Fungi in the class Leotiomycetes are ecologically diverse, including mycorrhizas, endophytes of roots and leaves, plant pathogens, aquatic and aero-aquatic hyphomycetes, mammalian pathogens, and saprobes. These fungi are commonly detected in cultures from diseased tissue and from environmental DNA extracts. The identification of specimens from such character-poor samples increasingly relies on DNA sequencing. However, the current classification of Leotiomycetes is still largely based on morphologically defined taxa, especially at higher taxonomic levels. Consequently, the formal Leotiomycetes classification is frequently poorly congruent with the relationships suggested by DNA sequencing studies. Previous class-wide phylogenies of Leotiomycetes have been based on ribosomal DNA markers, with most of the published multi-gene studies being focussed on particular genera or families. In this paper we collate data available from specimens representing both sexual and asexual morphs from across the genetic breadth of the class, with a focus on generic type species, to present a phylogeny based on up to 15 concatenated genes across 279 specimens. Included in the dataset are genes that were extracted from 72 of the genomes available for the class, including 10 new genomes released with this study. To test the statistical support for the deepest branches in the phylogeny, an additional phylogeny based on 3156 genes from 51 selected genomes is also presented. To fill some of the taxonomic gaps in the 15-gene phylogeny, we further present an ITS gene tree, particularly targeting ex-type specimens of generic type species. A small number of novel taxa are proposed: Marthamycetales ord. nov., and Drepanopezizaceae and Mniaeciaceae fams. nov. The formal taxonomic changes are limited in part because of the ad hoc nature of taxon and specimen selection, based purely on the availability of data. The phylogeny constitutes a framework for enabling future taxonomically targeted studies using deliberate specimen selection. Such studies will ideally include designation of epitypes for the type species of those genera for which DNA is not able to be extracted from the original type specimen, and consideration of morphological characters whenever genetically defined clades are recognized as formal taxa within a classification.

Recent molecular analyses separate Hymenoscyphus pseudoalbidus (causal agent of ash dieback in Europe) from the morphologically scarcely distinguishable H. albidus. Hymenoscyphus albidus was reported (as "Lambertella albida") on petioles of Fraxinus mandshurica in Japan. Phylogenetic analysis in the present study shows Japanese "L. albida" to be conspecific with H. pseudoalbidus but with a higher genetic variability compared to European isolates. The presence of croziers at the ascus base was found to be a clear distinguishing character of H. pseudoalbidus. Our phylogenetic analysis of the combined ITS and LSU-D1/D2 dataset supports Hymenoscyphus as more appropriate than Lambertella for H. pseudoalbidus. As the Hymenoscyphus clade includes members with two major characters (presence of substratal stroma and brown ascospores) currently used to circumscribe Lambertella, the generic delimitation of Lambertella requires redefinition.

Novel species of fungi described in this study include those from various countries as follows: Angola , Gnomoniopsis angolensis and Pseudopithomyces angolensis on unknown host plants. Australia , Dothiora corymbiae on Corymbia citriodora, Neoeucasphaeria eucalypti (incl. Neoeucasphaeria gen. nov.) on Eucalyptus sp., Fumagopsis stellae on Eucalyptus sp., Fusculina eucalyptorum (incl. Fusculinaceae fam. nov.) on Eucalyptus socialis, Harknessia corymbiicola on Corymbia maculata, Neocelosporium eucalypti (incl. Neocelosporium gen. nov., Neocelosporiaceae fam. nov. and Neocelosporiales ord. nov.) on Eucalyptus cyanophylla, Neophaeomoniella corymbiae on Corymbia citriodora , Neophaeomoniella eucalyptigena on Eucalyptus pilularis, Pseudoplagiostoma corymbiicola on Corymbia citriodora, Teratosphaeria gracilis on Eucalyptus gracilis, Zasmidium corymbiae on Corymbia citriodora. Brazil , Calonectria hemileiae on pustules of Hemileia vastatrix formed on leaves of Coffea arabica , Calvatia caatinguensis on soil, Cercospora solani-betacei on Solanum betaceum , Clathrus natalensis on soil, Diaporthe poincianellae on Poincianella pyramidalis , Geastrum piquiriunense on soil, Geosmithia carolliae on wing of Carollia perspicillata , Henningsia resupinata on wood, Penicillium guaibinense from soil, Periconia caespitosa from leaf litter, Pseudocercospora styracina on Styrax sp., Simplicillium filiforme as endophyte from Citrullus lanatus , Thozetella pindobacuensis on leaf litter, Xenosonderhenia coussapoae on Coussapoa floccosa. Canary Islands (Spain) , Orbilia amarilla on Euphorbia canariensis. Cape Verde Islands , Xylodon jacobaeus on Eucalyptus camaldulensis. Chile , Colletotrichum arboricola on Fuchsia magellanica. Costa Rica , Lasiosph...

