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)...

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.

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Novel species of fungi described in the present study include the following from Australia: Vermiculariopsiella eucalypti, Mulderomyces natalis (incl. Mulderomyces gen. nov.), Fusicladium paraamoenum, Neotrimmatostroma paraexcentricum, and Pseudophloeospora eucalyptorum on leaves of Eucalyptus spp., Anungitea grevilleae (on leaves of Grevillea sp.), Pyrenochaeta acaciae (on leaves of Acacia sp.), and Brunneocarpos banksiae (incl. Brunneocarpos gen. nov.) on cones of Banksia attenuata. Novel foliicolous taxa from South Africa include Neosulcatispora strelitziae (on Strelitzia nicolai), Colletotrichum ledebouriae (on Ledebouria floridunda), Cylindrosympodioides brabejum (incl. Cylindrosympodioides gen. nov.) on Brabejum stellatifolium, Sclerostagonospora ericae (on Erica sp.), Setophoma cyperi (on Cyperus sphaerocephala), and Phaeosphaeria breonadiae (on Breonadia microcephala). Novelties described from Robben Island (South Africa) include Wojnowiciella cissampeli and Diaporthe cissampeli (both on Cissampelos capensis), Phaeotheca salicorniae (on Salicornia meyeriana), Paracylindrocarpon aloicola (incl. Paracylindrocarpon gen. nov.) on Aloe sp., and Libertasomyces myopori (incl. Libertasomyces gen. nov.) on Myoporum serratum. Several novelties are recorded from La Réunion (France), namely Phaeosphaeriopsis agapanthi (on Agapanthus sp.), Roussoella solani (on Solanum mauritianum), Vermiculariopsiella acaciae (on Acacia heterophylla), Dothiorella acacicola (on Acacia mearnsii), Chalara clidemiae (on Clidemia hirta), Cytospora tibouchinae (on Tibouchina semidecandra), Diaporthe ocoteae (on Ocotea obtusata), Castanediella eucalypticola, Phaeophleospora eucalypticola and Fusicladium eucalypticola (on Eucalyptus robusta), Lareunionomyces syzygii (incl. Lareunionomyces gen. nov.) and Parawiesneriomyces syzygii (incl. Parawiesneriomyces gen. nov.) on leaves of Syzygium jambos. Novel taxa from the USA include Meristemomyces arctostaphylos (on Arctostaphylos patula), Ochroconis dracaenae (on Dracaena reflexa), Rasamsonia columbiensis (air of a hotel conference room), Paecilomyces tabacinus (on Nicotiana tabacum), Toxicocladosporium hominis (from human broncoalveolar lavage fluid), Nothophoma macrospora (from respiratory secretion of a patient with pneumonia), and Penidiellopsis radicularis (incl. Penidiellopsis gen. nov.) from a human nail. Novel taxa described from Malaysia include Prosopidicola albizziae (on Albizzia falcataria), Proxipyricularia asari (on Asarum sp.), Diaporthe passifloricola (on Passiflora foetida), Paramycoleptodiscus albizziae (incl. Paramycoleptodiscus gen. nov.) on Albizzia falcataria, and Malaysiasca phaii (incl. Malaysiasca gen. nov.) on Phaius reflexipetalus. Two species are newly described from human patients in the Czech Republic, namely Microascus longicollis (from toenails of patient with suspected onychomycosis), and Chrysosporium echinulatum (from sole skin of patient). Furthermore, Alternaria quercicola is described on leaves of Quercus brantii (Iran), Stemphylium beticola on leaves of Beta...

Novel species of fungi described in this study include those from various countries as follows: Antarctica: Cadophora antarctica from soil. Australia: Alfaria dandenongensis on Cyperaceae, Amphosoma persooniae on Persoonia sp., Anungitea nullicana on Eucalyptus sp., Bagadiella eucalypti on Eucalyptus globulus, Castanediella eucalyptigena on Eucalyptus sp., Cercospora dianellicola on Dianella sp., Cladoriella kinglakensis on Eucalyptus regnans, Cladoriella xanthorrhoeae (incl. Cladoriellaceae fam. nov. and Cladoriellales ord. nov.) on Xanthorrhoea sp., Cochlearomyces eucalypti (incl. Cochlearomyces gen. nov. and Cochlearomycetaceae fam. nov.) on Eucalyptus obliqua, Codinaea lambertiae on Lambertia formosa, Diaporthe obtusifoliae on Acacia obtusifolia, Didymella acaciae on Acacia melanoxylon, Dothidea eucalypti on Eucalyptus dalrympleana, Fitzroyomyces cyperi (incl. Fitzroyomyces gen. nov.) on Cyperaceae, Murramarangomyces corymbiae (incl. Murramarangomyces gen. nov., Murramarangomycetaceae fam. nov. and Murramarangomycetales ord. nov.) on Corymbia maculata, Neoanungitea eucalypti (incl. Neoanungitea gen. nov.) on Eucalyptus obliqua, Neoconiothyrium persooniae (incl. Neoconiothyrium gen. nov.) on Persoonia laurina subsp. laurina, Neocrinula lambertiae (incl. Neocrinulaceae fam. nov.) on Lambertia sp., Ochroconis podocarpi on Podocarpus grayae, Paraphysalospora eucalypti (incl. Paraphysalospora gen. nov.) on Eucalyptus sieberi, Pararamichloridium livistonae (incl. Pararamichloridium gen. nov., Pararamichloridiaceae fam. nov. and Pararamichloridiales ord. nov.) on Livistona sp., Pestalotiopsis dianellae on Dianella sp., Phaeosphaeria gahniae on Gahnia aspera, Phlogicylindrium tereticornis on Eucalyptus tereticornis, Pleopassalora acaciae on Acacia obliquinervia, Pseudodactylaria xanthorrhoeae (incl. Pseudodactylaria gen. nov., Pseudodactylariaceae fam. nov. and Pseudodactylariales ord. nov.) on Xanthorrhoea sp., Pseudosporidesmium lambertiae (incl. Pseudosporidesmiaceae fam. nov.) on Lambertia formosa, Saccharata acaciae on Acacia sp., Saccharata epacridis on Epacris sp., Saccharata hakeigena on Hakea sericea, Seiridium persooniae on Persoonia sp., Semifissispora tooloomensis on Eucalyptus dunnii, Stagonospora lomandrae on Lomandra longifolia, Stagonospora victoriana on Poaceae, Subramaniomyces podocarpi on Podocarpus elatus, Sympoventuria melaleucae on Melaleuca sp., Sympoventuria regnans on Eucalyptus regnans, Trichomerium eucalypti on Eucalyptus tereticornis, Vermiculariopsiella eucalypticola on Eucalyptus dalrympleana, Verrucoconiothyrium acaciae on Acacia falciformis, Xenopassalora petrophiles (incl. Xenopassalora gen. nov.) on Petrophile sp., Zasmidium dasypogonis on Dasypogon sp., Zasmidium gahniicola on Gahnia sieberiana. Brazil: Achaetomium lippiae on Lippia gracilis, Cyathus isometricus on decaying wood, Geastrum caririense on soil, Lycoperdon demoulinii (incl. Lycoperdon subg. Arenicola) on soil, Megatomentella cristata (incl. Megatomentella gen. nov.) on unidentified plant, Mutinus verrucosus on soil, Par...