The numbers of fungi: contributions from traditional taxonomic studies and challenges of metabarcoding

The global diversity of fungi has been estimated using several different approaches. There is somewhere between 2–11 million estimated species, but the number of formally described taxa is around 150,000, a tiny fraction of the total. In this paper, we examine 12 ascomycete genera as case studies to establish trends in fungal species descriptions, and introduce new species in each genus. To highlight the importance of traditional morpho-molecular methods in publishing new species, we introduce novel taxa in 12 genera that are considered to have low species discovery. We discuss whether the species are likely to be rare or due to a lack of extensive sampling and classification. The genera are Apiospora, Bambusicola, Beltrania, Capronia, Distoseptispora, Endocalyx, Neocatenulostroma, Neodeightonia, Paraconiothyrium, Peroneutypa, Phaeoacremonium and Vanakripa. We discuss host-specificity in selected genera and compare the number of species epithets in each genus with the number of ITS (barcode) sequences deposited in GenBank and UNITE. We furthermore discuss the relationship between the divergence times of these genera with those of their hosts. We hypothesize whether there might be more species in these genera and discuss hosts and habitats that should be investigated for novel species discovery.


Introduction
Fungi thrive in diverse environments and are involved in the decomposition and nutrient cycling of dead plant material in terrestrial and aquatic ecosystems (Wainwright et al. 2003;Bucher et al. 2004;Pointing et al. 2005;Jobard et al. 2010;Nagahama et al. 2011). The diversity in a particular area or ecosystem is usually expressed as the number of species in the system (Bermudez and Lindemann-Matthies 2020), highlighting the fundamental role of taxonomy in biodiversity assessment and biology . Some 150,600 fungal species have formally been described (http:// www. speci esfun gorum. org/; 8 December 2021), but this is only a fraction of the 2 to 11 million estimated species (Hawksworth and Lücking 2017;Lücking et al. 2021;Baldrian et al. 2021). Species estimates from metabarcoding data are highest, such as the 11.7-13.2 million species estimate of Wu et al. (2019). Tropical and warm-temperate areas seem particularly rich in unexplored fungal diversity Menolli and Sanchez-Garcia 2020). However, the most critical aspect of estimating fungal numbers is defining a species accurately Lücking et al. 2021). Thus, global biodiversity needs to be extensively studied to determine the realistic number of fungi (Wu et al. 2019). Several methods have been developed and used to identify and describe fungal species (Taylor et al. 2000;Aime et al. 2021;Bhunjun et al. 2021a;Chethana et al. 2021). Based on the currently accepted classification system, molecular approaches have greatly improved the understanding of evolutionary relationships in fungi (Naranjo-Ortiz and Gabaldón 2020). Evolution refers to the heritable genetic changes that accumulate during the lifetime through environmental adaptations, which result from natural selection, mutation, genetic drift, and migration (gene flow) (Andrews et al. 2012). Therefore, evolutionary studies can help to predict the next trend for fungal discovery.
In this study, we discuss species discovery, the likelihood of new species being discovered, the evolution with plant hosts, and the possibility of the use of metabarcoding for recognizing the "dark taxa" in selected genera (Taberlet et al. 2012;Ryberg and Nilsson 2018). The newly introduced taxa are justified and the potential for discovering additional species is discussed. A comparison of the number of ITS sequences with the number of species epithets listed in Species Fungorum (2021) is appraised. Metabarcoding indicates that fungal diversity is much higher when using a morpho-molecular approach. However, data from high throughput sequencing (HTS) studies cannot be used to describe these species formally as they lack holotypes. High throughput sequencing indicates that many largely unexplored habitats contain large numbers of undescribed taxa (Tedersoo et al. 2021). We also integrate data from different platforms for species introduction and compare the number of species in Species Fungorum with HTS data from UNITE database (Nilsson et al. 2019).

Sequence alignment and phylogenetic analyses
Consensus sequences were assembled using Geneious Prime 2021 (Biomatters Ltd., Auckland, New Zealand). Sequences of closely related strains were retrieved using BLASTn searches against GenBank. Sequences were aligned with MAFFT version 7 (Katoh et al. 2019), with minimal adjustment of any ambiguous nucleotides by visual examination and manually corrected in AliView version 1.26 (Larsson 2014). Leading or trailing gaps exceeding the primer binding site were trimmed from the alignments prior to tree building and the gaps in the alignment were treated as missing data. The concatenation of the multimarker datasets was created by using Sequence Matrix version 1.8 (Vaidya et al. 2011).
Individual gene phylogenetic analyses were performed to determine the compatibility and the best marker for species delineation. Phylogenetic analyses of the combined dataset were performed using maximum likelihood, maximum parsimony and Bayesian inference. Maximum likelihood analyses (ML), including 1000 bootstrap pseudoreplicates, were performed at the CIPRES web portal (Miller et al. 2017) using RAxML v. 8.2.12 (Stamatakis 2014). The general time reversible (GTR) model with a discrete gamma distribution plus invariant site (GTR + I + G) was used as the nucleotide substitution model. Maximum parsimony analysis was conducted using PAUP v.4.0b 10 with the heuristic search option and the number of replicates set to 1000 each (Swofford and Swofford 2002). The tree length (TL), composite consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were documented. The best model for each gene was determined in JModelTest version 2.1.10 (Darriba et al. 2012) for the Bayesian analysis. The Bayesian inference posterior probabilities (BPP) distribution (Zhaxybayeva and Gogarten 2002) was estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes 3.2.2 on XSEDE (Ronquist and Huelsenbeck 2003). Six simultaneous Markov chains were run for 1,000,000 to 10,000,000 generations, depending on individual settings for the fungal group and trees were sampled at every 100th or 1000th generation. Suitable burn-in thresholds were determined in Tracer version 1.7 (Rambaut et al. 2018). The first 10-25% of generated trees representing the burn-in phase of the analyses were discarded, while the remaining trees were used to calculate Bayesian posterior probabilities (BPP) in the majority rule consensus tree. The phylograms were visualized in FigTree version 1.4.0 (Rambaut 2014) and edited using Adobe Illustrator CS6 version 15.0 (Adobe Systems, USA).

Genera characteristics and taxonomy of introduced species
In this section, we introduce novel taxa in 12 genera and discuss species discovery and the likelihood of new species being discovered. We did a BLASTn search for the ITS region as it is the primary fungal barcode (Schoch et al. 2012). Taxa with high similarity and query cover (in the range of 90-100%) were considered as close relative to the novel species described in this study. To evaluate the amount of sequence data available in GenBank to the number of species epithets, the number of ITS sequences in each genus was compared with the number of species epithets listed in Species Fungorum (2021) until September 2021 (Table 1). The number of species hypotheses (SH) in UNITE (Nilsson et al. 2019) was also determined using the 98.5% threshold level. The species hypothesis represents the species-level for group of individuals that share a given set of observed characters among their OTUs.

Neodeightonia C. Booth
Botryosphaeriaceae was introduced by Theissen and Sydow (1918) to accommodate three genera, Botryosphaeria, Phaeobotryon and Dibotryon. Subsequently, Neodeightonia was introduced by Booth and Punithalingam (1969). Phillips et al. (2019) accepted 22 genera in Botryosphaeriaceae. Neodeightonia is a member of Botryosphaeriaceae and is characterized by hyaline, aseptate ascospores with polar apiculi surrounded by a membrane that swells and expands when mounted in water. In the asexual morphs, the conidia are initially hyaline, becoming brown, 1-septate at maturity, with smooth to finely roughened walls, or fine striations Wu et al. 2021). This genus was previously considered as a synonym of Botryosphaeria (von Arx and Müller 1975). However, Neodeightonia is distinguishable from Botryosphaeria based on dark, 1-septate ascospores as well as in phylogeny (Phillips et al. 2008). Longitudinal striations on the conidial wall are an additional characteristic feature of this genus (Phillips et al. 2008). Neodeightonia differs from Lasiodipolodia by the absence of conidiomatal paraphyses, and conidial striations distinguish it from Diplodia (Phillips et al. 2008).
Botryosphaeriaceae diverged from other families in Botryosphaeriales around 94 MYA in the late Cretaceous period (crown age of 61 MYA in the Paleogene period), in a period dominated by the expansion of angiosperms occupying environments previously dominated by conifers Batista et al. 2021). Botryosphaeriaceae are cosmopolitan in distribution, occurring on a wide range of hosts from tropical and temperate regions as, pathogens, endophytes or saprobes (Slippers and Wingfield 2007;Liu et al. 2012;Phillips et al. 2019). Palms (Arecales) and bamboo (Poales) diverged around 118-116 MYA in the early Cretaceous period and major lineages migrated between Eurasia, the Pacific and the Indian ocean (Baker and Couvreur 2013). Palms and bamboo species further diversified during the plate-driven breakup in the late Cretaceous period which was followed by the Cretaceous extinction event which resulted in the loss of around 70% of species (65.5 MYA, Cretaceous-Tertiary extinction) (Peace et al. 2020). Neodeightonia diverged from Lasiodiplodia around 19 MYA in the early Neogene period (crown age of 8 MYA in the late Neogene period) . Several land-dwelling mammals and plants known today were present during this period . Neodeightonia species have been found across Eurasia, mostly associated with specific plants such as palms, bamboo and eudicotyledons (Planchonia) Phillips et al. 2019). The distribution of Botryosphaeriaceae began around 94 MYA, which was after the diversification of Arecales over several continents (Baker and Couvreur 2013). This might suggest that Neodeightonia co-evolved as endophytes with these hosts to adapt to the new environmental conditions.

Neocatenulostroma Quaedvl. & Crous
Neocatenulostroma was introduced in Teratosphaeriaceae by Quaedvlieg et al. (2014), with N. microsporum as the type species. The genus includes endophytic, plant pathogenic and saprobic taxa (Markovskaja et al. 2016). Neocatenulostroma species have been isolated from a range of substrates, including rocks (Markovskaja et al. 2016). Neocatenulostroma species are characterised by globose to slightly subglobose ascomata and chains of longitudinal, cylindrical to Y-shaped or ellipsoidal irregularly branched conidia .
There are three epithets listed under Neocatenulostroma in Species Fungorum (2021) and all species have molecular data (Table 1). Table 1 shows the trend of taxa introduced in Neocatenulostroma according to the Species Fungorum (2021). There are 23 ITS sequences of Neocatenulostroma in GenBank. A BLASTn search of the ITS region of the type species Neocatenulostroma microsporum strain CBS 101951 (NR_145114) showed high similarity (90-100%) to 18 uncultured sequences and 16 unidentified isolates. There are three species hypotheses (comprising 137 sequences) that have high similarity in UNITE. Most Neocatenulostroma species are plant pathogens. For example, N. abietis causes diseases on a wide range of conifer hosts (firs, pines and junipers) (Markovskaja et al. 2016). Neocatenulostroma abietis has also been isolated from a range of substrates, commonly as saprobe or endophyte in pine needles . Neocatenulostroma microsporum Fig. 2 Phylogram generated from maximum likelihood analysis based on combined LSU, SSU, ITS and tef1-α sequence data of Neodeightonia. Seventeen taxa were included in the combined analyses, which comprised 2859 characters (LSU = 889 bases, SSU = 1061 bases, ITS = 603 bases, tef1-α = 306 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −5591.196172 is presented. Bootstrap support values for ML equal to or greater than 70% and BPP equal to or greater than 0.90 are given above the nodes. Diplodia seriata (CBS 112555) and D. magnoliigena (MFLUCC 18-1554) were used as outgroup taxa. The newly generated sequences are indicated in blue. The ex-type strains are indicated in bold  Konta et al. (2016) 1 3 causes diseases on leaves of Protea and Encephalartos, but not conifers . Neocatenulostroma germanicum is pathogenic on pine needles and it has also been isolated as a saprobe from rocks and epoxy resin (Pangallo et al. 2015). Neocatenulostroma abietis has been associated with different lifestyles in pines. This possibly suggests that Neocatenulostroma colonise pines as endophytes, and switch lifestyles at host senescence Ascomata 140 × 176 µm (n = 5) diam., pseudothecial, scattered to gregarious, uniloculate, subepidermal to erumpent, depressed, subglobose in section, dark brown to black, with central apical ostiole. Peridium 14-25 µm wide (x̅ = 16 μm, n = 8), thick at apex, thin-walled at the lower half, composed of several layers of pale brown to dark brown cells of textura angularis at the apex, textura prismatica at the sides, inner layer lined with 6-8 layers of subhyaline cells, thin at base. Hamathecium aparaphysate. Asci 38-60 × 11-15 μm (x̅ = 40 × 11 μm, n = 30), 8-spored, bitunicate, subsessile, ovoid to broadly ellipsoid, straight to slightly curved, with short pedicel, apically rounded, ocular chamber clearly visible when immature. Ascospores 10-16 × 3-6 μm (x̅ = 12 × 4 μm, n = 40), overlapping, biseriate to multiseriate, fusoid-ellipsoidal with obtuse ends, straight or slightly curved, medianly 1-septate, cell above septa larger than those below, thick-walled, hyaline, without persistent mucus sheath. Asexual morph: Mycelium producing chlamydospores and chlamydospore-like structures after two months, hyaline to dark brown, hyphae, septate, branched, verruculose, thick-walled, transformed into chlamydospores.
Culture characteristics: Ascospores germinating on PDA within 24-48 h. Germinating ascospores become either verruculose, brown and distorted, germ tubes developing from apical and basal cells. Appressorial-like structures are formed at the end of germ hyphae. Colonies on PDA slowgrowing, reaching 10 mm in diameter after four weeks of incubation at 25 °C. Colonies black, umbonate at the centre, with circular, friable, black margin; reverse black. Chlamydospore-like structures formed in culture. Notes: Neocatenulostroma castaneae is phylogenetically distinct but nests with Austroafricana and Neocatenulostroma in the maximum likelihood, maximum parsimony and Bayesian inference analyses with moderate bootstrap support (Fig. 4). BLASTn results of the LSU sequences showed 97% similarity to Pseudoteratosphaeria ohnowa (CBS 112896) across 99% of the query sequence which translates to over 96% similarity, and the ITS region was 93% similar to Pseudotaeniolina globosa (CBS 109889) across 99% of the query sequence which translates to over 92% similarity. Neocatenulostroma castaneae is distinct from Austroafricana and Neocatenulostroma, which are saprobes having depressed and subglobose ascomata, with only dark brown chlamydospore-like structures observed in culture (Markovskaja et al. 2016). The sexual morph of Neocatenulostroma castaneae is similar to members of Teratosphaeriaceae in their aparaphysate, subsessile, ovoid to broadly ellipsoid asci, and fusoid-ellipsoidal ascospores with obtuse ends, such as Austroafricana, Parateratosphaeria, Teratosphaeria, Teratosphaeriopsis and Xenoteratosphaeria (Quaedvlieg et al. 2012Crous et al. 2017;Abdollahzadeh et al. 2020). Chlamydospore-like structures are commonly found in Teratosphaeriaceae, as in Constantinomyces, Incertomyces and Monticola (Ruibal et al. 2018;Crous et al. 2019). Neocatenulostroma castaneae is a saprobe on dried branches, but appressorial pegs were noted at the tips or in between the hypha cells in the axenic culture (Fig. 3). This suggests an endophytic or pathogenic life stage . Neocatenulostroma castaneae is introduced as a new species as it forms a distinct lineage from Austroafricana and Neocatenulostroma species. The rpb2 gene is an important marker for Teratosphaeriaceae, therefore, the new taxon is not introduced as a new genus as it lacks the rpb2 gene.

Bambusicola D.Q. Dai & K.D. Hyde
Bambusicola was introduced by Dai et al. (2012) and is typified by B. massarinia. It belongs to Bambusicolaceae, Pleosporales, Dothideomycetes (Hyde et al. 2013;Dai et al. 2017;Hongsanan et al. 2020). Three genera are accepted in Bambusicolaceae, namely Bambusicola, Leucaenicola and Palmiascoma Liu et al. 2015;Jayasiri et al. 2019). Bambusicola is noticeable as black dots on the host surfaces, and only known from Bambusoideae or Ficus . Most Bambusicola taxa were isolated from bamboo as pathogen or saprobes, and they can decompose bamboo and woody material (Cai et al. 2006).
Bambusicola originally included four species discovered from bamboo in tropical areas   Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, and rpb2 sequence data representing the placement of Mycosphaerellales taxa. One hundred and thirty-nine taxa were included in the combined analyses, which comprised 1992 characters (LSU = 916 bases, ITS = 736 bases, rpb2 = 340 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −39830.487503 is presented. Bootstrap support values for ML equal to or greater than 50% and BPP equal to or greater than 0.70 are given above the nodes. Capnodium neocoffeicola (CBS 139614) and Capnodium paracoartatum (MFLUCC 14-0282) were used as outgroup taxa. The newly generated sequence is indicated in blue. The type-derived sequences are indicated in bold. Thick branches represent support values equal to or greater than 75% ML and BPP equal to or greater than 0.95 Bambusicolaceae based on morphology and molecular data (Wijesinghe et al. 2021). There are 17 ITS sequences annotated as Bambusicola in GenBank. A BLASTn search of the ITS region of the type species Bambusicola massarinia strain MFLUCC 11-0389 (NR_121548) showed high similarity and query cover (90-100%) to almost all Bambusicolaceae species (one sequence was annotated as Pleosporales sp. PSU-ES100 (JN116643) and one unidentified Phoma sp. 20_2 (KC354579)). There are ten species hypotheses (comprising 15 sequences) that show high similarity in the UNITE database. Bamboo is mainly distributed in tropical and subtropical areas (Lobovikov et al. 2007). Bambusicolaceae diverged around 44 MYA (58-15 MYA), and Bambusoideae is thought to have diversified around 42 (44-42) MYA (Guo et al. 2019;Hongsanan et al. 2020;Bhunjun et al. 2021b). It is likely that Bambusicolaceae species may have co-evolved with bamboo (Wysocki et al. 2015;Bhunjun et al. 2021b). Poaceae became diverse and ubiquitous in the Eocene period and the abundance of grass favoured the evolution of early grazing animals, such as Eohippus (Beaver 2019). This also resulted in the evolution of bamboo-eating mammals such as the red pandas (Ailurus fulgens) at around 39.9 MYA (Hu et al. 2017). In addition to Bambusoideae, Bambusicola species are also found on Ficus. Tree-dwellers such as monkeys, birds, and fruit bats can be hypothesized to have facilitated the diversification of Bambusicola species as Ficus fruits are an important part of their diet. The majority of studies on Bambusicola are confined to China and Thailand, therefore a large number of species is likely to be discovered as other countries are explored for Bambusicola diversity. More studies focusing on the endophytic lifestyle of fungi on Bambusoideae will advance our understanding of the host-specificity of bambusicolous fungi.   , vermiform to cylindrical, elongate, rounded at the ends, slightly curved, 2-3-septate, hyaline when young, pale brown to brown when mature, smooth-walled, guttulate. Microconidia 2.5-4.5 × 1.2-2.5 μm (x̅ = 3 × 2 μm, n = 20), globose or oblong to ellipsoidal, rounded at the ends, aseptate, hyaline to pale brown, guttulates.

