Biodiversity loss from disturbances caused by human activities means that species are disappearing at an ever increasing rate. The high number of species that have yet to be described have generated extreme crisis to the taxonomist. Therefore, more than in any other era, effective ways to discover and delimitate species are needed. This paper reviews the historically foremost approaches used to delimit species in Ascomycota, the most speciose phylum of Fungi. These include morphological, biological, and phylogenetic species concepts. We argue that a single property to delineate species boundaries has various defects and each species concept comes with its own advantages and disadvantages. Recently the rate of species discovery has increased because of the advancement of phylogenetic approaches. However, traditional phylogenetic methods with few gene regions lack species-level resolution, and do not allow unambiguous conclusions. We detail the processes that affect gene tree heterogeneity, which acts as barriers to delimiting species boundaries in classical low-rank phylogenies. So far, limited insights were given to the DNA-based methodologies to establish well-supported boundaries among fungal species. In addition to reviewing concepts and methodologies used to delimit species, we present a case study. We applied different species delimitation methods to understand species boundaries in the plant pathogenic and cryptic genus Phyllosticta (Dothideomycetes, Botryosphaeriales). Several DNA-based methods over-split the taxa while in some methods several taxa fall into a single species. These problems can be resolved by using multiple loci and coalescence-based methods. Further, we discuss integrative approaches that are crucial for understanding species boundaries within Ascomycota and provide several examples for ideal and pragmatic approaches of species delimitation.

Xylariomycetidae (Ascomycota) is a highly diversified group with variable stromatic characters. Our research focused on inconspicuous stromatic xylarialean taxa from China, Italy, Russia, Thailand and the United Kingdom. Detailed morphological descriptions, illustrations and combined ITS-LSU-rpb2-tub2-tef1 phylogenies revealed 39 taxa from our collections belonging to Amphisphaeriales and Xylariales. A new family (Appendicosporaceae), five new genera (Magnostiolata, Melanostictus, Neoamphisphaeria, Nigropunctata and Paravamsapriya), 27 new species (Acrocordiella photiniicola, Allocryptovalsa sichuanensis, Amphisphaeria parvispora, Anthostomella lamiacearum, Apiospora guiyangensis, A. sichuanensis, Biscogniauxia magna, Eutypa camelliae, Helicogermslita clypeata, Hypocopra zeae, Magnostiolata mucida, Melanostictus longiostiolatus, M. thailandicus, Nemania longipedicellata, N. delonicis, N. paraphysata, N. thailandensis, Neoamphisphaeria hyalinospora, Neoanthostomella bambusicola, Nigropunctata bambusicola, N. nigrocircularis, N. thailandica, Occultitheca rosae, Paravamsapriya ostiolata, Peroneutypa leucaenae, Seiridium italicum and Vamsapriya mucosa) and seven new host/geographical records are introduced and reported. Divergence time estimates indicate that Delonicicolales diverged from Amphisphaeriales + Xylariales at 161 (123–197) MYA. Amphisphaeriales and Xylariales diverged 154 (117–190) MYA with a crown age of 127 (92–165) MYA and 147 (111–184) MYA, respectively. Appendicosporaceae (Amphisphaeriales) has a stem age of 89 (65–117) MYA. Ancestral character state reconstruction indicates that astromatic, clypeate ascomata with aseptate, hyaline ascospores that lack germ slits may probably be ancestral Xylariomycetidae having plant-fungal endophytic associations. The Amphisphaeriales remained mostly astromatic with common septate, hyaline ascospores. Stromatic variations may have developed mostly during the Cretaceous period. Brown ascospores are common in Xylariales, but they first appeared in Amphisphaeriaceae, Melogrammataceae and Sporocadaceae during the early Cretaceous. The ascospore germ slits appeared only in Xylariales during the Cretaceous after the divergence of Lopadostomataceae. Hyaline, filiform and apiospores may have appeared as separate lineages, providing the basis for Xylariaceae, which may have diverged independently. The future classification of polyphyletic xylarialean taxa will not be based on stromatic variations, but the type of ring, the colour of the ascospores, and the presence or absence or the type of germ slit.

During our investigation of palm fungi in Thailand, two interesting taxa from Elaeis guineensis and Metroxylon sagu (Arecaceae) were collected. Based on phylogenetic analyses of a combined dataset of ITS, LSU, rpb2, and tub2 nucleotide sequences as well as unique morphological characteristics, we introduce the new genus Haploanthostomella within Xylariales, and a new species Endocalyx metroxyli. Additionally, in our study, the genus Endocalyx is transferred to the family Cainiaceae based on its brown conidia and molecular phylogenetic evidence.

Inaccurate taxonomic placement of fossils can lead to the accumulation of errors in molecular clock studies and their generated evolutionary lineages. There are limited fossil data that can be used in divergence time estimations. Therefore, reliable morphological characterization and taxonomical identification of fossil fungi are extremely important. Most fossils of Dothideomycetes and Sordariomycetes are from the early Cenozoic (66–23 Mya), with fewer from the late Mesozoic (174–145 Mya). However, it is hard to distinguish some fossil descriptions as photographs and illustrations are unclear; thus, the validity of using these fossils in calibrations of molecular clocks is problematic. This study brings scattered paleobiological data on selected fossil Ascomycota, using descriptions, fossil images and illustrations, coupled with recent age estimations, and taxonomic and phylogenetic affinity of extant species. As an integrated approach, this study summarizes a historical fossil outline with a reliable minimum age for 16 calibrating points viz. crown of Aigialus, Anzia, Aspergillus, Asterina, Calicium chlorosporum–C. nobile, Capnodiales, Chaenotheca, Colletotrichum, Diaporthales, Meliola, Ophiocordyceps, Microthyriales, Microthyrium, Muyocopron, Pezizomycotina and Stigmatomyces. A scheme of Ascomycota ancient lineages is also provided in order to improve divergence time estimations.

The deviation of conventional fungal niches is an important factor in the implications of hidden fungal diversity and global fungal numbers. The Xylariomycetidae (Sordariomycetes, Ascomycota), which is also referred to as xylarialean taxa, has a wide range of species that demonstrate a high degree of variation in their stromatic characteristics, showing either conspicuous or inconspicuous forms. In this study, samples were collected while focusing on temporal and spatial parameters and substrate characteristics. Based on internal transcribed spacer (ITS), 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and β-tubulin (TUB2) multigene phylogeny and morphology, five new species are introduced as Muscodor brunneascosporus, M. lamphunensis (Xylariaceae), Nigropunctata hydei, N. saccata (Incertae sedis), and Xenoanthostomella parvispora (Gyrotrichaceae). Plant substrates in the early stages of decay and attached to the host were feasible sample niches, with an emphasis on the collection of inconspicuous, hidden xylarialean species. The appearance of inconspicuous saprobic xylarialean forms during the rainy season may be linked to the change in nutritional mode, from endophytic mode during the dry season to saprobic in the wet. Therefore, it would be fascinating to concentrate future research on how seasonal fluctuations affect nutritional mode shifts, especially in northern Thailand, which would provide the optimal spatial characteristics. In order to establish a comprehensive linkage between endophytic and saprobic modes, it is imperative to have a substantial representation of endophytic isolate sequences resembling inconspicuous xylariaceous fungi within publicly accessible databases.