Or Operational Taxonomic Units Research Articles

Natrix2 – Improved amplicon workflow with novel Oxford Nanopore Technologies support and enhancements in clustering, classification and taxonomic databases

Sequencing of amplified DNA is the first step towards the generation of Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs) for biodiversity assessment and comparative analyses of environmental communities and microbiomes. Notably, the rapid advancements in sequencing technologies have paved the way for the growing utilization of third-generation long-read approaches in recent years. These sequence data imply increasing read lengths, higher error rates, and altered sequencing chemistry. Likewise, methods for amplicon classification and reference databases have progressed, leading to the expansion of taxonomic application areas and higher classification accuracy. With Natrix, a user-friendly and reducible workflow solution, processing of prokaryotic and eukaryotic environmental Illumina sequences using 16S or 18S is possible. Here, we present an updated version of the pipeline, Natrix2, which incorporates VSEARCH as an alternative clustering method with better performance for 16S metabarcoding approaches and mothur for taxonomic classification on further databases, including PR2, UNITE and SILVA. Additionally, Natrix2 includes the handling of Nanopore reads, which entails initial error correction and refinement of reads using Medaka and Racon to subsequently determine their taxonomic classification.

Improving FAIRness of eDNA and Metabarcoding Data: Standards and tools for European Nucleotide Archive data deposition

The advancements in sequencing technologies have promoted the generation of molecular data for cataloguing and describing biodiversity. The analysis of environmental DNA (eDNA) through the application of metabarcoding techniques enables comprehensive descriptions of communities and their function, being fundamental for understanding and preserving biodiversity. Metabarcoding is becoming widely used and standard methods are being generated for a growing range of applications with high scalability. The generated data can be made available in its unprocessed form, as raw data (the sequenced reads) or as interpreted data, including sets of sequences derived after bioinformatics processing (Amplicon Sequence Variants (ASVs) or Operational Taxonomic Units (OTUs)) and occurrence tables (tables that describe the occurrences and abundances of species or OTUs/ASVs). However, for this data to be Findable, Accessible, Interoperable and Reusable (FAIR), and therefore fully available for meaningful interpretation, it needs to be deposited in public repositories together with enriched sample metadata, protocols and analysis workflows (ten Hoopen et al. 2017). Metabarcoding raw data and associated sample metadata is often stored and made available through the International Nucleotide Sequence Database Collaboration (INSDC) archives (Arita et al. 2020), of which the European Nucleotide Archive (ENA, Burgin et al. 2022) is its European database, but it is often deposited with minimal information, which hinders data reusability. Within the scope of the Horizon 2020 project, Biodiversity Community Integrated Knowledge Library (BiCIKL), which is building a community of interconnected data for biodiversity research (Penev et al. 2022), we are working towards improving the standards for molecular ecology data sharing, developing tools to facilitate data deposition and retrieval, and linking between data types. Here we will present the ENA data model, showcasing how metabarcoding data can be shared, while providing enriched metadata, and how this data is linked with existing data in other research infrastructures in the biodiversity domain, such as the Global Biodiversity Information Facility (GBIF), where data is deposited following the guidelines published in Abarenkov et al. (2023). We will also present the results of our recent discussions on standards for this data type and discuss future plans towards continuing to improve data sharing and interoperability for molecular ecology.

EDNA Metabarcoding Data in the Global Biodiversity Information Facility (GBIF)

The Global Biodiversity Information Facility (GBIF) is an international network and data infrastructure that provides free and open access to biodiversity data from around the world, enabling scientists, policymakers, and the public to explore and analyze information about the Earth's living organisms. Originally developing at a distance from GBIF, metabarcoding of DNA has become a standard tool for detecting species in bulk samples or environmental samples such as soil, water, and air. Raw sequence data (fastq files) are often shared and deposited in dedicated repositories. Seen from a biodiversity documenting perspective, raw sequences have limited value, as several steps of bioinformatic processing and filtering are needed to arrive at a credible set of sequences that can be interpreted by comparing to a sequence reference database. Most often, such interpretated DNA metabarcoding data come in the shape a table with abundances of so-called Amplicon Sequence Variants (ASV) or Operational Taxonomic Units (OTU) across samples—a so-called ASV/OTU table—and some associated files, e.g., spatiotemporal and other sample metadata, and taxonomic inferences of sequences. In this session we present GBIF state-of-work and plans for developing and improving publishing and standardisation of OTU table-like biodiversity data for easier and broader reuse, including a prototype tool using the OTU table as a publishing model, mapping this research-familiar data format to the Darwin Core standard (Darwin Core Task Group 2009).

