembly is dictated by the consumed prey, and that different prey taxa may remodel the microbiota in drastically different ways.     

Bacterial and eukaryote microbiomes of mosquito habitats in dengue-endemic southern Taiwan

Mosquitoes interact with the microbiome of the waters where they oviposit in several ways. Past work suggests adult mosquitoes can detect certain microbes that stimulate oviposition. The presence or absence of certain microbes in water containers thus can attract or repel mosquito species to different containers. I hypothesized that these relationships could be detected via metagenomics. I focused on two container breeders that coexist in Southern Taiwan: the dengue vector Aedes aegypti and the less competent vector Ae. albopictus. In addition to culturing, I performed 16S and 18S rDNA metagenomics assays, the latter of which had never been applied to mosquito waters before, to identify the microbial diversity of artificial containers with and without mosquito larvae. I found no correlation between mosquito presence to any features of the containers or to their microbiomes, which instead correlated strongly with location. Microbial diversity across containers was highly variable, even within the same location, with multiple taxa only found in single containers. This variability is reasonable, because mosquito gut microbiomes are also extremely variable. The possibility remains that microbes in natural containers differ significantly from those in artificial containers, and that these differences drive Aedes preferences for human-associated containers. Broad, single-microbe experimental work is recommended to identify possible attractant or repellent microbial taxa.     

Temporal and Spatial Distribution of the Microbial Community of Winogradsky Columns

Winogradsky columns are model microbial ecosystems prepared by adding pond sediment to a clear cylinder with additional supplements and incubated with light. Environmental gradients develop within the column creating diverse niches that allow enrichment of specific bacteria. The enrichment culture can be used to study soil and sediment microbial community structure and function. In this study we used a 16S rRNA gene survey to characterize the microbial community dynamics during Winogradsky column development to determine the rate and extent of change from the source sediment community. Over a period of 60 days, the microbial community changed from the founding pond sediment population: Cyanobacteria, Chloroflexi, Nitrospirae, and Planctomycetes increased in relative abundance over time, while most Proteobacteria decreased in relative abundance. A unique, light-dependent surface biofilm community formed by 60 days that was less diverse and dominated by a few highly abundant bacteria. 67–72% of the surface community was comprised of highly enriched taxa that were rare in the source pond sediment, including the Cyanobacteria Anabaena, a member of the Gemmatimonadetes phylum, and a member of the Chloroflexi class Anaerolinea. This indicates that rare taxa can become abundant under appropriate environmental conditions and supports the hypothesis that rare taxa serve as a microbial seed bank. We also present preliminary findings that suggest that bacteriophages may be active in the Winogradsky community. The dynamics of certain taxa, most notably the Cyanobacteria, showed a bloom-and-decline pattern, consistent with bacteriophage predation as predicted in the kill-the-winner hypothesis. Time-lapse photography also supported the possibility of bacteriophage activity, revealing a pattern of colony clearance similar to formation of viral plaques. The Winogradsky column, a technique developed early in the history of microbial ecology to enrich soil microbes, may therefore be a useful model system to investigate both microbial and viral ecology.     

微生物组学研究的“淘金时代”

人体及动物肠道中生存着数量庞大的共生微生物;这些微生物无时无刻不参与着宿主的生命活动。揭示这些共生微生物在宿主体内的变化规律、与宿主之间的依存和博弈关系等,将使人类更加全面的认知高等生物体的生命本质。本专刊从肠道微生物与疾病、肠道微生物群落结构、肠道微生物与宿主互作、肠道微生物资源和肠道微生物研究方法 5个层面展示了我国科研工作者在肠道微生物研究领域的新进展及新观点。     

Anthropogenic activities change the relationship between microbial community taxonomic composition and functional attributes

