The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

The ecological significance of fungi occurring asymptomatically inside living plant leaves is poorly understood. Given the broad saprotrophic potential of many endophytic fungi, we hypothesized that they persist in decaying litter for an extended period of time after leaf abscission. Fungal assemblages were assessed by highthroughput sequencing in autumn leaves of beech (Fagus sylvatica) and in the corresponding leaf litter in 388 samples from 22 beech forest plots in three widely distant regions of Germany. A considerable proportion of the leafendophytic fungi was also found in 1-year-old litter. Cooccurrence networks revealed that the fungi formed unstructured assemblages inside the living leaves, rather than well-structured communities. Previously endophytic fungi constituted an integral part of the fungal litter community and were by far the most active fungi in 1-year-old litter. We therefore consider these endophytic occurrences to represent transient stages. Composition of the aboveground microbiome appears therefore to be closely connected to the process of litter decomposition. Considering the respective linked fungal habitat will facilitate predicting nutrient and carbon cycling and storage in forest ecosystems as well as elucidating the ecology of leaf microbiomes.

Cryptococcus flavescens and C. terrestris are phenotypically indistinguishable sister species that belong to the order Tremellales (Tremellomycetes, Basidiomycota) and which may be mistaken for C. laurentii based on phenotype. Phylogenetic separation between C. flavescens and C. terrestris was based on rDNA sequence analyses, but very little is known on their intraspecific genetic variability or propensity for sexual reproduction. We studied 59 strains from different substrates and geographic locations, and used a multilocus sequencing (MLS) approach complemented with the sequencing of mating type (MAT) genes to assess genetic variation and reexamine the boundaries of the two species, as well as their sexual status. The following five loci were chosen for MLS: the rDNA ITS-LSU region, the rDNA IGS1 spacer, and fragments of the genes encoding the largest subunit of RNA polymerase II (RPB1), the translation elongation factor 1 alpha (TEF1) and the p21-activated protein kinase (STE20). Phylogenetic network analyses confirmed the genetic separation of the two species and revealed two additional cryptic species, for which the names Cryptococcus baii and C. ruineniae are proposed. Further analyses of the data revealed a high degree of genetic heterogeneity within C. flavescens as well as evidence for recombination between lineages detected for this species. Strains of C. terrestris displayed higher levels of similarity in all analysed genes and appear to make up a single recombining group. The two MAT genes (STE3 and SXI1/SXI2) sequenced for C. flavescens strains confirmed the potential for sexual reproduction and suggest the presence of a tetrapolar mating system with a biallelic pheromone/receptor locus and a multiallelic HD locus. In C. terrestris we could only sequence STE3, which revealed a biallelic P/R locus. In spite of the strong evidence for sexual recombination in the two species, attempts at mating compatible strains of both species on culture media were unsuccessful.

b Kwoniella mangrovensis has been described as a sexual species with a bipolar mating system. Phylogenetic analysis of multiple genes places this species together with Kwoniella heveanensis in the Kwoniella clade, a sister clade to that containing two pathogenic species of global importance, Cryptococcus neoformans and Cryptococcus gattii, within the Tremellales. Recent studies defining the mating type loci (MAT) of species in these clades showed that, with the exception of C. neoformans and C. gattii, which are bipolar with a single biallelic multigene MAT locus, several other species feature a tetrapolar mating system with two unlinked loci (homeodomain [HD] and pheromone/receptor [P/R] loci). We characterized several strains from the original study describing K. mangrovensis; two MAT regions were amplified and sequenced: the STE20 gene (P/R locus) and the divergently transcribed SXI1 and SXI2 genes (HD locus). We identified five different mating types with different STE20/SXI allele combinations that together with results of mating experiments demonstrate that K. mangrovensis is not bipolar but instead has a tetrapolar mating system. Sequence and gene analysis for a 43-kb segment of the K. mangrovensis type strain MAT locus revealed remarkable synteny with the homologous K. heveanensis MAT P/R region, providing new insights into slower evolution of MAT loci in the Kwoniella compared to the Cryptococcus clade of the Tremellales. The study of additional isolates from plant substrates in Europe and Botswana using a combination of multilocus sequencing with MAT gene analysis revealed two novel sibling species that we name Kwoniella europaea and Kwoniella botswanensis and which appear to also have tetrapolar mating systems. Fungal mating-type loci (MAT) are specialized regions of the genome that determine sexual identity of haploid cells and progression through the sexual cycle (1). There is considerable interest in the genetic characterization of MAT loci due to their central role in fungal life cycles, their connection to lifestyle and virulence (viz., in human or plant-pathogenic taxa), and the impact of sexual recombination on population genetics and speciation. The discovery of remarkable convergence in the structure of sex-determining genomic regions from studies in animals, plants, and fungi illuminates the forces that shape the evolutionary trajectories of MAT loci or sex chromosomes in eukaryotes (2).In basidiomycetes, two major types of MAT loci have so far been recognized (3). The tetrapolar mating system of the corn smut Ustilago maydis or the mushroom Coprinopsis cinerea is governed by two small (Ͻ10-kb) unlinked loci: one encodes members of the homeodomain (HD) family of transcription factors, which heterodimerize upon mating to generate an active transcription regulator (HD locus), and the other encodes lipopeptide pheromone precursors and 7-transmembrane pheromone receptors that mediate intercellular signaling (P/R locus). Alleles at both loci must differ for mating to occur, and in many cases each...