Phylotypic Diversity of Bacteria Associated with Speleothems of a Silicate Cave in a Guiana Shield Tepui.

The diversity of microorganisms associated with speleological sources has mainly been studied in limestone caves, while studies in silicate caves are still under development. Here, we profiled the microbial diversity of opal speleothems from a silicate cave in Guiana Highlands. Bulk DNAs were extracted from three speleothems of two types, i.e., one soft whitish mushroom-like speleothem and two hard blackish coral-like speleothems. The extracted DNAs were amplified for sequencing the V3–V4 region of the bacterial 16S rRNA gene by MiSeq. A total of 210,309 valid reads were obtained and clustered into 3184 phylotypes or operational taxonomic units (OTUs). The OTUs from the soft whitish speleothem were mostly affiliated with Acidobacteriota, Pseudomonadota (formerly, Proteobacteria), and Chloroflexota, with the OTUs ascribed to Nitrospirota being found specifically in this speleothem. The OTUs from the hard blackish speleothems were similar to each other and were mostly affiliated with Pseudomonadota, Acidobacteriota, and Actinomycetota (formerly, Actinobacteria). These OTU compositions were generally consistent with those reported for limestone and silicate caves. The OTUs were further used to infer metabolic features by using the PICRUSt bioinformatic tool, and membrane transport and amino acid metabolism were noticeably featured. These and other featured metabolisms may influence the pH microenvironment and, consequently, the formation, weathering, and re-deposition of silicate speleothems.

A database and checklist of geometrid moths (Lepidoptera) from Colombia.

BackgroundMolecular DNA sequence data allow unprecedented advances in biodiversity assessments, monitoring schemes and taxonomic works, particularly in poorly-explored areas. They allow, for instance, the sorting of material rapidly into operational taxonomic units (such as BINs - Barcode Index Numbers), sequences can be subject to diverse analyses and, with linked metadata and physical vouchers, they can be examined further by experts. However, a prerequisite for their exploitation is the construction of reference libraries of DNA sequences that represent the existing biodiversity. To achieve these goals for Geometridae (Lepidoptera) moths in Colombia, expeditions were carried out to 26 localities in the northern part of the country in 2015–2019. The aim was to collect specimens and sequence their DNA barcodes and to record a fraction of the species richness and occurrences in one of the most biodiversity-rich countries. These data are the beginning of an identification guide to Colombian geometrid moths, whose identities are currently often provisional only, being morpho species or operational taxonomic units (OTUs). Prior to the current dataset, 99 Geometridae sequences forming 44 BINs from Colombia were publicly available on the Barcode of Life Data System (BOLD), covering 20 species only.New informationWe enrich the Colombian Geometridae database significantly by including DNA barcodes, two nuclear markers, photos of vouchers and georeferenced occurrences of 281 specimens of geometrid moths from different localities. These specimens are classified into 80 genera. Analytical tools on BOLD clustered 157 of the mentioned sequences to existing BINs identified to species level, identified earlier by experts. Another 115 were assigned to BINs that were identified to genus or tribe level only. Eleven specimens did not match any existing BIN on BOLD and are, therefore, new additions to the database. It is likely that many BINs represent undescribed species. Nine short sequences (< 500bp) were not assigned to BINs, but identified to the lowest taxonomic category by expert taxonomists and with comparisons of type material photos. The released new genetic information will help to further progress the systematics of Geometridae. An illustrated catalogue of all new records allows validation of our identifications; it is also the first document of this kind for Colombian Geometridae. All specimens are deposited at the Museo de Zoología of Universidad de Sucre (MZUS), North Colombia. DNA BINs are reported in this study through dx.doi.org/10.5883/DS-GEOCO, the species occurrences are available on SIB Colombia https://sibcolombia.net/ and the Global Biodiversity Information Facility (GBIF) https://www.gbif.org/ through https://doi.org/10.15472/ucfmkh.

