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)...

Summary Although the molecular phylogeny, evolution and biodiversity of arbuscular mycorrhizal fungi (AMF) are becoming clearer, phylotaxonomically reliable sequence data are still limited. To fill this gap, a data set allowing resolution and environmental tracing across all taxonomic levels is provided. Two overlapping nuclear DNA regions, totalling c. 3 kb, were analysed: the small subunit (SSU) rRNA gene (up to 1800 bp) and a fragment spanning c. 250 bp of the SSU rDNA, the internal transcribed spacer (ITS) region (c. 475–520 bp) and c. 800 bp of the large subunit (LSU) rRNA gene. Both DNA regions together could be analysed for 35 described species, the SSU rDNA for c. 76 named and 18 as yet undefined species, and the ITS region or LSU rDNA, or a combination of both, for c. 91 named and 16 as yet undefined species. Present phylogenetic analyses, based on the three rDNA markers, provide reliable and robust resolution from phylum to species level. Altogether, 109 named species and 27 cultures representing as yet undefined species were analysed. This study provides a reference data set for molecular systematics and environmental community analyses of AMF, including analyses based on deep sequencing.

The publication of a large number of taxon names at all levels within the arbuscular mycorrhizal fungi (Glomeromycota) has resulted in conflicting systematic schemes and generated considerable confusion among biologists working with these important plant symbionts. A group of biologists with more than a century of collective experience in the systematics of Glomeromycota examined all available molecular-phylogenetic evidence within the framework of phylogenetic hypotheses, incorporating morphological characters when they were congruent. This study is the outcome, wherein the classification of Glomeromycota is revised by rejecting some new names on the grounds that they are founded in error and by synonymizing others that, while validly published, are not evidence-based. The proposed "consensus" will provide a framework for additional original research aimed at clarifying the evolutionary history of this important group of symbiotic fungi.

Summary At present, molecular ecological studies of arbuscular mycorrhizal fungi (AMF) are only possible above species level when targeting entire communities. To improve molecular species characterization and to allow species level community analyses in the field, a set of newly designed AMF specific PCR primers was successfully tested. Nuclear rDNA fragments from diverse phylogenetic AMF lineages were sequenced and analysed to design four primer mixtures, each targeting one binding site in the small subunit (SSU) or large subunit (LSU) rDNA. To allow species resolution, they span a fragment covering the partial SSU, whole internal transcribed spacer (ITS) rDNA region and partial LSU. The new primers are suitable for specifically amplifying AMF rDNA from material that may be contaminated by other organisms (e.g., samples from pot cultures or the field), characterizing the diversity of AMF species from field samples, and amplifying a SSU‐ITS‐LSU fragment that allows phylogenetic analyses with species level resolution. The PCR primers can be used to monitor entire AMF field communities, based on a single rDNA marker region. Their application will improve the base for deep sequencing approaches; moreover, they can be efficiently used as DNA barcoding primers.

Summary Currently, no official DNA barcode region is defined for the Fungi. The COX1 gene DNA barcode is difficult to apply. The internal transcribed spacer (ITS) region has been suggested as a primary barcode candidate, but for arbuscular mycorrhizal fungi (AMF; Glomeromycota) the region is exceptionably variable and does not resolve closely related species. DNA barcoding analyses were performed with datasets from several phylogenetic lineages of the Glomeromycota. We tested a c. 1500 bp fragment spanning small subunit (SSU), ITS region, and large subunit (LSU) nuclear ribosomal DNA for species resolving power. Subfragments covering the complete ITS region, c. 800 bp of the LSU rDNA, and three c. 400 bp fragments spanning the ITS2, the LSU‐D1 or LSU‐D2 domains were also analysed. Barcode gap analyses did not resolve all species, but neighbour joining analyses, using Kimura two‐parameter (K2P) distances, resolved all species when based on the 1500 bp fragment. The shorter fragments failed to separate closely related species. We recommend the complete 1500 bp fragment as a basis for AMF DNA barcoding. This will also allow future identification of AMF at species level based on 400 or 1000 bp amplicons in deep sequencing approaches.

Summary• Glomus intraradices-like fungi are the most intensely studied arbuscular mycorrhizal (AM) fungi. However, there are several AM fungi named as G. intraradices that may not be conspecific. Therefore, the hypothesis was tested that DAOM197198 and similar AM fungi, such as BEG195, correspond to the type of G. intraradices.• The G. intraradices isotype material, a descendant (INVAM FL208) of the type culture, and a morphologically corresponding AM fungus (MUCL49410) isolated from the type locality were studied and compared with several cultures of DAOM197198 and BEG195.• Phylogenetic analyses of the partial small subunit (SSU), complete internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA regions revealed two clades, one including G. intraradices FL208 and MUCL49410, the other containing DAOM197198 and BEG195.• The two clades were clearly separated by sequence analyses, despite the high intraspecific and intrasporal ITS region sequence divergence of up to > 23%. We conclude that the AM fungi with the identifiers DAOM197198 and BEG195 are not G. intraradices, but fall in a clade that contains the recently described species G. irregulare.

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Due to the potential of arbuscular mycorrhizal fungi (AMF, Glomeromycota) to improve plant growth and soil quality, the influence of agricultural practice on their diversity continues to be an important research question. Up to now studies of community diversity in AMF have exclusively been based on nuclear ribosomal gene regions, which in AMF show high intra-organism polymorphism, seriously complicating interpretation of these data. We designed specific PCR primers for 454 sequencing of a region of the largest subunit of RNA polymerase II gene, and established a new reference dataset comprising all major AMF lineages. This gene is known to be monomorphic within fungal isolates but shows an excellent barcode gap between species. We designed a primer set to amplify all known lineages of AMF and demonstrated its applicability in combination with high-throughput sequencing in a long-term tillage experiment. The PCR primers showed a specificity of 99.94% for glomeromycotan sequences. We found evidence of significant shifts of the AMF communities caused by soil management and showed that tillage effects on different AMF taxa are clearly more complex than previously thought. The high resolving power of high-throughput sequencing highlights the need for quantitative measurements to efficiently detect these effects.