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Recent advances in sequencing technologies now permit the analyses of plant DNA from fossil samples (ancient plant DNA, plant aDNA), and thus enable the molecular reconstruction of palaeofloras.Hitherto, ancient frozen soils have proved excellent in preservingDNAmolecules, and have thus been the most commonly used source of plant aDNA. However, DNA from soil mainly represents taxa growing a fewmetres fromthe sampling point. Lakes have larger catchment areas and recent studies have suggested that plant aDNAfromlake sediments is a more powerful tool for palaeofloristic reconstruction. Furthermore, lakes can be found globally in nearly all environments, and are therefore not limited to perennially frozen areas. Here,we review the latest approaches and methods for the study of plant aDNA from lake sediments and discuss the progressmade up to the present.Weargue that aDNAanalyses add newand additional perspectives for the study of ancient plant populations and, in time, will provide higher taxonomic resolution and more precise estimation of abundance. Despite this, key questions and challenges remain for such plant aDNA studies. Finally, we provide guidelines on technical issues, including lake selection, and we suggest directions for future research on plant aDNA studies in lake sediments.

• Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. • We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. • Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. • Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.

• Disturbances have altered community dynamics in boreal forests with unknown consequences for belowground ecological processes. Soil fungi are particularly sensitive to such disturbances; however, the individual response of fungal guilds to different disturbance types is poorly understood. • Here, we profiled soil fungal communities in lodgepole pine forests following a bark beetle outbreak, wildfire, clear-cut logging, and salvage-logging. Using Illumina MiSeq to sequence ITS1 and SSU rDNA, we characterized communities of ectomycorrhizal, arbuscular mycorrhizal, saprotrophic, and pathogenic fungi in sites representing each disturbance type paired with intact forests. We also quantified soil fungal biomass by measuring ergosterol. • Abiotic disturbances changed the community composition of ectomycorrhizal fungi and shifted the dominance from ectomycorrhizal to saprotrophic fungi compared to intact forests. The disruption of the soil organic layer with disturbances correlated with the decline of ectomycorrhizal and the increase of arbuscular mycorrhizal fungi. Wildfire changed the community composition of pathogenic fungi but did not affect their proportion and diversity. Fungal biomass declined with disturbances that disrupted the forest floor. • Our results suggest that the disruption of the forest floor with disturbances, and the changes in C and nutrient dynamics it may promote, structure the fungal community with implications for fungal biomass–C.

Abstract Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization. Microbial ecology of industrial Kombucha fermentations.

Background The utility of environmental DNA (eDNA) metabarcoding surveys to accurately detect species depends on the degree of DNA dispersal. Multiple marine studies have observed only minimal eDNA transport by horizontal water movement across small spatial scales, leading to the conclusion that spatially specific eDNA signals accurately resemble in‐field species assemblages along a horizontal axis. Marine communities, however, are also structured vertically according to depth. In marine environments displaying permanent water stratification, vertical zonation patterns may be more apparent and present on smaller spatial scales (i.e., meters) than horizontal community structuring. The scale at which eDNA signals differ along a vertical transect and the accuracy of eDNA metabarcoding in revealing naturally stratified communities have yet to be assessed. Methods and results In this study, we determined the ability of eDNA metabarcoding surveys to distinguish vertically localized community assemblages. To test this, we sampled three vertical transects along a steep rock wall at three depths (0 m, 4 m, 15 m), covering two distinct communities that were separated by near‐permanent water column stratification in the form of a strong halocline at ~3 m. Using three metabarcoding assays, our eDNA metabarcoding survey detected 54 taxa, across 46 families and 7 phyla, including 19 fish, 15 crustacean, and 8 echinoderm species. Ordination and cluster analyses show distinct eDNA signals across the halocline for all three replicate transects, suggesting that vertical dispersal of eDNA between communities was limited. Furthermore, eDNA signals of individual taxa were only retrieved within their observed vertical distribution, providing biological validation for the obtained results. Our results demonstrate, for the first time, the need to take into consideration oceanographic (e.g. water column stratification) and biological processes (e.g. vertical community structuring) when designing sampling strategies for marine eDNA metabarcoding surveys. In this study, we determined the ability of eDNA metabarcoding surveys to distinguish vertically localized community assemblages. Our results demonstrate, for the first time, the need to take into consideration oceanographic (e.g., water column stratification) and biological processes (e.g., vertical community structuring) when designing sampling strategies for marine eDNA metabarcoding surveys.