Allopatric differentiation in the Enteromius anoplus complex in South Africa, with the revalidation of Enteromius cernuus and Enteromius oraniensis, and description of a new species, Enteromius mandelai (Teleostei: Cyprinidae).

The chubbyhead barb, Enteromius anoplus, as currently described, is the most widely distributed freshwater fish in South Africa. The species occurs in almost all the major river systems across the country, with the exception of the small coastal drainages on the south coast. The use of a comprehensive data set of mitochondrial (mtDNA) cytochrome b (cyt b) sequences uncovered the presence of four distinct lineages or operational taxonomic units (OTUs) within E. anoplus: (a) the Gouritz lineage endemic to the Gouritz River system, (b) the Olifants lineage endemic to the Olifants-Doring River system, (c) the Orange lineage endemic to the Orange River system and (d) the Eastern Cape lineage which is distributed across six river systems from the Great Fish to the Buffalo rivers in the Eastern Cape Province of South Africa. The present study provides a new diagnosis for E. anoplus s.s. (the Gouritz lineage), revalidates Enteromius cernuus (the Olifants lineage) and Enteromius oraniensis (the Orange lineage) as distinct species. The study also provides a description for a new species, Enteromius mandelai sp. nov (the Eastern Cape lineage). E. cernuus and E. mandelai differ from both E. anoplus and E. oraniensis by having a complete lateral line (vs. an incomplete lateral line in the latter species). E. cernuus further differs from the other three species by having long maxillary barbels which reach or exceed the vertical through the middle of the eye and the lowest number of circumpendicular scales (10-12 vs. 12-16 for the other three species). E. oraniensis is distinctive from the other three species by having inconspicuous barbels. These findings add to the growing body of literature that shows that the freshwater fishes of the Cape Fold and adjacent freshwater ecoregions have narrow geographic ranges. This has ramifications for conservation planning and management, as well as the understanding of the evolutionary history of the stream fishes in these global endemic hotspots.

Global Geographic and Temporal Analysis of SARS-CoV-2 Haplotypes Normalized by COVID-19 Cases During the Pandemic.

Since the identification of SARS-CoV-2, a large number of genomes have been sequenced with unprecedented speed around the world. This marks a unique opportunity to analyze virus spreading and evolution in a worldwide context. Currently, there is not a useful haplotype description to help to track important and globally scattered mutations. Also, differences in the number of sequenced genomes between countries and/or months make it difficult to identify the emergence of haplotypes in regions where few genomes are sequenced but a large number of cases are reported. We propose an approach based on the normalization by COVID-19 cases of relative frequencies of mutations using all the available data to identify major haplotypes. Furthermore, we can use a similar normalization approach to tracking the temporal and geographic distribution of haplotypes in the world. Using 171,461 genomes, we identify five major haplotypes or operational taxonomic units (OTUs) based on nine high-frequency mutations. OTU_3 characterized by mutations R203K and G204R is currently the most frequent haplotype circulating in four of the six continents analyzed (South America, North America, Europe, Asia, Africa, and Oceania). On the other hand, during almost all months analyzed, OTU_5 characterized by the mutation T85I in nsp2 is the most frequent in North America. Recently (since September), OTU_2 has been established as the most frequent in Europe. OTU_1, the ancestor haplotype, is near to extinction showed by its low number of isolations since May. Also, we analyzed whether age, gender, or patient status is more related to a specific OTU. We did not find OTU’s preference for any age group, gender, or patient status. Finally, we discuss structural and functional hypotheses in the most frequently identified mutations, none of those mutations show a clear effect on the transmissibility or pathogenicity.

Improved normalization of species count data in ecology by scaling with ranked subsampling (SRS): application to microbial communities.