Bambusicola nanensis
Culture characteristics: Macroconidia germinated on PDA within 12 h from single spores. Both ends produced germ tubes. Colony diameter reached 15-20 mm after 4 weeks at 26 °C on PDA, circular, with entire margin, flat, cottony, white from above, yellow from below.

Paraconiothyrium Verkley
Paraconiothyrium (Didymosphaeriaceae) was introduced by Munk (1953), and it is considered one of the most species-rich pleosporalean families (Hyde et al. 2013;Hongsanan et al. 2020;Dissanayake et al. 2021). The sexual morphs of Didymosphaeriaceae are characterized by uni-septate ascospores with trabeculate pseudoparaphyses , while asexual morphs are fusicladium-like or phoma-like (Hyde et al. 2013;Dissanayake et al. 2021). Paraconiothyrium is an asexual genus introduced by Verkley et al. (2004) to accommodate four taxa, P. brasiliense, P. cyclothyrioides, P. fungicola, and P. estuarinum Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, and SSU sequence data. Related sequences were taken from Genbank and Brahmanage et al. (2020). Twenty taxa were included in the combined analyses, which comprised 2433 characters (LSU = 815 bases, ITS = 819 bases, SSU = 799 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −7,163.929087 is presented. Maximum likelihood bootstrap support values equal to or greater than 75% and BPP equal to or greater than 0.95 are given above the nodes. Camarosporium aureum (MFLUCC 14-0620) and C. caraganicola (MFLUCC 14-0605) were used as outgroup taxa. The newly generated sequence is indicated in blue and the type-derived sequences are given in bold 1 3 (type species). Members of Paraconiothyrium are characterized by eustromatic, pycnidial conidiomata, phialidic or percurrent conidiogenous cells, and aseptate to 1-septate, hyaline to brown conidia (Verkley et al. 2004;Gonçalves et al. 2016). The sexual morph of Paraconiothyrium was considered as Paraphaeosphaeria (Verkley et al. 2004).
There are 20 epithets listed in Species Fungorum (2021) under Paraconiothyrium (Table 1) and over 500 ITS sequences annotated as Paraconiothyrium in Gen-Bank. A BLASTn search of the ITS region of the type species Paraconiothyrium estuarinum strain CBS 109850 (NR_166007) showed a high similarity and query cover (90-100%) to several Microsphaeropsis and Paraconiothyrium species as well as one Fusarium species which could be a misidentification (MN644543). In 2020, around 100 sequences of Paraconiothyrium were deposited, but the phylogenetic placement of some of these taxa are not confirmed. There are 37 species hypotheses (comprising 708 sequences) with high similarity to Paraconiothyrium in UNITE. However, as the genus has high diversity, the ITS region alone is not sufficient to demarcate species. Seven taxa were synonymized under this genus. Some Paraconiothyrium species were synonymized under Paraphaeosphaeria (for example, Paraconiothyrium minitans under Paraphaeosphaeria minitans and Paraconiothyrium sporulosum under Paraphaeosphaeria sporulosa, Verkley et al. 2014). Paracamarosporium and Pseudocamarosporium species have similar morphology, such as pycnidial conidiomata and enteroblastic and phialidic conidiogenesis (percurrent proliferation) with muriform conidia (Wijayawardene et al. 2014). Pseudocamarosporium was introduced by Wijayawardene et al. (2014) to accommodate camarosporium-like species. Pseudocamarosporium and Paracamarosporium show closer affinities, but they differ in morphology by the presence of paraphyses in Paracamarosporium .
Pathogenic, saprobic and endophytic Paraconiothyrium species are associated with different plant hosts and substrates . Most Paraconiothyrium species have been recorded on monocotyledons, such as grasses , mosses and clubmosses (Budziszewska et al. 2011;de Gruyter et al. 2013), eudicots (Verkley et al. 2004;Ariyawansa et al. 2014Ariyawansa et al. , 2015, soil and estuarine sediments (Verkley et al. 2004) and are also human pathogens (Verkley et al. 2014). A large number of species is likely to be discovered as they can be found on a wide range of hosts.
The crown age of the suborder Massarineae is estimated at 130 MYA in the Cretaceous period, and Didymosphaeriaceae diverged at around 100-75 MYA in the Cretaceous period (Phukhamsakda et al. 2016). The earliest moss and clubmoss fossils are reported from the Permian period and the crown age was estimated by Wikström and Kenrick (2001) as 298.9-251.9 MYA. Monocotyledons and eudicots (modern seed-bearing plants) diversified in the Cretaceous period (135-130 MYA), while grasses (Poaceae) diversified around 65 MYA in the late Cretaceous period (Chen et al. 2017). Didymosphaeriaceae diverged before the Cretaceous extinction event. The conditions of high humidity and reduced solar insolation after the extinction event favoured an increase of saprobic fungi that flourished on the detritus (Vajda and McLoughlin 2004). It can be hypothesised that Didymosphaeriaceae species diversified to adapt to various hosts that were dominant following the extinction event, such as Poaceae (Peace et al. 2020). Paraconiothyrium species are widespread and are associated with several lifestyles on a wide range of hosts. We would expect the number of species to increase with the study of hosts which diversified following the extinction event. This is supported by the new species introduced in this study from Ficus sp. (eudicots), which has a crown age of around 75-48.5 MYA . A new species, Paraconiothyrium fici, is introduced based on morphology and multi-marker phylogenetic analyses.  150-190 × 200-280 µm (x ̅ = 175 × 235 µm, n = 10) pycnidial, solitary or aggregated, scattered, immersed, uni-loculate, globose to subglobose, black, lacking ostioles. Conidiomatal wall 9-17 μm wide, comprising 4-5 cell-layers of thickwalled, hyaline to pale brown cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 1.9-4 × 2.2-3 µm (x ̅ = 2.7 × 2.9 µm, n = 10), enteroblastic, ampulliform to subcylindrical, phialidic with periclinal wall thickening or percurrent proliferations near the apex. Conidia 5.5-7.4 × 2.8-3.7 µm (x ̅ = 6.5 × 3.2 µm, n = 20), globose to cylindrical, initially hyaline, becoming pale to dark brown when mature, thin-walled, smooth, aseptate, with 1-3 small guttules.

