Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations.
ABSTRACT: High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n?=?883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards.IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.
Project description:The composition of phytoplankton community is the basis for environmental monitoring and assessment of the ecological status of aquatic ecosystems. Community composition studies of phytoplankton have been based on time-consuming and expertise-demanding light microscopy analyses. Molecular methods have the potential to replace microscopy, but the high copy number variation of ribosomal genes and the lack of universal primers for simultaneous amplification of prokaryotic and eukaryotic genes complicate data interpretation. In this study, we used our previously developed directional primer-independent high-throughput sequencing (HTS) approach to analyze 16S and 18S rRNA community structures. Comparison of 83 boreal lake samples showed that the relative abundances of eukaryotic phytoplankton at class level and prokaryotic cyanobacteria at order level were consistent between HTS and microscopy results. At the genus level, the results had low correspondence, mainly due to lack of sequences in the reference library. HTS was superior to identify genera that are extensively represented in the reference databases but lack specific morphological characteristics. Targeted metatranscriptomics proved to be a feasible method to complement the microscopy analysis. The metatranscriptomics can also be applied without linking the sequences to taxonomy. However, direct indexing of the sequences to their environmental indicator values needs collections of more comprehensive sample sets, as long as the coverage of molecular barcodes of eukaryotic species remains insufficient.
Project description:In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacter clade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments.
Project description:The structure of microbial communities, microalgae, heterotrophic protozoa and fungi contributes to characterize food webs and productivity and, from an anthropogenic point of view, the qualitative characteristics of water bodies. Traditionally, in freshwater environments many investigations have been directed to the study of pelagic microalgae ("phytoplankton") and periphyton (i.e., photosynthetic and mixotrophic protists) through the use of light microscopy (LM). While the number of studies on bacterioplankton communities have shown a substantial increase after the advent of high-throughput sequencing (HTS) approaches, the study of the composition, structure, and spatio-temporal patterns of microbial eukaryotes in freshwater environments was much less widespread. Moreover, the understanding of the correspondence between the relative phytoplankton abundances estimated by HTS and LM is still incomplete. Taking into account these limitations, this study examined the biodiversity and seasonality of the community of eukaryotic microplankton in the epilimnetic layer of a large and deep perialpine lake (Lake Garda) using HTS. The analyses were carried out at monthly frequency during 2014 and 2015. The results highlighted the existence of a rich and well diversified community and the presence of numerous phytoplankton taxa that were never identified by LM in previous investigations. Furthermore, the relative abundances of phytoplankton estimated by HTS and LM showed a significant relationship at different taxonomic ranks. In the 2 years of investigation, the temporal development of the whole micro-eukaryotic community showed a clear non-random and comparable distribution pattern, with the main taxonomic groups coherently distributed in the individual seasons. In perspective, the results obtained in this study highlight the importance of HTS approaches in assessing biodiversity and the relative importance of the main protist groups along environmental gradients, including those caused by anthropogenic impacts (e.g., eutrophication and climate change).
Project description:Understanding biotic versus abiotic forces that shape community structure is a fundamental aim of microbial ecology. The acidic and heavy metal extreme Río Tinto (RT) in southwestern Spain provides a rare opportunity to conduct an ecosystem-wide biodiversity inventory at the level of all three domains of life, because diversity there is low and almost exclusively microbial. Despite improvements in high-throughput DNA sequencing, environmental biodiversity studies that use molecular metrics and consider entire ecosystems are rare. These studies can be prohibitively expensive if domains are considered separately, and differences in copy number of eukaryotic ribosomal RNA genes can bias estimates of relative abundances of phylotypes recovered. In this study we have overcome these barriers (1) by targeting all three domains in a single polymerase chain reaction amplification and (2) by using a replicated sampling design that allows for incidence-based methods to extract measures of richness and carry out downstream analyses that address community structuring effects. Our work showed that combined bacterial and archaeal richness is an order of magnitude higher than eukaryotic richness. We also found that eukaryotic richness was highest at the most extreme sites, whereas combined bacterial and archaeal richness was highest at less extreme sites. Quantitative community phylogenetics showed abiotic forces to be primarily responsible for shaping the RT community structure. Canonical correspondence analysis revealed co-occurrence of obligate symbionts and their putative hosts that may contribute to biotic forces shaping community structure and may further provide a possible mechanism for persistence of certain low-abundance bacteria encountered in the RT.