BackgroundAnalysis of species count data in ecology often requires normalization to an identical sample size. Rarefying (random subsampling without replacement), which is the current standard method for normalization, has been widely criticized for its poor reproducibility and potential distortion of the community structure. In the context of microbiome count data, researchers explicitly advised against the use of rarefying. Here we introduce a normalization method for species count data called scaling with ranked subsampling (SRS) and demonstrate its suitability for the analysis of microbial communities.MethodsSRS consists of two steps. In the scaling step, the counts for all species or operational taxonomic units (OTUs) are divided by a scaling factor chosen in such a way that the sum of scaled counts equals the selected total number of counts Cmin. The relative frequencies of all OTUs remain unchanged. In the subsequent ranked subsampling step, non-integer count values are converted into integers by an algorithm that minimizes subsampling error with regard to the population structure (relative frequencies of species or OTUs) while keeping the total number of counts equal Cmin. SRS and rarefying were compared by normalizing a test library representing a soil bacterial community. Common parameters of biodiversity and population structure (Shannon index H’, species richness, species composition, and relative abundances of OTUs) were determined for libraries normalized to different size by rarefying as well as SRS with 10,000 replications each. An implementation of SRS in R is available for download (https://doi.org/10.20387/BONARES-2657-1NP3).ResultsSRS showed greater reproducibility and preserved OTU frequencies and alpha diversity better than rarefying. The variance in Shannon diversity increased with the reduction of the library size after rarefying but remained zero for SRS. Relative abundances of OTUs strongly varied among libraries generated by rarefying, whereas libraries normalized by SRS showed only negligible variation. Bray–Curtis index of dissimilarity among replicates of the same library normalized by rarefying revealed a large variation in species composition, which reached complete dissimilarity (not a single OTU shared) among some libraries rarefied to a small size. The dissimilarity among replicated libraries normalized by SRS remained negligibly low at each library size. The variance in dissimilarity increased with the decreasing library size after rarefying, whereas it remained either zero or negligibly low after SRS.ConclusionsNormalization of OTU or species counts by scaling with ranked subsampling preserves the original community structure by minimizing subsampling errors. We therefore propose SRS for the normalization of biological count data.

Differential Gut Scfa Microbial Taxa Correlated With Blood Pressure Status In African American Collegiate Athletes

INTRODUCTION: The gut microbiome and reduced short-chain fatty acid (SCFA) producing microbes have been related to hypertension status in sedentary individuals. Hypertension is common amongst athletes and epidemiologic data reports that cardiovascular sudden death is more common in African Americans (AA) (5-fold), compared to whites, and is related to the elevated prevalence of hypertension independently in athletes and in AA. Exercise is generally known to reduce blood pressure (BP) and stimulates beneficial changes in the gut microbiome to promote gut health (increasing gut SCFA), but it is unknown whether there are differential gut microbial characteristics related to BP status in athletes. PURPOSE: To determine gut microbial characteristics related to gut SCFA in AA collegiate athletes with and without hypertension and identify specific microbial taxa related to BP status. METHODS: The present work included 30 AA collegiate athletes stratified by normal BP (systolic BP (SBP) ≤129 mmHg; n=15) and high BP (SBP ≥130 mmHg; n=15) and we performed 16S rRNA gene sequencing on fecal samples. RESULTS: Relative to BP status, we did not observe any significant differences in alpha or beta diversity, or operational taxonomic units (OTUs). However, we observed that SCFA producing microbes were differentially abundant between the 2 groups and the relative abundance of some microbes were significantly correlated with systolic BP (g_Lactococcus, R=0.5 p=0.0074; g_Aldercruetzia, R=0.59 p=0.001; g_Paraprevotella, R=-0.38 p=0.044; g_cc_115, R=0.41 p=0.29). CONCLUSION: We report that SCFA producing microbes were differentially abundant in AA collegiate athletes stratified by BP status. Although exercise training broadly increases SCFA microbes in the gut, identification of microbial community characteristics and specific taxa will provide insight into gut microbial functional profiles related to greater BP in AA collegiate athletes.

Emerging threat ofEimeriaoperational taxonomic units (OTUs) on poultry production

Coccidiosis is an antagonistic poultry disease which negatively impacts animal welfare and productivity. The disease is caused by an obligate, intracellular protozoon known as Eimeria. Several Eimeria species known to infect chickens have been well documented. However, recent studies have elucidated the emergence of three novel genetic variants or operational taxonomic units (OTUs). The discovery of OTUx, OTUy and OTUz complicates the identification and diagnosis of coccidiosis. OTUs are clusters of unknown or uncultivated organisms that are grouped according to a similarity in DNA sequence to a set of specific gene markers. OTUs have been reported in the Earth's Southern Hemisphere, including Australia, Venezuela, India, Zambia, Uganda, Tanzania, China and Ghana. Elucidating their impact on the poultry industry is fundamental in preventing anticoccidial resistance and to access the potential of OTUs as vaccine candidates to provide cross-protection against similar Eimeria species. The identification of OTUs further decreases the risk of false negative coccidial diagnosis. Therefore, this article reviews the importance and risk imposed by OTUs, coupled with their prevalence and geographical distribution in chickens globally.

Take care of the environment: housing conditions affect the interplay of nutritional interventions and intestinal microbiota in broiler chickens

BackgroundThe intestinal microbiota is shaped by many interactions between microorganisms, host, diet, and the environment. Exposure to microorganisms present in the environment, and exchange of microorganisms between hosts sharing the same environment, can influence intestinal microbiota of individuals, but how this affects microbiota studies is poorly understood. We investigated the effects of experimental housing circumstances on intestinal microbiota composition in broiler chickens, and how these effects may influence the capacity to determine diet related effects in a nutrition experiment. A cross-sectional experiment was conducted simultaneously in a feed research facility with mesh panels between pens (Housing condition 1, H1), in an extensively cleaned stable with floor pens with solid wooden panels (H2), and in isolators (H3). In H1 and H2 different distances between pens were created to assess gut microbiota exchange between pens. Feed with and without a blend of medium-chain fatty acids (MCFA) was used to create differences in cecal microbiota between pens or isolators within the same housing condition. Male one-day-old Ross broiler chickens (n = 370) were randomly distributed across H1, H2, and H3. After 35 days cecal microbiota composition was assessed by 16S ribosomal RNA gene amplicon sequencing. Metabolic functioning of cecal content was assessed based on high-performance liquid chromatography.ResultsMicrobial alpha diversity was not affected in broilers fed +MCFA in H1 but was increased in H2 and H3. Based on weighted UniFrac distances, the nutritional intervention explained 10%, whereas housing condition explained 28% of cecal microbiota variation between all broilers. The effect size of the nutritional intervention varied within housing conditions between 11, 27, and 13% for H1, H2, and H3. Furthermore, performance and metabolic output were significantly different between housing conditions. The distance between pens within H1 and H2 did not influence the percentage of shared genera or operational taxonomic units (OTUs).ConclusionsThe cecal microbiota of broilers was modifiable by a nutritional intervention, but the housing condition affected microbiota composition and functionality stronger than the diet intervention. Consequently, for interpretation of intestinal microbiota studies in poultry it is essential to be aware of the potentially large impact of housing conditions on the obtained results.

Quantifying and Understanding Well-to-Well Contamination in Microbiome Research.