Paraconiothyrium fici
Culture characteristics: Conidia germinating on PDA within 24 h, reaching 20-25 mm in 2 weeks at 25 °C. Germ tubes produced from the basal and apical cells of conidia. Mycelia superficial, circular, with entire margin, flat, smooth, from above white; reverse, pale yellow to light brown.
The family Herpotrichiellaceae comprises 16 genera and more than 260 species (Wijayawardene et al. 2020). Capronia is the largest genus in Herpotrichiellaceae with over 80 extant species classified based on morphology and/ or phylogeny ). There are 81 epithets listed in Species Fungorum (2021, Table 1). Species have been investigated through multi-marker phylogenetic analyses based on ITS, tef1-α, tub2 and act and occasionally other markers Untereiner et al. 2011;Teixeira et al. 2017;Phookamsak et al. 2019;Sánchez et al. 2019). The ITS region has proven valuable in delimiting species in Herpotrichiellaceae and also used to support sexual-asexual morph connections within Herpotrichiellaceae (Uijthof 1996;Uijthof et al. 1998;de Hoog 1999;Rogers et al. 1999). The combined ITS and LSU dataset provides useful resolution within the clades (Untereiner and Naveau 1999;Sánchez et al. 2019;Wan et al. 2021). There are 112 ITS sequences annotated as Capronia in GenBank. A BLASTn search of the ITS region of Capronia lijiangensis strain CGMCC 3.20501 (as the molecular data of the type species C. sexdecimspora is not available) showed a high similarity and query cover (90-100%) to several unidentified names such as Ascomycete sp. (ZGZII03175) and one uncultured Capronia sp. (MOTU18). There are 52 species hypotheses (comprising 571 sequences) that have high similarity to Capronia in UNITE. Herpotrichiellaceae harbours a great diversity of polyphyletic asexual morphs, and Capronia is a homothallic sexual genus covering all asexual members. Species belonging to this family have different ecological preferences and exhibit highly diversified lifestyles (de Hoog , 2014. Most of them are opportunistic pathogens of human and cool-blooded animals or saprobic on wood, parasitic on fungi or lichens, and also have thermo-tolerance behaviour, but the species are generally not host-specific (Untereiner et al. 1995;Untereiner 2000;Crous et al. 2007;Tsurykau and Etayo 2017;Sánchez et al. 2019). We would expect the number of species to increase as they can be found from a wide range of hosts.
Culture characteristics: Colonies on PDA reaching 40-45 mm diam., after 4 weeks at 25 ± 2 °C, slow growing, colonies circular, umbonate, dull to rough with entire edge, sparse; colony from above: whitish to nyanza; from below, black; not producing pigment in PDA media.
Notes: Capronia lijiangensis formed a well-supported sister clade to C. camelliae-yunnanensis and C. pilosella with 78% ML and 1.00 BPP support (Fig. 10). Capronia lijiangensis differs by 18 bases in the ITS region, three bases in the LSU gene and two bases in the SSU gene compared to C. camelliae-yunnanensis. Capronia lijiangensis differs from C. pilosella by eight bases in the ITS region, six bases in the LSU and one base in the SSU genes. Capronia lijiangensis produces wider and shorter conidia as compared to those in C. pilosella (9.5-15 × 3.5-5 μm vs. 7.5-17 × 2.5-4 μm) (Müller et al. 1987) while C. camelliae-yunnanensis was described in the asexual state . Capronia lijiangensis is the first species in Capronia known to have both sexual and asexual morphs, but asci were not observed due to limited and dried ascomata on the natural substrate.

Apiospora Sacc.
Apiospora was introduced by Saccardo (1875) and belongs to Apiosporaceae (Hyde et al. 2020a;Wijayawardene et al. 2020;Pintos and Alvarado 2021). Apiosporaceae was established by Hyde et al. (1998) and is typified by Apiospora (Ellis 1971;Feng et al. 2021). The asexual morph Arthrinium has been linked to the sexual morph of Apiospora (Ellis 1971;Seifert et al. 2011). Crous and Groenewald (2013) synonymized Apiospora under Arthrinium based on the one fungus-one name principle, but the genera are regarded as distinct based on molecular data (Pintos and Alvarado 2021). Six genera are recognized in Apiosporaceae, viz. Appendicospora, Arthrinium, Apiospora, Dictyoarthrinium, Endocalyx and Nigrospora (Hyde et al. 2020a;Wijayawardene et al. 2020;Pintos and Alvarado 2021). The sexual morph of Apiospora is similar to Khuskia and Nigrospora (Pintos and Alvarado 2021). The asexual morphs of Apiospora and Arthrinium sensu stricto are also similar in having basauxic conidiogenesis cells (Hughes 1953;Pintos and Alvarado 2021). Apiospora is a distinct lineage in the phylogenetic analyses and is morphologically distinct from the other genera in Apiosporaceae.
There are 85 epithets listed under Apiospora in Species Fungorum (2021) with two new species introduced by Crous et al. (2021). Table 1 shows the species discovery trend in Apiospora based on Species Fungorum (2021). Arthrinium has "Papularia"-like asexual morphs with basauxic conidiogenesis cells and "Apiospora"-like sexual morphs (Smith et al. 2003;Crous and Groenewald 2013;Jiang et al. 2019;Pintos and Alvarado 2021). Arthrinium and Apiospora have no clearly defined differences in morphology, although most species of Arthrinium are more diverse in the shapes of conidia than those in Apiospora (Hawksworth et al. 2011;Crous and Groenewald 2013;Pintos and Alvarado 2021). Most of the species were previously described based on morphology, but DNA sequence analyses have increased the discovery rate of Apiospora species. There are 63 ITS sequences annotated as Apiospora in GenBank. A BLASTn search of the ITS region of Apiospora tropica strain MFLUCC 21-0056 (as the molecular data of the type species A. montagnei is not verified) showed a high similarity and query cover (90-100%) to almost all Apiospora/Arthrinium species. Few of the sequences were annotated as Pleosporales sp. (PBC4) or fungal sp. (BMP3011). Thus, we may miss some of the Apiospora taxa as we did not include the unidentified sequences in the phylogenetic analysis. There is one UNITE species hypothesis (comprising 12 sequences) that show high similarity to Apiospora. Apiospora species are saprobes, for example, A. setostroma Kwon et al. 2021), and they also act as endophytes, pathogens on invasive plants or opportunistic pathogens on humans, such as, A. sacchari and A. paraphaeosperma (Ramos et al. 2010;Sharma et al. 2014). Apiospora species have been mostly found on Poaceae, but also from other host families (Pintos and Alvarado 2021). A large number of species will likely be discovered as some species appear to be host genus or family specific. For example, A. pterosperma has only been associated with Cyperaceae (Pintos and Alvarado 2021). Fifty-five species of Arthrinium were classified as Apiospora by Pintos and Alvarado (2021) based on multigene phylogenetic analysis. Sixty-six taxa were validated by morphological characters and molecular data (Pintos and Alvarado 2021). Multi-marker datasets based on LSU, ITS, tub2, and tef1-α markers are important to separate Apiospora and Arthrinium as two distinct clades within Apiosporaceae (Hyde et al. 1998;Smith et al. 2003;Pintos and Alvarado 2021).
Apiosporaceae taxa have a widespread distribution mainly with terrestrial habitats on a variety of hosts and do not appear to be host-specific Hyde et al. 2020a;Pintos and Alvarado 2021). The species of Apiosporaceae are not only found in temperate, cold or alpine regions Feng et al. 2021;Kwon et al. 2021), but have also been reported in tropical or subtropical areas Tang et al. 2020;Tian et al. 2021). Most Apiosporaceae species however, occur on monocotyledons such as Arecaceae and Poaceae, while some species were found associated with algae, herbaceous dicotyledons and soil (Sharma et al. 2014;Jiang et al. 2019;Kwon et al. 2021). The divergence estimate of Apiosporaceae is around 18 MYA, whereas the ancestor lineage evolved around 94 MYA (Hyde et al. 2020a). This was during the Cenomanian-Turonian extinction event (94 MYA) when global warming caused increased temperatures in the oceans and resulted in low plant nutrients (Pearce et al. 2009). There was peak abundances of green algal groups due to an increase in oxygen deficiency and total organic carbon content in the ocean (Pearce et al. 2009 More studies focusing on the endophytic lifestyle of fungi on Arecaceae will probably help us understand the hostspecificity of Apiosporaceae fungi. A new species, Apiospora tropica was found on dead bamboo culms in Thailand and is described based on morphology and phylogenetic analyses (Figs. 11,12).  Fig. 11 Etymology: epithet "tropica" means humid tropical. Holotype: MFLU 21-0084 Saprobic on dead bamboo culms. Sexual morph: Ascostromata raised, immersed to erumpent, raised areas on the host surface, with ascostromata breaking through raised cracks at the black centre, multi-loculate, forming groups in stromata, gregarious, fusiform. Locules 136-188 μm diam., 87-103 μm high, immersed in stromata, perithecial, arranged in a row, ampulliform to subglobose, brown to reddish-brown, ostioles with periphyses. Peridium comprising 3-5 layers, thick-walled, composed of dark brown cells of textura angularis. Hamathecium composed of long, 5-6.5 μm wide, septate, unbranched paraphyses, not anastomosing. Asci 83-98 × 15-18 μm (x̅ = 88.5 × 16.5 μm, n = 15), 8-spored, unitunicate, clavate, apically rounded, sessile to subsessile. Ascospores 21-26 × 5.5-10 μm (x̅ = 24 × 7 μm, n = 20), biseriate, partly overlapping, reniform, rarely straight, hyaline, septate, slightly constricted at the septum, sometimes with an indistinct septum when mature, usually 2-celled, with a large upper cell and a smaller lower cell, mostly curved at the lower cell, smooth-walled, surrounded by a large entire sheath, becoming shallow when dry. Asexual morph: Undetermined.