Project description:We used fingerprinting and cloning-sequencing to study the spatiotemporal dynamics and diversity of Planctomycetes in two perialpine lakes with contrasting environmental conditions. Planctomycetes, which are less-abundant bacteria in freshwater ecosystems, appeared to be structured in the same way as the entire bacterial community in these ecosystems. They were more diversified and displayed fewer temporal variations in the hypolimnia than in the epilimnia. Like the more-abundant bacterial groups in aquatic systems, Planctomycetes communities seem to be composed of a very small number of abundant and widespread operational taxonomic units (OTUs) and a large number of OTUs that are present at low abundance. This indicates that the concept of "abundant or core" and "rare" bacterial phylotypes could also be applied to less-abundant freshwater bacterial phyla. The richness and diversity of Planctomycetes were mainly driven by pH and were similar in both of the lakes studied, whereas the composition of the Planctomycetes community seemed to be determined by a combination of factors including temperature, pH, and nutrients. The relative abundances of the dominant OTUs varied over time and were differently associated with abiotic factors. Our findings demonstrate that less-abundant bacterial phyla, such as Planctomycetes, can display strong spatial and seasonal variations linked to environmental conditions and suggest that their functional role in the lakes studied might be attributable mainly to a small number of phylotypes and vary over space and time in the water column.
Project description:The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity.
Project description:We analysed the 16S rRNA gene diversity within the bacterioplankton community in the water column of the ornamental fish Pterophyllum scalare and Archocentrus nigrofasciatus aquaria during a 60-day growth experiment in order to detect any dominant bacterial species and their possible association with the rearing organisms. The basic physical and chemical parameters remained stable but the bacterial community at 0, 30 and 60 days showed marked differences in bacterial cell abundance and diversity. We found high species richness but no dominant phylotypes were detected. Only few of the phylotypes were found in more than one time point per treatment and always with low relative abundance. The majority of the common phylotypes belonged to the Proteobacteria phylum and were closely related to Acinetobacter junii, Pseudomonas sp., Nevskia ramosa, Vogesella perlucida, Chitinomonas taiwanensis, Acidovorax sp., Pelomonas saccharophila and the rest belonged to the ?-Proteobacteria, Bacteroidetes, Actinobacteria, candidate division OP11 and one unaffiliated group. Several of these phylotypes were closely related to known taxa including Sphingopyxis chilensis, Flexibacter aurantiacus subsp. excathedrus and Mycobacterium sp. Despite the high phylogenetic diversity most of the inferred ecophysiological roles of the found phylotypes are related to nitrogen metabolism, a key process for fish aquaria.
Project description:The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.
Project description:The growth rate is a fundamental characteristic of bacterial species, determining its contributions to the microbial community and carbon flow. High-throughput sequencing can reveal bacterial diversity, but its quantitative inaccuracy precludes estimation of abundances and growth rates from the read numbers. Here, we overcame this limitation by normalizing Illumina-derived amplicon reads using an internal standard: a constant amount of Escherichia coli cells added to samples just before biomass collection. This approach made it possible to reconstruct growth curves for 319 individual OTUs during the grazer-removal experiment conducted in a freshwater reservoir ?ímov. The high resolution data signalize significant functional heterogeneity inside the commonly investigated bacterial groups. For instance, many Actinobacterial phylotypes, a group considered to harbor slow-growing defense specialists, grew rapidly upon grazers' removal, demonstrating their considerable importance in carbon flow through food webs, while most Verrucomicrobial phylotypes were particle associated. Such differences indicate distinct life strategies and roles in food webs of specific bacterial phylotypes and groups. The impact of grazers on the specific growth rate distributions supports the hypothesis that bacterivory reduces competition and allows existence of diverse bacterial communities. It suggests that the community changes were driven mainly by abundant, fast, or moderately growing, and not by rare fast growing, phylotypes. We believe amplicon read normalization using internal standard (ARNIS) can shed new light on in situ growth dynamics of both abundant and rare bacteria.
Project description:Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities.