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Fungal metabarcoding of substrates such as soil, wood, and water is uncovering an unprecedented number of fungal species that do not seem to produce tangible morphological structures and that defy our best attempts at cultivation, thus falling outside the scope of the International Code of Nomenclature for algae, fungi, and plants. The present study uses the new, ninth release of the species hypotheses of the UNITE database to show that species discovery through environmental sequencing vastly outpaces traditional, Sanger sequencing-based efforts in a strongly increasing trend over the last five years. Our findings challenge the present stance of some in the mycological community - that the current situation is satisfactory and that no change is needed to "the code" - and suggest that we should be discussing not whether to allow DNA-based descriptions (typifications) of species and by extension higher ranks of fungi, but what the precise requirements for such DNA-based typifications should be. We submit a tentative list of such criteria for further discussion. The present authors hope for a revitalized and deepened discussion on DNA-based typification, because to us it seems harmful and counter-productive to intentionally deny the overwhelming majority of extant fungi a formal standing under the International Code of Nomenclature for algae, fungi, and plants. [ABSTRACT FROM AUTHOR]

Recent molecular surveys revealed an unexpected diversity of mycoplankton in lakes and oceans. The early diverging fungal lineages are known to be prominent parasites of phytoplankton. However, due to missing fungal reference data, their identity and ecology remain mostly unknown. To overcome this problem, we combined single-spore-based DNA barcoding during one sampling season of the eutrophic Lake Inba (Japan), which is dominated by two diatoms. By linking microscopically picked single fungal spore on two diatoms to subsequent DNA barcoding of ribosomal maker genes, we identified 12 distinct lineages, affiliated not only to the known parasitic phylum Chytridiomycota (chytrids), but also to the enigmatic phyla Rozellomycota and Aphelidiomycota. The detected Rozellomycota could be a hyperparasite of parasitic chytrid infecting diatoms. Host specificity appeared to be different among clades. However, the barcoding of single-spore DNA could not clearly prove host specificity due to the limited number of samples and resolution in the targeted gene regions. The degree of host specificity was thus confirmed by the cross-infection experiments. Five chytrid strains were generalists infecting all four diatoms strains, while the remaining three strains of chytrids were specialists exhibiting host preferences. Additional growth experiments indicated a trade-off between host specificity and growth rate, so that generalists with more possible hosts grew slower than specialists with one host. Our results suggest that the combination of microscopy with single-spore-based barcoding is a promising approach to evaluate the complex host-parasite interactions, while infection experiments can verify the interactions and shed light on the underlying ecological principles. [ABSTRACT FROM AUTHOR]

Sequence comparison and analysis of the various ribosomal genetic markers are the dominant molecular methods for identification and description of fungi. However, new environmental fungal lineages known only from DNA data reveal significant gaps in our sampling of the fungal kingdom in terms of both taxonomy and marker coverage in the reference sequence databases. To facilitate the integration of reference data from all of the ribosomal markers, we present three sets of general primers that allow for amplification of the complete ribosomal operon from the ribosomal tandem repeats. The primers cover all ribosomal markers: ETS, SSU, ITS1, 5.8S, ITS2, LSU and IGS. We coupled these primers successfully with third‐generation sequencing (PacBio and Nanopore sequencing) to showcase our approach on authentic fungal herbarium specimens (Basidiomycota), aquatic chytrids (Chytridiomycota) and a poorly understood lineage of early diverging fungi (Nephridiophagidae). In particular, we were able to generate high‐quality reference data with Nanopore sequencing in a high‐throughput manner, showing that the generation of reference data can be achieved on a regular desktop computer without the involvement of any large‐scale sequencing facility. The quality of the Nanopore generated sequences was 99.85%, which is comparable with the 99.78% accuracy described for Sanger sequencing. With this work, we hope to stimulate the generation of a new comprehensive standard of ribosomal reference data with the ultimate aim to close the huge gaps in our reference datasets. [ABSTRACT FROM AUTHOR]

Motivation Correct taxonomic identification of DNA sequences is central to studies of biodiversity using both shotgun metagenomic and metabarcoding approaches. However, no genetic marker gives sufficient performance across all the biological kingdoms, hampering studies of taxonomic diversity in many groups of organisms. This has led to the adoption of a range of genetic markers for DNA metabarcoding. While many taxonomic classification software tools can be re-trained on these genetic markers, they are often designed with assumptions that impair their utility on genes other than the SSU and LSU rRNA. Here, we present an update to Metaxa2 that enables the use of any genetic marker for taxonomic classification of metagenome and amplicon sequence data. Results We evaluated the Metaxa2 Database Builder on 11 commonly used barcoding regions and found that while there are wide differences in performance between different genetic markers, our software performs satisfactorily provided that the input taxonomy and sequence data are of high quality. Availability and implementation Freely available on the web as part of the Metaxa2 package at http://microbiology.se/software/metaxa2/. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Most studies of aquatic plankton focus on either macroscopic or microbial communities, and on either eukaryotes or prokaryotes. This separation is primarily for methodological reasons, but can overlook potential interactions among groups. Here we tested whether DNA metabarcoding of unfractionated water samples with universal primers could be used to qualitatively and quantitatively study the temporal dynamics of the total plankton community in a shallow temperate lake. Significant changes in the relative proportions of normalized sequence reads of eukaryotic and prokaryotic plankton communities over a 3-month period in spring were found. Patterns followed the same trend as plankton estimates measured using traditional microscopic methods. The bloom of a conditionally rare bacterial taxon belonging to Arcicella was characterized, which rapidly came to dominate the whole lake ecosystem and would have remained unnoticed without metabarcoding. The data demonstrate the potential of universal DNA metabarcoding applied to unfractionated samples for providing a more holistic view of plankton communities. [ABSTRACT FROM AUTHOR]