Vanakripa Bhat, W.B. Kendr. & Nag Raj
Vanakripa was introduced by Bhat and Kendrick (1993), with V. gigaspora as the type species. The genus is considered incertae sedis in Pezizomycotina (Wijayawardene et al. 2020), and a generic key was provided by Mota et al. (2008). Vanakripa has a special clavate to vermiform, hyaline, separating cells attached to the conidia (Bhat and Kendrick 1993).
There are nine epithets listed under Vanakripa in Species Fungorum (2021) ( Table 1). The latest addition was V. inexpectata by Leão-Ferreira et al. (2013) and V. chinensis by Zhang et al. (2021), and they are the only two species introduced within the last decade. A BLASTn search of the ITS region of Vanakripa chiangmaiense strain MFLUCC 21-0158 (as the molecular data of the type species V. gigaspora is not available) showed high similarity and query cover (90-100%) to Vanakripa minutiellipsoidea strain CBS 112523. The ITS region also showed high similarity (> 90%) to members of Conioscyphales, but the query cover of the results was lower than 50%. There are two species hypotheses (comprising two sequences) that show high similarity to Vanakripa in UNITE. There are only four ITS sequences annotated as Vanakripa in GenBank as most species were described based only on morphology. Vanakripa minutiellipsoidea is the only other Vanakripa species with molecular data and was isolated from a submerged dead petiole of Eleiodoxa conferta (Pinnoi 2003). Thus, four species were confirmed based on morphology and molecular data. Vanakripa species are mostly found on submerged wood in aquatic environments in the tropical zone (Mota et al. 2008). The other species were isolated from undetermined hosts, and their host specificity needs to be further investigated. A new species, Vanakripa chiangmaiense is introduced from an undetermined decaying wood in Thailand.
Culture characteristics: Conidia germinating on PDA within 24 h. Colonies on PDA floccose, rounded, sallow to yellow, white aerial hyphae at the surface; reverse yellow from the centre with light yellow at the rim.
Material examined: Thailand, Chiang Mai Province, saprobic on decaying wood, 16 December 2020, X.G. Tian, U4-4 (HKAS 21-122165, holotype); ex-type living culture, MFLUCC 21-0158. Fig. 12 Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, tef1-α and tub2 sequence data from Apiosporaceae and related families in the order Amphisphaeriales. Eighty-four taxa were included in the combined analyses which comprised 3500 characters (LSU = 863 bases, ITS = 634 bases, tef1-α = 1020 bases, tub2 = 983 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −31266.866896 is presented. Bootstrap support values for ML equal to or greater than 75% and BPP equal to or greater than 0.95 are given at the nodes. Seiridium phylicae strains CPC 19962 and CPC 19965 were used as outgroup taxa. The newly generated sequence is indicated in blue. The type-derived sequences are given in bold ◂ 1 3 GenBank accession numbers: LSU = OL606152, SSU = OL606141, ITS = OL753684.
Notes: Vanakripa chiangmaiense is closely related to V. minutiellipsoidea (0.99 BPP, Fig. 14), but the classification of Conioscypha and Vanakripa needs further studies. Vanakripa chiangmaiense has ellipsoid, smooth or verruculose conidia, and V. minutiellipsoidea has ellipsoid to broadly clavate, smooth conidia (Pinnoi 2003). The closest match of the ITS sequence of Vanakripa chiangmaiense was V. minutiellipsoidea (CBS 112523) with 88.04% similarity across 80% of the query sequence, which translates into 70.43% similarity based on a BLASTn search. The closest match of the LSU sequence of V. chiangmaiense was V. minutiellipsoidea (CBS 112523) with 98.10% similarity across 100% of the query sequence, which translates into 98.10% similarity. Therefore, we introduce V. chiangmaiense as a new species based on phylogenetic and morphological evidence.
Distoseptispora is a well-studied genus with 30 species listed in Species Fungorum. Unusually, all species have sequence data available in GenBank (Table 1). There are 74 ITS sequences annotated as Distoseptispora in GenBank. A BLASTn search using the ITS region of the type species Distoseptispora fluminicola strain MFLUCC 15-0417 (NR_154041) showed high similarity and query cover (90-100%) to Distoseptispora and a single uncultured fungus clone C1_AF3. There are 52 species hypotheses (comprising 101 sequences) that have high similarity to Distoseptispora in UNITE. There are over 490 species epithets listed as Sporidesmium in Index Fungorum but there are only 32 ITS sequences annotated as Sporidesmium in Gen-Bank and 12 species hypotheses (comprising 16 sequences) in UNITE. Therefore, some Sporidesmium epithets listed in Index Fungorum could represent Distoseptispora species   freshwater and terrestrial habitats (Su et al. 2016a, b;Hyde et al. , 2019aHyde et al. , b, 2020aYang et al. 2018;Luo et al. 2018Luo et al. , 2019Sun et al. 2020;Monkai et al. 2020;Song et al. 2020). Distoseptispora species can be found in a wide range of habitats, therefore, we would expect the number of species to increase with extensive studies. Hyde et al. (2020a) estimated the crown age of Distoseptisporaceae at around 44.21 MYA, followed by the Cretaceous-Paleogene extinction event (65 MYA). After this extinction event, there was a mass extinction of threequarters of plant and animal species (Ward et al. 2005). The conditions of high humidity and reduced solar insolation after the extinction event favoured saprobic fungi such as Distoseptispora that flourished on the detritus (Vajda and McLoughlin 2004). It can be hypothesised that Distoseptispora species diversified to adapt to various hosts, that were prevalent during this period (Peace et al. 2020). Etymology: Referring to the cylindrical conidia of this fungus Holotype: HKAS 115796 Saprobic on submerged, decaying wood in freshwater. Sexual morph: Undetermined. Asexual morph: Colonies on the substratum superficial, effuse, scattered, gregarious, hairy, brown to dark brown. Mycelium mostly immersed, composed of branched, septate, brown to dark brown, smooth hyphae. Conidiophores 105-157 × 6.5-8.5 μm (x̅ = 131 × 7.5 μm, n = 25), macronematous, mononematous, erect, cylindrical, mostly in a small group of 2-7, rarely solitary, straight or slightly flexuous, unbranched, 18 to 30-septate, smooth, dark brown. Conidiogenous cells 6-8.5 × 5.5-7.5 μm (x̅ = 7.3 × 6.7 μm, n = 25), holoblastic, monoblastic, integrated, determinate, terminal, cylindrical, brown to dark brown. Conidia 136.5-278 × 8.5-11 μm (x̅ = 207 × 9.5 μm, n = 25), acrogenous, solitary, dry, cylindrical to elongated, straight or slightly curved, truncated at the base, rounded at the apex, 20-65-distoseptate, smooth, greenish-brown to dark brown, hyaline at the apex, smooth, thick-walled.
Culture characteristics: Colonies on PDA attaining 3.5 cm diam., after 4 weeks at room temperature, greyish brown to brown at above, circular, smooth, velvety; reverse black, brown to dark brown.
Notes: Distoseptispora cylindricospora can be easily distinguished from other Distoseptispora species by its conidiophores. The conidiophores of D. cylindricospora mostly occur in small groups and the conidiophores can be up to 20-65-septate. However, the conidiophores of other Distoseptispora species are usually solitary and have fewer than 10 septa (Su et al. 2016b;Hyde et al. , 2019a2020a, b, c, d;Xia et al. 2017;Yang et al. 2018;Luo et al. 2018Luo et al. , 2019Sun et al. 2020;Monkai et al. 2020;Song et al. 2020).

Fig. 18
Phylogram generated from maximum likelihood analysis based on combined LSU and ITS sequence data representing species of Beltraniaceae. Related sequences were taken from Hyde et al. (2020a). Thirty-one taxa were included in the combined analyses, which comprised 1452 characters (LSU = 859 bases and ITS = 593 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −4008.944784 is presented. Bootstrap support values for ML equal to or greater than 60% and BPP equal to or greater than 0.95 are given near the nodes. Castanediella couratarii (CBS 579.71) was used as the outgroup taxon. The newly generated sequence is indicated in blue. The type-derived sequences are given in bold Beltrania was introduced with a single species, B. rhombica in 1882, and B. querna was added to the genus after two years (Harkness 1884). Without molecular methods, the identification of new collections was relatively slow, and it took nearly 40 years until another species -B. malaiensis was identified in 1931 (Table 1). After 2010, taxonomic studies of Beltrania were relatively well-studied because the type species B. rhombica was sequenced (Shirouzu et al. 2010;Rajeshkumar et al. 2016;Lin et al. 2017b). Zheng et al. (2020) introduced B. sinensis based on morphological and phylogenetic analysis and accepted 16 species in Beltrania in their taxonomic key. From 2019 to 2021, another six taxa were introduced, including two varieties of B. hasaneana Patil 2019, 2021;Hyde et al. 2020a;Quevedo et al. 2020;Zheng et al. 2020). Only six species of Beltrania (from China, Japan, Malaysia, New Zealand, Thailand) have been sequenced (Shirouzu et al. 2010;Crous et al. 2014Crous et al. , 2015aLin et al. 2017b;Tibpromma et al. 2018). The phylogeny of Beltrania and beltrania-like taxa is not well-resolved.