Functional redundancy is considered common in microbial systems, but recent studies have challenged this idea. The mechanism for this contradictory result is not clear. However, in this study, we hypothesize that strong environmental filtering which links to the anthropogenic activities is able to weaken microbial functional redundancy. We used metagenome and 16S rRNA gene high-throughput sequencing to investigate planktonic microbial communities in a subtropical river. We found that the weak anthropogenic activities might result in a low selection pressure in the river upstream area. Therefore, the microbial community functional attributes were stable although the community composition changed with the water temperature and NO₃-N in upstream area (this indicates functional redundancy). However, the strong anthropogenic activities in river downstream area selected pollutant-degraded functions (e.g. nitrogen metabolism, toluene, xylenes and ethylbenzene degradation) and potentially pollutant-degraded (tolerant) microbes, and therefore caused the microbial community composition synchronously changed with the variation of community functional attributes. Our results reveal that strong environmental filtering which associates with the anthropogenic activities not only has effects on microbial community composition and community functional attributes but also on their relationships. These results provide a new insight to refine the functional redundancy idea.     

Microbial community development on model particles in the deep sulfidic waters of the Black Sea

Microorganisms attached to particles have been shown to be different from free-living microbes and to display diverse metabolic activities. However, little is known about the ecotypes associated with particles and their substrate preference in anoxic marine waters. Here, we investigate the microbial community colonizing particles in the anoxic and sulfide-rich waters of the Black Sea. We incubated beads coated with different substrates in situ at 1000 and 2000 m depth. After 6 h, the particle-attached microbes were dominated by Gamma- and Alpha-proteobacteria, and groups related to the phyla Latescibacteria, Bacteroidetes, Planctomycetes and Firmicutes, with substantial variation across the bead types, indicating that the attaching communities were selected by the substrate. Further laboratory incubations for 7 days suggested the presence of a community of highly specialized taxa. After incubation for 35 days, the microbial composition across all beads and depths was similar and primarily composed of putative sulfur cycling microbes. In addition to the major shared microbial groups, subdominant taxa on chitin and protein-coated beads were detected pointing to specialized microbial degraders. These results highlight the role of particles as sites for attachment and biofilm formation, while the composition of organic matter defined a secondary part of the microbial community.     

Threatened Corals Provide Underexplored Microbial Habitats

Contemporary in-depth sequencing of environmental samples has provided novel insights into microbial community structures, revealing that their diversity had been previously underestimated. Communities in marine environments are commonly composed of a few dominant taxa and a high number of taxonomically diverse, low-abundance organisms. However, studying the roles and genomic information of these “rare” organisms remains challenging, because little is known about their ecological niches and the environmental conditions to which they respond. Given the current threat to coral reef ecosystems, we investigated the potential of corals to provide highly specialized habitats for bacterial taxa including those that are rarely detected or absent in surrounding reef waters. The analysis of more than 350,000 small subunit ribosomal RNA (16S rRNA) sequence tags and almost 2,000 nearly full-length 16S rRNA gene sequences revealed that rare seawater biosphere members are highly abundant or even dominant in diverse Caribbean corals. Closely related corals (in the same genus/family) harbored similar bacterial communities. At higher taxonomic levels, however, the similarities of these communities did not correlate with the phylogenetic relationships among corals, opening novel questions about the evolutionary stability of coral-microbial associations. Large proportions of OTUs (28.7–49.1%) were unique to the coral species of origin. Analysis of the most dominant ribotypes suggests that many uncovered bacterial taxa exist in coral habitats and await future exploration. Our results indicate that coral species, and by extension other animal hosts, act as specialized habitats of otherwise rare microbes in marine ecosystems. Here, deep sequencing provided insights into coral microbiota at an unparalleled resolution and revealed that corals harbor many bacterial taxa previously not known. Given that two of the coral species investigated are listed as threatened under the U.S. Endangered Species Act, our results add an important microbial diversity-based perspective to the significance of conserving coral reefs.     

