Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative proteincoding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter-and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.DNA barcoding | fungal biodiversity T he absence of a universally accepted DNA barcode for Fungi, the second most speciose eukaryotic kingdom (1, 2), is a serious limitation for multitaxon ecological and biodiversity studies. DNA barcoding uses standardized 500-to 800-bp sequences to identify species of all eukaryotic kingdoms using primers that are applicable for the broadest possible taxonomic group. Reference barcodes must be derived from expertly identified vouchers deposited in biological collections with online metadata and validated by available online sequence chromatograms. Interspecific variation should exceed intraspecific variation (the barcode gap), and barcoding is optimal when a sequence is constant and unique to one species (3, 4). Ideally, the barcode locus would be the same for all kingdoms. A region of the mitochondrial gene encoding the cytochrome c oxidase subunit 1 (CO1) is the barcode for animals (3, 4) and the default marker adopted by the Consortium for the Barcode of Life for all groups of organisms, including fungi (5). In Oomycota, part of the kingdom Stramenopila historically studied by mycologists, the de facto barcode internal transcribed spacer (ITS) region is suitable for identification, but the default CO1 marker is more reliable in a few clades of closely related species (6)...

Dark septate endophytic (DSE) fungi represent a frequent root-colonizing fungal group common in environments with strong abiotic stress, such as (semi)arid ecosystems. This work aimed to study the DSE fungi colonizing the plants of semiarid sandy grasslands with wood steppe patches on the Great Hungarian Plain. As we may assume that fungi colonizing both invasive and native species are generalists, root associated fungi (RAF) were isolated from eight native and three invasive plant species. The nrDNA sequences of the isolates were used for identification. To confirm that the fungi were endophytes an artificial inoculation system was used to test the isolates: we considered a fungus as DSE if it colonized the roots without causing a negative effect on the plant and formed microsclerotia in the roots. According to the analyses of the ITS sequence of nrDNA the 296 isolates clustered into 41 groups. We found that 14 of these 41 groups were DSE, representing approximately 60% of the isolates. The main DSE groups were generalist and showed no specificity to area or season and colonized both native and invasive species, demonstrating that exotic plants are capable of using the root endophytic fungi of the invaded areas. The DSE community of the region shows high similarity to those found in arid grasslands of North America. Taking into account a previous hypothesis about the common root colonizers of those grasslands and our results reported here, we hypothesize that plants of (semi)arid grasslands share common dominant members of the DSE fungal community on a global scale.

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Dark septate endophytes (DSE) are a form-group of root endophytic fungi with elusive functions. Here, the genomes of two common DSE of semiarid areas, Cadophora sp. and Periconia macrospinosa were sequenced and analyzed with another 32 ascomycetes of different lifestyles. Cadophora sp. (Helotiales) and P. macrospinosa (Pleosporales) have genomes of 70.46 Mb and 54.99 Mb with 22,766 and 18,750 gene models, respectively. The majority of DSE-specific protein clusters lack functional annotation with no similarity to characterized proteins, implying that they have evolved unique genetic innovations. Both DSE possess an expanded number of carbohydrate active enzymes (CAZymes), including plant cell wall degrading enzymes (PCWDEs). Those were similar in three other DSE, and contributed a signal for the separation of root endophytes in principal component analyses of CAZymes, indicating shared genomic traits of DSE fungi. Number of secreted proteases and lipases, aquaporins, and genes linked to melanin synthesis were also relatively high in our fungi. In spite of certain similarities between our two DSE, we observed low levels of convergence in their gene family evolution. This suggests that, despite originating from the same habitat, these two fungi evolved along different evolutionary trajectories and display considerable functional differences within the endophytic lifestyle.

Dark septate endophytes (DSE) are distributed worldwide as root-colonising fungi, and frequent in environments with strong abiotic stress. DSE is not a taxon, but constitutes numerous fungal taxa belonging to several orders of Ascomycota. In this study we investigate three unidentified DSE lineages belonging to Pleosporales that were found previously in semiarid sandy grasslands. For molecular phylogenetic studies seven loci (ITS, partial 18S nrRNA, 28S nrRNA, actin, calmodulin, transcription-elongation factor 1- α and ß -tubulin genes) were amplified and sequenced. Based on morphology and the resulting molecular phylogeny these isolates were found to represent three novel genera within the Pleosporales, namely Aquilomyces, Flavomyces and Darksidea, with eight novel species. Molecular data revealed that monotypic Aquilomyces belongs to Morosphaeriaceae, monotypic Flavomyces represents an incertae sedis lineage related to Massarinaceae, and Darksidea, with six new species, is allied to the Lentitheciaceae. During this study we tested numerous conditions to induce sporulation, and managed for the first time to induce several DSE to form their sexual morphs.