Under the rules for the naming of fungi with pleomorphic life-cycles adopted in July 2011, the nomenclaturally correct name for the fungus causing the current ash dieback in Europe is determined to be Hymenoscyphus fraxineus, with the basionym Chalara fraxinea, and Hymenoscyphus pseudoalbidus as a taxonomic synonym of H. fraxineus.

Five species of Hymenoscyphus that fruit on black stromatized parts of dead leaves of deciduous trees are presented, giving details on their morphological and ecological characteristics. Several of these species have previously been misplaced in rutstroemiaceous genera because of the presence of a substratal stroma. However, the heteropolar, scutuloid ascospores with an often hook-like lateral protrusion at the rounded apex and the ascus apical ring of the Hymenoscyphus-type represent two reliable morphological characteristics that, together with molecular data, provide clear evidence for their placement in the genus Hymenoscyphus (Helotiaceae). Among the species treated is Hymenoscyphus fraxineus (=Hymenoscyphus pseudoalbidus), the causal agent of the European ash dieback disease. Since 1992 this species started within Europe to replace the rather uncommon Hymenoscyphus albidus, which is likewise confined to leaves of Fraxinus. Hy. fraxineus has been recorded already since 1990 in Eastern Asia (Japan, Korea, northeast of China), where it had been initially misidentified as Lambertella albida (≡Hy. albidus). In these regions, it occurs as a harmless saprotroph on Fraxinus mandshurica and Fraxinus rhynchophylla, suggesting that those populations are native while the European ash dieback disease has a recent Eastern Asiatic origin. The distinctly higher genetic diversity found in Japanese Hy. fraxineus in contrast to European Hy. fraxineus supports this view. Genetic similarities between Japanese Hy. fraxineus and European Hy. albidus suggest that also Hy. albidus might be a descendant of Asian Hy. fraxineus, though having invaded Europe much earlier. However, consistent genetic deviation between European and Asian Hy. fraxineus at two nucleotide positions of the ITS region indicates that the European ash disease originates from a region different from the presently known areas in Eastern Asia.Our results underline the importance of detailed morphological studies in combination with molecular work. Hy. fraxineus was described from Europe as a cryptic species that differed from Hy. albidus by molecular data alone. However, the Hy. albidus/Hy. fraxineus species complex represents one of many examples within the ascomycetes in which subtle microscopic differences between closely related species, in this case the presence or absence of croziers at the ascus base, are strictly correlated with molecular characteristics. Two species that closely resemble Hy. albidus and Hy. fraxineus form pseudosclerotia in Aesculus leaves and again differ from each other mainly in the ascus base: Hymenoscyphus aesculi on Aesculus hippocastanum from Europe lacks croziers, whereas Hymenoscyphus honshuanus from Japan on Aesculus turbinata possesses croziers. Other taxa treated here include Hymenoscyphus vacini, a European species growing on stromatized net veins of skeletonized leaves of Acer, and Hymenoscyphus torquatus, a Chinese species on unidentified herbaceous stems. An equivalent stroma-forming North American species on leaves of Fra...