Microbial sequences inferred as belonging to one sample may not have originated from that sample. Such contamination may arise from laboratory or reagent sources or from physical exchange between samples. This study seeks to rigorously assess the behavior of this often-neglected between-sample contamination. Using unique bacteria, each assigned a particular well in a plate, we assess the frequency at which sequences from each source appear in other wells. We evaluate the effects of different DNA extraction methods performed in two laboratories using a consistent plate layout, including blanks and low-biomass and high-biomass samples. Well-to-well contamination occurred primarily during DNA extraction and, to a lesser extent, in library preparation, while barcode leakage was negligible. Laboratories differed in the levels of contamination. Extraction methods differed in their occurrences and levels of well-to-well contamination, with plate methods having more well-to-well contamination and single-tube methods having higher levels of background contaminants. Well-to-well contamination occurred primarily in neighboring samples, with rare events up to 10 wells apart. This effect was greatest in samples with lower biomass and negatively impacted metrics of alpha and beta diversity. Our work emphasizes that sample contamination is a combination of cross talk from nearby wells and background contaminants. To reduce well-to-well effects, samples should be randomized across plates, samples of similar biomasses should be processed together, and manual single-tube extractions or hybrid plate-based cleanups should be employed. Researchers should avoid simplistic removals of taxa or operational taxonomic units (OTUs) appearing in negative controls, as many will be microbes from other samples rather than reagent contaminants.IMPORTANCE Microbiome research has uncovered magnificent biological and chemical stories across nearly all areas of life science, at times creating controversy when findings reveal fantastic descriptions of microbes living and even thriving in what were once thought to be sterile environments. Scientists have refuted many of these claims because of contamination, which has led to robust requirements, including the use of controls, for validating accurate portrayals of microbial communities. In this study, we describe a previously undocumented form of contamination, well-to-well contamination, and show that this sort of contamination primarily occurs during DNA extraction rather than PCR, is highest with plate-based methods compared to single-tube extraction, and occurs at a higher frequency in low-biomass samples. This finding has profound importance in the field, as many current techniques to "decontaminate" a data set simply rely on an assumption that microbial reads found in blanks are contaminants from "outside," namely, the reagents or consumables.

Identification of harmful protozoa in outdoor cultivation of Chlorella and the use of ultrasonication to control contamination

Contaminating organisms in mass cultivation present one of the major challenges that must be overcome for successful commercialization of algal biofuels. The present study identified a range of contaminating organisms in Chlorella cultures cultivated in outdoor raceway ponds at the Arizona Center for Algae Technology and Innovation (Mesa, AZ). Nineteen organisms or Operational Taxonomic Units (OTU) in the Chlorella culture were identified by a combination of microscopic observation and 18S rRNA denaturing gradient gel electrophoresis (DGGE). More detailed analyses identified these contaminating organisms as 2 fungi, 7 flagellates, 3 amoebae, 4 ciliates, 1 rotifer, and 2 large insects. Among them Poterioochromonas sp., a small flagellate, appeared to be one of the most harmful causing culture collapse. In order to control Poterioochromonas sp., various operational parameters and application strategies of ultrasonic treatment were investigated. During sixteen-day consecutive Chlorella cultivation in batch mode, the ultrasonication conditions of 6 L min−1 flow rate with the power of 495 W at 100% amplitude and a treatment frequency of once for 1 h every day, was proven to be the most effective in preventing Poterioochromonas outbreak in Chlorella culture with volume of 60 L. The above ultra-sonication method was also effective at destroying an unknown fungus, an amoeba (Acanthocystis sp.), and ciliates (a member of the family Orchitophryidae). Our findings can serve as a technical foundation for the application of ultrasonication to control some of the contaminating microorganisms in mass cultivation of microalgae including Chlorella.

Molecular Identification of Bacteria in Intra-abdominal Abscesses Using Deep Sequencing