Epichloë endophytes associated with cool-season grass species can protect their hosts from herbivory and can suppress mycorrhizal colonization of the hosts’ roots. However, little is known about whether or not Epichloë endophyte infection can also change the foliar fungal assemblages of the host. We tested 52 grassland study sites along a land-use intensity gradient in three study regions over two seasons (spring vs. summer) to determine whether Epichloë infection of the host grass Lolium perenne changes the fungal community structure in leaves. Foliar fungal communities were assessed by Next Generation Sequencing of the ITS rRNA gene region. Fungal community structure was strongly affected by study region and season in our study, while land-use intensity and infection with Epichloë endophytes had no significant effects. We conclude that effects on non-systemic endophytes resulting from land use practices and Epichloë infection reported in other studies were masked by local and seasonal variability in this study’s grassland sites.

Nine yeast species belonging to genera Candida, Cryptococcus, Phaffomyces, Rhodotorula and Wickerhamomyces, and one species of Aureobasidium genus were isolated from the cloaca of migratory birds. Candida glabrata and C. inconspicua were the species most frequently isolated and Wickerhamomyces sylviae, which has recently been described as a new species isolated from bird cloaca, was again found. The majority of isolates showed the ability to grow up to 40 °C and/or at pH 3.0, two environmental conditions typical of the digestive tract of birds. The phylogenetic analysis of the D1/D2 domain of 26S rRNA gene placed the cultures of Phaffomyces in a new lineage that differed from the closest species, P. opuntiae, by 13 nucleotide substitutions. The new species was able to grow at 40 °C and at pH 2.5, which suggests a possible adaptation to the bird cloaca. Moreover, the ability to grow in the presence of digitonin at pH 3.7 and the assimilation of ethyl acetate indicates a potential cactophilic origin. For the first time, the presence of yeasts belonging to the Phaffomyces clade in Europe and also in non-cactus environments is reported. The new species is formally described as P. usticensis sp. nov. (PYCC 6346(T) = CBS 12958(T)).

Nitrate pollution of water resources is a global problem (Almasri, 2007). In some regions, the guideline values of groundwater and drinking water regulations are considerably exceeded in surface waters, but also in groundwater (Carrey et al., 2014). Several European Union member states, including Germany, were penalized by the European Court of Justice for non-compliance with the Nitrate Directive (European Court of Justice, 2010Justice, , 2018. However, further infringements and sanctions are to be expected. This contamination results mainly from anthropogenic N fertilizers used to increase agricultural productivity (Hosono et al., 2013). Excess N enters groundwater as NO 3 − . Due to a geogenic NO 3 − degradation capacity of most aquifers (sulfide minerals and organic C), part of the NO 3 − can be degraded (Rivett et al., 2008). Based on sulfide-S and organic C contents of the aquifer, groundwater recharge and NO 3 − concentration, the remaining time until a NO 3 − breakthrough to drinking water production wells may be calculated (e.g., Ortmeyer, Volkova, et al., 2021). Overall, a link between hydrologic and water quality models is important because including transit times can improve understanding of NO 3 − transport in aquifers (Hrachowitz et al., 2016). In the future, considerable increases in NO 3 − concentrations and NO 3 − breakthroughs to raw water wells are expected, as this degradation capacity is decreasing and finite (Knowles, 1982;Schwientek et al., 2017). Climate change is expected to enhance this deterioration of water resource quality and quantity (Fleck et al., 2017;Ortmeyer, Mas-Pla, et al., 2021). In addition to a decrease in groundwater recharge and a drop in water levels, Stuart et al. (2011) point to possible increasing rates of NO 3 − leaching under future climate scenarios. The thickness of the unsaturated zone is crucial for reaching NO 3 − peaks at the groundwater table (Wang et al., 2012). Nitrate storage in the vadose zone is also important, but often not considered in

Forest litter harbors complex networks of microorganisms whose major components are bacteria, fungi and protists. Protists, being highly selective consumers of bacteria and fungi could influence decomposition processes by shifting competitive microbial interactions. We investigated the eukaryotic diversity from 18 samples of one-year beech (Fagus sylvatica) leaf litter by RNA-based high-throughput sequencing of the small-subunit ribosomal RNA gene. By applying a metatranscriptomics approach, we avoided biases inherent to PCR-based methods, and could therefore focus on elusive protistan groups. We obtained 14 589 eukaryotic assembled sequences (contigs) representing 2223 unique taxa. Fungi dominated the eukaryotic assemblage, followed by an equal proportion of protists and plants. Among protists, the phylum Amoebozoa clearly dominated, representing more than twice the proportion of Alveolata (mostly ciliates) and Rhizaria (mostly Cercozoa), which are often retrieved as the dominant protistan groups in soils, revealing potential primer biases. By assigning functional traits to protists, we could assess that the proportion of free-living and heterotrophs was much higher than that of parasites and autotrophs, opening the way to a better understanding of the role played by the protistan communities and how biodiversity interacts with decomposition processes.