Biogeography and ecological diversity patterns of rare and abundant bacteria in oil‐contaminated soils

Revealing the biogeographies and ecologies of rare and abundant microorganisms is crucial to understand ecosystem diversity and function. In this study, we investigated the biogeographic assemblies and ecological diversity patterns of rare and abundant bacteria in long‐term oil‐contaminated soils at intervals of 46–360 km by performing high‐throughput sequencing of 16S rRNA genes. The results clearly revealed distinct distribution patterns for rare and abundant bacteria in soil samples. Rare taxa were unevenly distributed; however, abundant taxa were ubiquitous across all samples. Both rare and abundant subcommunities showed significant distance–decay relationships, and their assemblies were driven by different factors. The rare subcommunity primarily exhibited a spatially structured distribution (i.e., stochastic processes), while edaphic factors (i.e., deterministic processes) largely contributed to the structure of the abundant subcommunity. A network analysis revealed closer relationships between abundant bacteria and their heightened influence on other co‐occurrences in the community compared with rare species. In conclusion, rare microbial taxa may play potential roles in maintaining ecosystem diversity, although they do not appear to be central to microbial networks. Abundant microbes are vital for microbial co‐occurrences in oil‐contaminated soils, and high relative abundance and ubiquitous distribution suggest potential roles in the degradation of organic pollutants.     

The Western English Channel contains a persistent microbial seed bank

Robust seasonal dynamics in microbial community composition have previously been observed in the English Channel L4 marine observatory. These could be explained either by seasonal changes in the taxa present at the L4 site, or by the continuous modulation of abundance of taxa within a persistent microbial community. To test these competing hypotheses, deep sequencing of 16S rRNA from one randomly selected time point to a depth of 10 729 927 reads was compared with an existing taxonomic survey data covering 6 years. When compared against the 6-year survey of 72 shallow sequenced time points, the deep sequenced time point maintained 95.4% of the combined shallow OTUs. Additionally, on average, 99.75%±0.06 (mean±s.d.) of the operational taxonomic units found in each shallow sequenced sample were also found in the single deep sequenced sample. This suggests that the vast majority of taxa identified in this ecosystem are always present, but just in different proportions that are predictable. Thus observed changes in community composition are actually variations in the relative abundance of taxa, not, as was previously believed, demonstrating extinction and recolonization of taxa in the ecosystem through time.     

包头泉山金矿浸矿酸性微生物群落优势度变化的比较研究

【目的】研究不同矿石组成对微生物群落结构的影响。【方法】选取包头泉山金矿的酸性矿坑水(Acid mine drainage,AMD)对4种不同组成的矿石样品进行浸矿,采用16S rRNA-PCR和RFLP相结合的方法,研究浸矿前后浸矿微生物种群优势度的变化情况。【结果】所选取的3个酸性矿坑水考查点之间浸矿微生物的种类及数量相似度较大,浸矿微生物结构相对变化度不大。H71和F11相对其他两个样品的群落结构差异较大,而J72和S71几乎一样,说明在钾长型和石英型矿石的选择压力下,浸矿微生物的被选择趋势是大致相同的。【结论】浸矿前后样品中浸矿微生物的系统发育分析结果表明,所考查的克隆子的序列可分为两大分支:Acidithiobacillus菌属和一个相对独立的菌属,且这两个分支内部菌株之间的发育距离较近。     

Heterogeneity of the rice microbial community of the Chinese centuries-old Honghe Hani rice terraces system

The Honghe Hani rice terraces system (HHRTS) is a traditional rice cultivation system where Hani people cultivate remarkably diverse rice varieties. Recent introductions of modern rice varieties to the HHRTS have significantly increased the severity of rice diseases within the terraces. Here, we determine the impacts of these recent introductions on the composition of the rice-associated microbial communities. We confirm that the HHRTS contains a range of both traditional HHRTS landraces and introduced modern rice varieties and find differences between the microbial communities of these two groups. However, this introduction of modern rice varieties has not strongly impacted the overall diversity of the HHRTS rice microbial community. Furthermore, we find that the rice varieties (i.e. groups of closely related genotypes) have significantly structured the rice microbial community composition (accounting for 15%–22% of the variance) and that the core microbial community of HHRTS rice plants represents less than 3.3% of all the microbial taxa identified. Collectively, our study suggests a highly diverse HHRTS rice holobiont (host with its associated microbes) where the diversity of rice hosts mirrors the diversity of their microbial communities. Further studies will be needed to better determine how such changes might impact the sustainability of the HHRTS.