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)...

DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequence similarity searches with NR_prefixed accession numbers. A set of standards and protocols is proposed to improve the data quality of new sequences, and we suggest how type and other reference sequences can be used to improve identification of Fungi.Database URL: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353

The lichen-forming fungal genus Cladonia is species-rich with approximately 500 described species. The accepted barcode for fungi (ITS rDNA) often fails in identifying Cladonia spp. In order to find other markers that, in combination with the ITS rDNA region can be used for species identification in Cladonia, we studied the loci IGS rDNA, ef1α, rpb2 and cox1. A total of 782 sequences from 36 species have been analyzed. PCR amplification success rate, intraspecific and interspecific genetic distance variation, calculated using the K2P model, and the correct identification percentage (PCI) were taken into account to assess possible barcode regions. The marker showing the least intraspecific genetic distance range was cox1, followed by ITS rDNA and ef1α. Of the five studied markers only cox1 showed a barcoding gap. The rpb2 locus showed the highest PCI values, but it was the most difficult to amplify. The highest correct identification rates using blast method were obtained with rpb2.

Nomenclatural type definitions are one of the most important concepts in biological nomenclature. Being physical objects that can be re-studied by other researchers, types permanently link taxonomy (an artificial agreement to classify biological diversity) with nomenclature (an artificial agreement to name biological diversity). Two proposals to amend the International Code of Nomenclature for algae, fungi, and plants (ICN), allowing DNA sequences alone (of any region and extent) to serve as types of taxon names for voucherless fungi (mainly putative taxa from environmental DNA sequences), have been submitted to be voted on at the 11th International Mycological Congress (Puerto Rico, July 2018). We consider various genetic processes affecting the distribution of alleles among taxa and find that alleles may not consistently and uniquely represent the species within which they are contained. Should the proposals be accepted, the meaning of nomenclatural types would change in a fundamental way from physical objects as sources of data to the data themselves. Such changes are conducive to irreproducible science, the potential typification on artefactual data, and massive creation of names with low information content, ultimately causing nomenclatural instability and unnecessary work for future researchers that would stall future explorations of fungal diversity. We conclude that the acceptance of DNA sequences alone as types of names of taxa, under the terms used in the current proposals, is unnecessary and would not solve the problem of naming putative taxa known only from DNA sequences in a scientifically defensible way. As an alternative, we highlight the use of formulas for naming putative taxa (candidate taxa) that do not require any modification of the ICN.

The diversity of lichen photobionts is not fully known. We studied here the diversity of the photobionts associated with Cladonia, a sub-cosmopolitan genus ecologically important, whose photobionts belong to the green algae genus Asterochloris. The genetic diversity of Asterochloris was screened by using the ITS rDNA and actin type I regions in 223 specimens and 135 species of Cladonia collected all over the world. These data, added to those available in GenBank, were compiled in a dataset of altogether 545 Asterochloris sequences occurring in 172 species of Cladonia. A high diversity of Asterochloris associated with Cladonia was found. The commonest photobiont lineages associated with this genus are A. glomerata, A. italiana, and A. mediterranea. Analyses of partitioned variation were carried out in order to elucidate the relative influence on the photobiont genetic variation of the following factors: mycobiont identity, geographic distribution, climate, and mycobiont phylogeny. The mycobiont identity and climate were found to be the main drivers for the genetic variation of Asterochloris. The geographical distribution of the different Asterochloris lineages was described. Some lineages showed a clear dominance in one or several climatic regions. In addition, the specificity and the selectivity were studied for 18 species of Cladonia. Potentially specialist and generalist species of Cladonia were identified. A correlation was found between the sexual reproduction frequency of the host and the frequency of certain Asterochloris OTUs. Some Asterochloris lineages co-occur with higher frequency than randomly expected in the Cladonia species.