Beltrania aquatica
Culture characteristics: Colonies on PDA, colony irregular, reaching 30 mm diam., after 20 days at room temperature (25-30 °C), surface rough, with sparse mycelia, velvety, dry, raised in the middle from the side view, edge entire; from above and below, subhyaline at the margin, white at the middle; not producing pigmentation in culture.

Phaeoacremonium camporesii
Culture characteristics: Ascospores germinating on PDA within 24 h. Germ tubes produced from the basal and apical cells of ascospores. Colonies growing on MEA, reaching 20-25 mm in 2 weeks at 16 °C. Mycelia superficial, circular, with entire margin, flat, smooth, from above white; reverse, whitish light brown. Phylogram generated from maximum likelihood analysis based on combined LSU, ITS, tub2 and act sequence data representing species of Phaeoacremonium. Sequences were taken from Huang et al. (2018) and Calabon et al. (2021). The combined analyses comprised 103 taxa and 2108 characters (LSU = 892 bases, ITS = 472 bases, tub2 = 481 bases, act = 263 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −19257.268076 is presented. Bootstrap support values for ML equal to or greater than 70% and BPP equal to or greater than 0.95 are given at the nodes. Pleurostoma ootheca (CBS 115329) and Pleurostoma richardsiae (CBS 270.33) were used as outgroup taxa. The newly generated sequence is indicated in blue. The typederived sequences are given in bold Material examined: Italy, Forlì-Cesena Province, Tontola-Predappio city, on a dead hanging branch of Corylus avellana L. (Betulaceae), 30 January 2019, Erio Camporesi, IT4202 (MFLU 19-0506, holotype); ex-type living culture, MFLUCC 21-0224.

Endocalyx Berk. & Broome
There are eight epithets listed under Endocalyx in Species Fungorum (2021), with only two new species introduced since 2000 (Species Fungorum 2021) (Table 1). Molecular data is only available for E. cinctus and E. metroxyli (from Japan and Thailand; Okada et al. 2017;Konta et al. 2021). Introductions of new species should rely on morphological comparison and molecular data.
Only one ITS sequence is annotated as Endocalyx in Gen-Bank. A BLASTn search of the ITS region of Endocalyx ptychospermatis strain ZHKUCC 21-0008 (as the molecular data of the type species E. thwaitesii is not available) showed a high similarity and query cover (90-100%) to two Xylariales species. There is only one species hypothesis (comprising one sequence) that has high similarity in UNITE. Most records of Endocalyx are saprobes. Cainiaceae species are usually found in monocotyledons, mainly grasses . The host-specificity remains to be studied, but most Endocalyx species occur on Arecaceae, indicating an endophytic lifestyle (Rashmi et al. 2019). Therefore, we would expect the number of species to increase with extensive study of poorly studied hosts. Samarakoon et al. (2016) reported that Amphisphaeriales and Xylariales diverged during 252-66 MYA. The stem age of Cainiaceae is estimated at around 85.12 MYA, and the crown age is around 48.64 MYA . Cainiaceae diverged before the Cretaceous extinction event. It can be hypothesised that Cainiaceae species diversified to adapt to various hosts that were dominant following the extinction event, such as monocotyledons (Peace et al. 2020). The present study introduces Endocalyx ptychospermatis, a novel taxon isolated from a palm tree in Guangdong, China.
Culture characteristics: Colonies on PDA reaching 6 cm diam., after seven days at 25 °C, white at first, irregular, raised, undulate, rough, after maturity, smooth at the margin, white from above, pale-brown from below.

Peroneutypa Berl.
Diatrypaceae was proposed by Nitschke (1869) with Diatrype as the type genus. Diatrypaceae is characterised by Fig. 22 Phylogram generated from maximum likelihood analysis based on combined LSU, ITS and SSU sequence data from Cainiaceae and related families in Xylariales. Eighteen taxa were included in the combined analyses, which comprised 2547 characters (LSU = 908 bases, ITS = 571 bases, SSU = 1068 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −8340.507794 is presented. Bootstrap support values for ML equal to or greater than 75% and BPP equal to or greater than 0.95 are given above the nodes. The tree is rooted with Acrocordiella omanensis (SQUCC 15091). The newly generated sequences are indicated in blue and the type-derived sequences are indicated in bold  (HKAS 113189, holotype). a Appearance of stromata on unidentified dead wood. b Close-up of erumpent stromata. c-d Vertical section of ascomata (including the long neck). e Section of the neck. f Peridium. g Group of asci. h-k Individual asci. l Ascospores. m Germinating ascospore. n, o Colonies on PDA after 30 days (n = colony from above, o = colony from below). Scale bars: d = 100 µm, e-f = 50 µm, g = 30 µm, h-k = 5 µm, l-m = 10 µm 1 3 perithecial ascomata, usually embedded in a black stroma, cylindric-clavate to clavate with long pedicellate asci and allantoid ascospores (Glawe and Rogers 1984;de Almeida et al. 2016). Peroneutypa is a monophyletic genus in Diatrypaceae introduced by Berlese (1902) together with the species P. bellula, P. corniculata and P. heteracantha, but without assigning a type species (Shang et al. 2018;Dayarathne et al. 2020). Rappaz (1987) proposed P. bellula as the type species as the herbarium was in good condition. The genus was synonymized under Eutypella whereas Peroneutypa is characterized by valsoid stroma, long prominent necks, sessile to long stalks, small asci with truncate apices  Shang et al. (2018). Sixty-five taxa were included in the combined analyses, which comprised 1100 characters (ITS = 639 bases, tub2 = 461 bases) after alignment. The best scoring RAxML tree with a final log likelihood score of −12477.423944 is presented. Bootstrap support values for ML equal to or greater than 75% and BPP equal to or greater than 0.95 are given at the nodes. Kretzschmaria deusta (CBS 826.72) and Xylaria hypoxylon (CBS 122620) were used as outgroup taxa. The newly generated sequence is indicated in blue. The type-derived sequences are indicated in bold and black; type species are denoted with TS after the species name 1 3 and allantoid ascospores, but no asexual morph has been reported for this genus (Radu 2013;Raymundo et al. 2014;de Almeida et al. 2016;Shang et al. 2017).
There are 29 epithets listed in Species Fungorum under Peroneutypa. Carmarán et al. (2006) resurrected eight species from Echinomyces and Eutypella and accommodated them under Peroneutypa. Shang et al. (2018) revised Peroneutypa based on multi-gene analyses with an updated key to Peroneutypa. Two new species were also introduced by Dayarathne et al. (2020) and three species were moved to other genera (Table 1). Over 65 ITS sequences are annotated as Peroneutypa in GenBank. A BLASTn search of the ITS region showed a high similarity (88.61%) and query cover (> 90%) to Peroneutypa scoparia (MG980578). There are 18 species hypotheses (comprising 41 sequences) that have high similarity to Peroneutypa in UNITE. Members of this genus are mainly saprobes and pathogens, and they are widely distributed in terrestrial habitats (Lumbsch and Huhndorf 2010;Maharachchikumbura et al. 2015;Shang et al. 2017;Hyde et al. 2019a). As endophytes, some Peroneutypa species appear to be host-specific. For example, the endophyte Peroneutypa scoparia has only been isolated from Garcinia sp. (Phongpaichit et al. 2007), but this could also be due to the lack of sampling. There is likely to be a large number of species that will be discovered as some species appear to be host-specific.
Culture characteristics: Ascospores germinated within 12 h on PDA, colonies grow rapidly on PDA at room temperature, reaching around 50 mm diam., after 4 weeks. Colonies on medium appear circular to irregular, medium dense, flat or effuse, with fimbriate edge, colonies from above and below white to greyish when immature, slight radish pigments visible from above; reverse pale grey to reddishbrown at maturity. Yellow-brown pigment diffused in PDA.
Notes: The morphology of Peroneutypa kunmingensis fits within the generic concept of Peroneutypa with its long prominent necks, small asci, and allantoid ascospores (Carmarán et al. 2006). BLASTn results of ITS and tub2 sequence data showed similarities to Peroneutypa scoparia (MW448609, 88.61% similarity across 95% of the query sequence, which translates into 84.18%) and P. scoparia (MG586231, 81.38% similarity across 96% of the query sequence, which translates into 78.12%). Peroneutypa kunmingensis differs from P. scoparia which has solitary ascomata and small ostioles (Carmarán et al. 2006). Our new species formed a well-separated clade inside Peroneutypa with 93% ML and 1.00 BPP support (Fig. 24). We introduce P. kunmingensis as a new species based on morphological and phylogenetic support.