Intra-abdominal abscesses are localized collections of pus, which generally arise from a breach in the normal mucosal defense barrier that allows bacteria from gastrointestinal tract, and less commonly from the gynecologic or urinary tract, to induce inflammation, resulting in an infection. The microbiology of these abscesses is usually polymicrobial, associated with the primary disease process. However, the microbial identity, diversity and richness in intra-abdominal abscesses have not been well characterized, due in part to the difficulty in cultivating commensal organisms using standard culture-based techniques. We used culture-independent 16S rRNA Illumina sequencing to characterize bacterial communities in intra-abdominal abscesses collected by percutaneous drainage. A total of 43 abscess samples, including 19 (44.2%) Gram stain and culture-negative specimens, were analyzed and compared with results from conventional microbiologic cultures. Microbial composition was determined in 8 of 19 culture-negative samples and 18 of 24 culture-positive samples, identifying a total of 221 bacterial taxa or operational taxonomic units (OTUs) and averaging 13.1 OTUs per sample (interquartile range, 8-16.5 OTUs). Microbial richness for monomicrobial and polymicrobial samples was significantly higher than culture-negative samples (17 and 15.2 OTUs vs 8 OTUs, respectively), with a trend toward a higher microbial diversity (Shannon diversity index of 0.87 and 1.18 vs 0.58, respectively). The bacterial consortia identified by cultures correlated poorly with the microbial composition determined by 16S rRNA sequencing, and in most cases, the cultured isolates were minority constituents of the overall abscess microbiome. Intra-abdominal abscesses were generally polymicrobial with a surprisingly high microbial diversity, but standard culture-based techniques failed to reveal this diversity. These data suggest that molecular-based approaches may be helpful for documenting the presence of bacteria in intra-abdominal abscesses where standard cultures are unrevealing, particularly in the setting of prior antibiotic exposure.

Which of soil microbes is in positive correlation to yields of maize (Zea mays L.)?

Soil microorganisms are critical to maintain soil function, enhance plant health and increase crop yields. This study investigated the effects of organic matter on soil microbial community and assessed which of soil microbes were in positive correlation to maize yields. The results showed that different fertilizer treatments shaped specific microbial communities in the same soils. The most abundant beneficial soil microbes were found in treatments with organic fertilizer produced from cattle manure, return of wheat straw and 70% NPK admixture fertilizers treatment. The correlation analysis revealed that maize yields were in no correlation both to the shifts of soil microbial community structure and to the number of sequences or operational taxonomic units (OTUs) in soil microbes. However, maize yields were in positive correlation to microbial community structure shifts at the species level. 35 bacteria OTUs from 19 orders in 14 classes in 9 phyla were in positive correlation to yields of maize, while in fungi only one OTU&lt;sub&gt;25&lt;/sub&gt; belonging to Sordariales was in positive correlation. Our results indicate that the long-term application of organic and inorganic amendments could enrich the soil bacterial and fungal community and promote its diversity.

Bayesian Nonparametric Ordination for the Analysis of Microbial Communities

ABSTRACTHuman microbiome studies use sequencing technologies to measure the abundance of bacterial species or Operational Taxonomic Units (OTUs) in samples of biological material. Typically the data are organized in contingency tables with OTU counts across heterogeneous biological samples. In the microbial ecology community, ordination methods are frequently used to investigate latent factors or clusters that capture and describe variations of OTU counts across biological samples. It remains important to evaluate how uncertainty in estimates of each biological sample’s microbial distribution propagates to ordination analyses, including visualization of clusters and projections of biological samples on low-dimensional spaces. We propose a Bayesian analysis for dependent distributions to endow frequently used ordinations with estimates of uncertainty. A Bayesian nonparametric prior for dependent normalized random measures is constructed, which is marginally equivalent to the normalized generalized Gamma process, a well-known prior for nonparametric analyses. In our prior, the dependence and similarity between microbial distributions is represented by latent factors that concentrate in a low-dimensional space. We use a shrinkage prior to tune the dimensionality of the latent factors. The resulting posterior samples of model parameters can be used to evaluate uncertainty in analyses routinely applied in microbiome studies. Specifically, by combining them with multivariate data analysis techniques we can visualize credible regions in ecological ordination plots. The characteristics of the proposed model are illustrated through a simulation study and applications in two microbiome datasets. Supplementary materials for this article are available online.

Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys.

Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.

Algorithmic single-locus species delimitation: effects of sampling effort, variation and nonmonophyly in four methods and 1870 species of beetles.