• There is no consensus barcoding region for determination of arbuscular mycorrhizal fungal (AMF) taxa. To overcome this obstacle, we have developed an approach to sequence an AMF marker within the ribosome-encoding operon (rDNA) that covers all three widely applied variable molecular markers. • Using a nested PCR approach specific to AMF, we amplified a part (c. 2.5 kb) of the rDNA spanning the majority of the small subunit rRNA (SSU) gene, the complete internal transcribed spacer (ITS) region and a part of the large subunit (LSU) rRNA gene. The PCR products were sequenced on the PacBio platform utilizing Single Molecule Real Time (SMRT) sequencing. • Employing this method for selected environmental DNA samples, we were able to describe complex AMF communities consisting of various glomeromycotan lineages. • We demonstrate the applicability of this new 2.5 kb approach to provide robust phylogenetic assignment of AMF lineages without known sequences from pure cultures and to consolidate information about AMF taxon distributions coming from three widely used barcoding regions into one integrative dataset.

Next generation sequencing (NGS) technologies have led to a ubiquity of molecular sequence data. This data avalanche is particularly challenging in metagenetics, which focuses on taxonomic identification of sequences obtained from diverse microbial environments. Phylogenetic placement methods determine how these sequences fit into an evolutionary context. Previous implementations of phylogenetic placement algorithms, such as the evolutionary placement algorithm (EPA) included in RAxML, or PPLACER, are being increasingly used for this purpose. However, due to the steady progress in NGS technologies, the current implementations face substantial scalability limitations. Herein, we present EPA-NG, a complete reimplementation of the EPA that is substantially faster, offers a distributed memory parallelization, and integrates concepts from both, RAxML-EPA and PPLACER. EPA-NG can be executed on standard shared memory, as well as on distributed memory systems (e.g., computing clusters). To demonstrate the scalability of EPA-NG, we placed 1 billion metagenetic reads from the Tara Oceans Project onto a reference tree with 3748 taxa in just under 7 h, using 2048 cores. Our performance assessment shows that EPA-NG outperforms RAxML-EPA and PPLACER by up to a factor of 30 in sequential execution mode, while attaining comparable parallel efficiency on shared memory systems. We further show that the distributed memory parallelization of EPA-NG scales well up to 2048 cores. EPA-NG is available under the AGPLv3 license: https://github.com/Pbdas/epa-ng.

Multiple sequence alignment is a prerequisite for many evolutionary analyses. Multiple Alignment of Coding Sequences (MACSE) is a multiple sequence alignment program that explicitly accounts for the underlying codon structure of protein-coding nucleotide sequences. Its unique characteristic allows building reliable codon alignments even in the presence of frameshifts. This facilitates downstream analyses such as selection pressure estimation based on the ratio of nonsynonymous to synonymous substitutions. Here, we present MACSE v2, a major update with an improved version of the initial algorithm enriched with a complete toolkit to handle multiple alignments of protein-coding sequences. A graphical interface now provides user-friendly access to the different subprograms.

The study of environmental DNA (eDNA) has the potential to revolutionize biodiversity science and conservation action by enabling the census of species on a global scale in near real time. To achieve this promise, technical challenges must be resolved. In this review, we explore the main uses of eDNA as well as the complexities introduced by its misuse. Current eDNA methods require refinement and improved calibration and validation along the entire workflow to lessen false positives negatives. Moreover, there is great need for a better understanding of the \"natural history\" of eDNA-its origins, state, lifetime, and transportation-and for more detailed insights concerning the physical and ecological limitations of eDNA use. Although eDNA analysis can provide powerful information, particularly in freshwater and marine environments, its impact is likely to be less significant in terrestrial settings. The broad adoption of eDNA tools in conservation will largely depend on addressing current uncertainties in data interpretation.

Fish mitochondrial genome (mitogenome) data form a fundamental basis for revealing vertebrate evolution and hydrosphere ecology. Here, we report recent functional updates of MitoFish, which is a database of fish mitogenomes with a precise annotation pipeline MitoAnnotator. Most importantly, we describe implementation of MiFish pipeline for metabarcoding analysis of fish mitochondrial environmental DNA, which is a fast-emerging and powerful technology in fish studies. MitoFish, MitoAnnotator, and MiFish pipeline constitute a key platform for studies of fish evolution, ecology, and conservation, and are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed April 7th, 2018).