New species introduction versus GenBank depositions
We provide 12 case studies for different ascomycete genera (Fig. 25), with new species introduced in each. In these genera, 288 species have been introduced in the last 25 years while sequence data from 1467 entities (based on ITS sequences) were added during this period. Several studies have shown that the ITS region alone cannot resolve species level relationships, but it does indicate the possible number of species Maharachchikumbura et al. 2021;Pem et al. 2021;Bhunjun et al. 2022).
Fungi have generally been poorly studied for novel medicinal compounds. Many of the genera discussed in this study produce medicinal compounds, and this aspect has barely been researched. Beltrania rhombica has been reported to have antibacterial and antifungal properties against Staphylococcus aureus and Candida albicans, respectively (Rukachaisirikul et al. 2005). Several antifungal bioactive compounds against pathogenic fungal species (Reátegui et al. 2006;Silva et al. 2017) and other compounds such as naphthalenone and phytotoxins p-hydoxybenzaldehyde (Evidente et al. 2000;Tabacchi et al. 2000;Abou-Mansour et al. 2004) have been identified in Phaeoacremonium species. Species of Apiospora have also been used as antifungal agents in the pharmaceutical industry (Hyde et al. 2019b). Over 95 novel secondary metabolites have been reported from Paraconiothyrium . Paraconiothyrium produces sesquiterpenes, which has several biological activities such as anti-tumour, anti-inflammatory and antibacterial . Paraconiothyrium produces a variety of hydrolytic and oxidative active enzymes, including cellulases, hemicellulases, and lignin-degrading accessory enzymes .
Apiospora has the largest number of epithets listed in Index Fungorum (2021) among the selected genera in this study. Most Apiospora species have been found on monocotyledonous plants from a range of climates (Pintos and Alvarado 2021). The ITS sequence data for about 30 taxa have been deposited each year for the last two years, and the  Fig. 25 The estimates of fungal numbers based on taxonomy, the number of ITS region in GenBank (including uncultured fungi with high similarity) and the number of species hypotheses from environmental samples (UNITE) are illustrated for each entry number of species is likely to increase due to their diverse lifestyles and diverse host range. This is evident in several recent studies (Sharma et al. 2014;Jiang et al. 2019). Most Bambusicola species have been collected on Bambusoideae which are found mostly in tropical and subtropical areas (Lobovikov et al. 2007). The number of species reported in Bambusicola and the number of sequences for taxa deposited in GenBank as Bambusicola every year has remained low since the genus was introduced. The number of species is expected to increase as a large number of novel species continue to be introduced every year from Bambusoideae. The discovery of new taxa in Beltrania was relatively low until the application of molecular data in 1990. This resulted in 13 new species being introduced in 31 years as compared to 13 species in 100 years before that (Index Fungorum 2021). The number of ITS sequences deposited as Beltrania in Gen-Bank has also increased every decade, which would suggest an increase in the number of species. Among the selected genera, Capronia has the second largest number of epithets listed in Index Fungorum (2021) after Apiospora, and none of these genera is host-specific (Sánchez et al. 2019;Pintos and Alvarado 2021). Molecular data has improved species resolution in Capronia, which resulted in the discovery of 65 epithets in the 30 years since 1990 compared to 26 species in over 100 years before that (Index Fungorum 2021). The majority of Capronia species are saprotrophs on decaying wood, bark and leaves, while some may be fungicolous (Untereiner et al. 2011). Therefore, a large number of species is expected to be introduced in this genus. Distoseptispora species have mainly been reported as saprobes from a range of hosts, but only from China and Thailand (Song et al. 2020). This could suggest geographic specificity or could be due to the lack of studies from other countries. The genus has over 70 ITS sequences in GenBank. Endocalyx and Vanakripa are less collected genera as there are less than five species that have been introduced in the last ten years with a small amount of sequence data in Gen-Bank. Host-specificity of taxa remains to be determined, but Endocalyx species are usually found on monocotyledons. Of the species introduced in this paper, Neodeightonia and Vanakripa have the lowest number of species epithets listed in Index Fungorum (2021). Most Neodeightonia species have been recorded recently as saprobes in Thailand , and there are over 60 ITS sequences in GenBank. Paraconiothyrium has 20 species listed in Species Fungorum (2021), but over 500 ITS sequences are available in GenBank. Paraconiothyrium species have a worldwide distribution in diverse habitats . We expect that several species remain to be discovered and Paraconiothyrium is likely to attract more attention in future studies as it can produce several novel secondary metabolites . Peroneutypa species are associated with various lifestyles and are widely distributed (Hyde et al. 2019a). Eighteen Peroneutypa species have been introduced in the last 31 years, and 65 ITS sequences were deposited in GenBank over the same period. There are over 60 epithets of Phaeoacremonium listed in Index Fungorum (2021), and there are over 400 ITS sequences in GenBank. Phaeoacremonium species have been recorded from a wide range of hosts, including monocotyledons and dicotyledons. They have worldwide distribution with reports from America, Europe, Scandinavia, Ukraine, the Middle East and Africa (Gramaje et al. 2015). Several novel secondary metabolites have been identified from Phaeoacremonium species, and are likely to attract several extensive studies in the future. The potential for taxonomic novelties is presumably very large. Neocatenulostroma species are associated with various lifestyles such as endophytes, plant pathogens and saprobes (Markovskaja et al. 2016). There are no new species that have been introduced in Neocatenulostroma since it was introduced in 2014, but 23 ITS sequences were deposited in GenBank over the same interval. The ITS region has been suggested to represent the universal barcode for fungi (Schoch et al. 2012).
Most species in this study have been found in humid/ tropical/subtropical climates such as in Thailand and the southern part of China. Gramineae-inhabiting genera such as Apiospora and Bambusicola were more frequently discovered from warm climate zones, thus further studies in tropical regions would probably result in the discovery of novel taxa Pintos and Alvarado 2021). Although several previous studies have targeted fungal diversity on these hosts, we suspect that a large number of species remain to be discovered.
Another way to look at fungal diversity is to examine taxa from a single habitat, host and substrate, for example, studies of Dai et al. (2017), Tibpromma et al. (2018), Mapook et al. (2020) and Phukhamsakda et al. (2020). Overlapping taxa from each host in these studies are low. This indicates that there is some degree of host-specificity among these taxa at host, genus or family levels. Studies will increase the knowledge of taxa on these hosts and help answer how the environment affects the evolutionary development of fungal communities.

New genus introductions
Approximately 150,000 extant fungi and fungus-like taxa are presently recognized (Wijayawardene et al. 2020), representing 3.5-7% of the 2.2-3.8 million estimated species (Species Fungorum 2021; Hyde et al. 2020aHyde et al. , 2021. The large number of species discovered has also led to the introduction of several new genera every year. Figure 26 shows the trend of new genera introduced every decade from 1690 to 2020 based on data from Index Fungorum (2021). Since the 1900s, over 1000 genera were introduced every decade, except in the 1930s and 1940s as a result of World War II (van Goethem and van Zanden 2021). There was a peak of over 2200 new genera introduced from 2010-2019 (Fig. 26). This is due to a large number of novel taxa being discovered from extensive studies based on a specific location or host (Lücking and Hawksworth 2018), notably Mapook et al. (2020) and Phukhamsakda et al. (2020), each introducing 12 genera,  with six new genera and Yuan et al. (2020) with five new genera. Molecular advancements have increased the rate of species discovery, and they have, furthermore, provided important insights to understand the diversity of fungi, identify rare species and establish conservation goals (Willis 2018). High-throughput sequencing has added a new dimension to the assessment of fungal diversity-one that dramatically increased the estimated number to 11.7-13.2 million species (Wu et al. 2019). It seems clear that Sanger-based molecular mycology and high-throughput sequencing-powered metabarcoding effectively target different species. This paper explores the potential of Sangerbased mycology to unravel new species.  Observations on the potential of environmental sequencing for mycology Sanger sequencing has increased the number of species discovered tremendously, but there is also a large amount of data that have been recovered from metabarcoding efforts (Nilsson et al. 2019). The GlobalFungi database contains over 600 million fungal metabarcoding reads across over 17,000 samples with geographical locations and additional metadata from 178 studies (Větrovský et al. 2020;Tedersoo et al. 2021). It is important to integrate these data into the current taxonomic framework to be able to target the large diversity of hitherto poorly explored taxa, including the so-called dark taxa. These are species and lineages that are known only from sequence data and that at present cannot be linked to any physical specimen, culture, or resolved taxonomic name (Ryberg and Nilsson 2018). The challenge for the future will be to develop specific ways (media, baits) to target these taxa and to consider protocols for naming species known only from DNA sequences. Another challenge is that many environmental sequencing efforts have targeted Europe and North America, which has left many countries in other parts of the world significantly under-sampled in this regard. The majority of the new species in the present study are described from Asia, which offers an outlook on the potential to discover new species from environmental samples from Asia.
Beltrania, Capronia, Distoseptispora, Peroneutypa and Vanakripa are expected to have a high diversity in environmental samples as they are commonly found as saprotrophs. This is reflected in the large number of species hypotheses recorded for Capronia and Distoseptispora with 571 and 101 sequences, respectively in UNITE ( Table 4). The highest number of sequences were recorded in Paraconiothyrium with over 700 sequences classified as 37 species hypotheses. The lowest number of species hypotheses was recorded in Endocalyx (one SH) and Vanakripa (two SHs). Apiospora was recently synonymised with Arthrinium, and there are 72 species hypotheses classified as Arthrinium representing some Apiospora species. The low number of species hypotheses in some genera could be due to the lack of available ITS sequences in GenBank or because metabarcoding sequences are usually not deposited in GenBank (Hawksworth and Lücking 2017). All in all, it seems reasonable that a considerable number of new species are recoverable through environmental sequencing, and the present study hopes to motivate researchers to look beyond species that form fruiting bodies and those might not be straightforward to raise in culture.