The vast number of undescribed species and the fast rate of biodiversity loss call for new approaches to speed up alpha taxonomy. A plethora of methods for delimiting species or operational taxonomic units (OTUs) based on sequence data have been published in recent years. We test the ability of four delimitation methods (BIN, ABGD, GMYC, PTP) to reproduce established species boundaries on a carefully curated DNA barcode data set of 1870 North European beetle species. We also explore how sampling effort, intraspecific variation, nearest neighbour divergence and nonmonophyly affect the OTU delimitations. All methods produced approximately 90% identity between species and OTUs. The effects of variation and sampling differed between methods. ABGD was sensitive to singleton sequences, while GMYC showed tendencies for oversplitting. The best fit between species and OTUs was achieved using simple rules to find consensus between discordant OTU delimitations. Using several approaches simultaneously allows the methods to compensate for each other's weaknesses. Barcode-based OTU-picking is an efficient way to delimit putative species from large data sets where the use of more sophisticated methods based on multilocus or genomic data is not feasible.

Phylogenetic Profiling and Diversity of Bacterial Communities in the Death Valley, an Extreme Habitat in the Atacama Desert.

The Atacama Desert, one of the driest deserts in the world, represents a unique extreme environmental ecosystem to explore the bacterial diversity as it is considered to be at the dry limit for life. A 16S rRNA gene (spanning the hyper variable V3 region) library was constructed from an alkaline sample of unvegetated soil at the hyperarid margin in the Atacama Desert. A total of 244 clone sequences were used for MOTHUR analysis, which revealed 20 unique phylotypes or operational taxonomic units (OTUs). V3 region amplicons of the 16S rRNA were suitable for distinguishing the bacterial community to the genus and specie level. We found that all OTUs were affiliated with taxa representative of the Firmicutes phylum. The extremely high abundance of Firmicutes indicated that most bacteria in the soil were spore-forming survivors. In this study we detected a narrower diversity as compared to other ecological studies performed in other areas of the Atacama Desert. The reported genera were Oceanobacillus (representing the 69.5% of the clones sequenced), Bacillus, Thalassobacillus and Virgibacillus. The present work shows physical and chemical parameters have a prominent impact on the microbial community structure. It constitutes an example of the communities adapted to live in extreme conditions caused by dryness and metal concentrations .

Multiple genetic lineages challenge the monospecific status of the West African endemic frog family Odontobatrachidae.

BackgroundCorrect species identification is crucial in different fields of biology, and in conservation. The endemic West African frog family Odontobatrachidae currently contains a single described species, Odontobatrachus natator. From western Guinea to western Côte d'Ivoire it inhabits forests around waterfalls or cascades. Based on more than 130 specimens from 78 localities, covering the entire distribution, we investigated the molecular diversity of these frogs.ResultsOur analyses included mitochondrial and nuclear genes, with a concatenated alignment of 3527 base pairs. We detected high level of genetic differentiation with five distinct lineages or operational taxonomic units (OTUs). These OTUs were also identified by two different species delimitation approaches, Generalized Mixed Yule Coalescent (GMYC) and cluster algorithm. All OTUs occur in parapatry in the Upper Guinean forests. One OTU, assigned to the “true” Odontobatrachus natator, covers the largest distribution, ranging from the border region of western Sierra Leone-Guinea to eastern Liberia. Two OTUs are restricted to western Guinea (Fouta Djallon and foothills), while two others occur in eastern Guinea and the border region of Guinea-Liberia-Côte d'Ivoire. The OTU representing O. natator consists of two divergent subclades: one restricted to the Freetown Peninsula (Sierra Leone) and the other covering all populations further inland. Environmental niche models indicated that the restricted Freetown Peninsula population is separated by unsuitable habitat from remaining populations.ConclusionGeographic isolation of OTUs and molecular differences comparable to species level differentiation in other frog families indicate that O. natator contains cryptic species diversity. Respective distribution patterns most probably resulted from repeated changes of forest cover (contraction and expansion) over evolutionary timescales. The survival within forest refugia that have persisted through multiple drier periods and subsequent dispersal during wetter times may best explain the observed geographic distributions of OTUs. According to the IUCN Red List range criteria each OTU should be classified as “Endangered.” If the Freetown Peninsula “natator” population is recognized as a distinct species it would warrant recognition as “Critically Endangered.” The identification of cryptic lineages highlights the urgent need to protect these frogs, all of which are endemic to small areas within the Upper Guinean biodiversity hotspot.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0346-9) contains supplementary material, which is available to authorized users.