Evolution
Divergence time studies suggest that almost all true fungi have a single common ancestor. It is hypothesised that the earliest fungi may have evolved about 1010-890 MYA (Fig. 27) and lived at the mouth of a river (Loron et al. 2019). They were probably aquatic organisms with a flagellum before integrating into the terrestrial environment (Heckman et al. 2001). The fossil record shows that fungi were abundant, and they may have been the dominant life form on earth about 250 MYA (Loron et al. 2019). Taxa of Apiosporaceae diverged around 18 MYA, and the ancestor diverged around 94 MYA, which correlates with the estimated diversification of Poaceae in the Cretaceous-Paleogene period (Hyde et al. 2020a). Apiospora species are mainly associated with Poaceae and also with a variety of other hosts (Sharma et al. 2014;Pintos and Alvarado 2021). Bambusicolaceae and the Bambusoideae hosts diversified around 44-42 MYA , which could explain the host-specificity in most Bambusicolaceae taxa. Herpotrichiellaceae emerged during or after the Cretaceous-Paleogene extinction event at 75-50 MYA (Teixeira et al. 2017). Herpotrichiellaceae species have different ecological preferences and exhibit highly diversified lifestyles, which could be the result of adaptation to new or different hosts after the extinction event. Distoseptisporaceae diverged around 44.21 MYA, following the Tertiary-Cretaceous extinction event, which resulted in the loss of the majority of plant and animal species Hyde et al. 2020a). This may have favoured the saprotrophic lifestyles of Distoseptispora and allowed them to associate with several hosts.
There are approximately 60,000 species of monocotyledons, including the most prominent monocotyledon families Arecaceae (palms) and Poaceae (true grasses). Numerous fungal species from palms presumably remain to be discovered in Bartalinia, Neodeightonia, and the lesser-known genus Endocalyx. These genera are estimated to have diversified in the Paleogene or late Cretaceous. The evolution and diversification of Arecaceae (106.6-89.8MYA) and Poaceae (65 MYA) is estimated to have occurred during major diversification events of the monocotyledons, which appeared in Fig. 27 An overview of fungal diversification. The fungal evolution data were retrieved from Liu et al. (2017), Hongsanan et al. (2020), Hyde et al. (2020a, b, c, d); the Pangaea dispersal data were retrieved from Peace et al. (2020); the relationship of different kingdom and plants were retrieved from Dahlgren et al. (2012), Baker and Couvreur (2013) and Eguchi and Tamura (2016); Insect divergence estimates were retrieved from Condamine et al. (2016), Shin et al. (2021); fish divergence estimates from Nelson et al. (2006) and Long et al. (2014); reptile divergence estimates from Sues (2019); mammal divergence estimates from Senft and Macfalan (2021); timeline for Earth's: Burgess et al. (2014); chronostratigraphic Chart: Cohen et al. (2012Cohen et al. ( , 2013. E = Early, M = Middle, L = Late. The pictures (fungi and moss) were from the authors. The drawings (animal and plants) were retrieved from Microsoft power point graphic and the figure was illustrated with Adobe Illustrator CC 2019 (Adobe Inc.) ◂ the late Cretaceous (150-140 MYA to late Jurassic-early Cretaceous) (Soltis and Soltis 2004). This was a time of great productivity in the world's oceans and the main diversification of angiosperm plants, and also the time when several continents separated due to plate tectonics (Soltis and Soltis 2004). Therefore, this might suggest that extensive sampling of monocotyledons in different continents is likely to yield a large number of species as they evolved 150-140 MYA. The loss of biodiversity is ongoing due to various factors such as extinction events, loss of natural habitat, the overuse of natural resources, overpopulation, global warming, and human-induced pollution (Dunlap and Jorgenson 2012). The introduction of foreign plants by accident (e.g., weeds) such as trade and animal migration has furthermore led to the loss of species in agriculture, forestry and industry (e.g., eucalyptus and rubber) (Thuiller et al. 2008). The impact of global warming has prolonged the growing season of plants in terrestrial ecosystems (Linderholm 2006). As a result, plants could alter the community composition and deplete soil nutrients, which would make the soil uninhabitable for other plants, animals, insects and micro-organisms (Jobbagy and Jackson 2001;Wagg et al. 2014;Pugnaire et al. 2019).
The climate change effects on biodiversity have resulted in a need for more effective ways to discover species (Volodin et al. 2013). Novel algorithmic approaches for analyzing sequence data combined with rapidly expanding DNA barcode libraries could provide a potential solution (Kokkonen 2015). There is a need to establish how many species are likely to be discovered before some host plants become extinct (Costello et al. 2013). This study suggests that there is some level of host-specificity as a result of the co-evolution of fungal groups and their respective hosts. Therefore, extensive studies of hosts that co-evolved with fungal groups are likely to warrant the discovery of a large diversity of novel fungi. An important factor in predicting fungal numbers is whether species are host-specific or generalists (Zhou and Hyde 2001). Some species in this study are ubiquitous, whereas some show some level of specificity. Determining host-specificity based on the knowledge of 150,000 fungal species is not likely to be reliable as it represents only 10% of the most conservative estimate of 1.5 million species (Species Fungorum 2021). A large diversity of fungi is likely to be discovered in unstudied countries and hosts, even in small genera such as Atrocalyx, Endocalyx Lignosphaeria, Okeanomyces, Rhamphoriopsis and Vanakripa. There is a need for studies in hot and humid climates which could increase the number of species in many genera.

Concluding remarks
Fungi have been found from all habitats, and many new species have been found in historically understudied regions and habitats (Hyde et al. 2020a, b, c, d). A great diversity is likely to be discovered in tropical and warm-temperate areas (Hillebrand 2004). New fungal species are also likely to be found in areas that have been extensively studied. Thailand has been studied extensively, and yet it continues to be one of the top countries for fungal discoveries Index Fungorum 2021). Metabarcoding has revealed a much larger diversity of fungi than traditional methods, which only targets taxa with visible fruiting bodies or species cultured on artificial media (Wu et al. 2019). A large diversity of undescribed species will likely continue to be discovered by metabarcoding, even in environments that have been extensively studied using traditional methods (Martin-Sanchez and Sáiz-Jiménez 2012; Lindahl et al. 2013;Su et al. 2015;Wang et al. 2015;Wu et al. 2019).
Most fungal species evolved following the great extinction events during the Triassic-Jurassic extinction, where 66-80% of biodiversity on earth became extinct. Much of the evolved diversity followed the Pangaea plate disintegration that started in the Middle Jurassic and formed continental amalgamation in the early Cretaceous. The finally shaped supercontinent was detected in the Cretaceous to Cenozoic, and the event resulted in independent speciation of the modern taxa (Peace et al. 2020). The Cretaceous-Paleogene mass extinction resulted in the loss of biodiversity especially in the vertebrate groups and 80% species-level extinctions of land plants (Wilf and Johnson 2004). The surviving mammals, birds, frogs, and fishes rapidly diversified in the early Cenozoic. During this time, other organisms also independently evolved with an estimated 925,000 insect species, 6,495 mammal species, 32,500 fish species and 18,000 bird species (Burgin et al. 2018;Willis 2018;Cheek et al. 2020).
Fungi have the second-largest estimated species numbers after insects (2-11 million estimated species). Known species of fungi are less than plants, yet the estimates are much higher. Due to the lack of studies and few morphological differences, fungi that evolved with their hosts are likely to be many more fungal species than currently known. There is no reason that fungi should not have been subjected to the same evolutionary pressures and that the numbers of fungi that have evolved are greater than the present evidence has revealed. Fungi have limited morphological characters to differentiate them, and thus large numbers of cryptic species are likely to exist in most genera. We can only establish accurate fungal diversity estimates with extensive molecular studies of fungal species.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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