Revision of Bionectriaceae and acremonium-like fungi in Hypocreales

Revision of Bionectriaceae and acremonium-like fungi in Hypocreales Lin Zhao 2025 Revision of Bionectriaceae and acremonium-like fungi in Hypocreales Lin Zhao 17 September 2025 Lin Zhao Revision of Bionectriaceae and acremonium-like fungi in Hypocreales PhD thesis, Utrecht University, Utrecht, the Netherlands (2025) ISBN: 978-94-6510-796-7 Cover design: Lin Zhao, Marjan Vermaas Layout design: Lin Zhao Printed by: ProefschriftMaken, Utrecht - https://www.proefschriftmaken.nl/ Copyright © 2025 by Lin Zhao All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without written permission of the author, or when applicable, of the publishers of the scientific papers. Revision of Bionectriaceae and acremonium-like fungi in Hypocreales Revisie van Bionectriaceae en acremonium-achtige schimmels in Hypocreales (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. ir. W. Hazeleger, ingevolge het besluit van het College voor Promoties in het openbaar te verdedigen op woensdag 17 september 2025 des ochtends te 10.15 uur door Lin Zhao geboren op 20 april 1995 te Fujian, China Promotor: Prof. dr. P.W. Crous Copromotor: Dr. J.Z. Groenewald Beoordelingscommissie: Dr. ir. A.D. van Diepeningen Prof. dr. E.E. Kuramae Prof. dr. M. Thines Prof. dr. ir. R.P. de Vries Prof. dr. H.A.B. Wösten To my family, my friends and myself The research described in this thesis was performed at the Evolutionary Phytopathology Group, Westerdijk Fungal Biodiversity Institute (formerly CBS-KNAW), Utrecht, the Netherlands and supported by the China Scholarship Council (CSC student number: 202006510014) CONTENTS Chapter 1 General introduction 8 Chapter 2 Revising Clonostachys and allied genera in Bionectriaceae 24 Chapter 3 Bionectriaceae: a poorly known family of hypocrealean fungi with major commercial potential 126 Chapter 4 Revision of acremonium-like fungi in Hypocreales: A taxonomic and phylogenetic perspective 266 Chapter 5 Summary and general discussion 404 Appendix English summary 420 Nederlandse samenvatting 422 Curriculum vitae 424 List of publications 425 Acknowledgements 426 Chapter 1 General introduction 15 1 Fig. 3. Morphological characteristics of sexual morphs of Bionectriaceae. A–N. Perithecial ascomata. A–D. Perithecia: Clonostachys aurantiaca (A), Parageonectria arachispora (B), Hydropisphaera solani (C), Clavatomyces korfii (D). E, F. Section through perithecium: Clonostachys fusca (E), Parageonectria arachispora (F). G, H. Lateral perithecial wall showing two regions: Clonostachys aurantiaca (G), Hydropisphaera armeniaca (H). I–K. Asci: Hydropisphaera solani (I), Hydropisphaera armeniaca (J), Clonostachys fusca (K). L–N. Ascospores: Hydropisphaera solani (L), Hydropisphaera armeniaca (M), Parageonectria arachispora (N). O–R. Cleistothecial ascomata. O, P. Cleistothecium (Emericellopsis salmosynnemata). Q. Asci (Emericellopsis soli). R. Ascospores (Emericellopsis mexicana). Scale bars: E, F = 100 μm; G–R = 10 μm. Chapter 1 16 Fig. 4. Morphological characteristics of asexual morphs of Bionectriaceae. A–C. Acremonium-like: Protocreopsis polyphialidica (A), Sesquicillium thailandense (B), Acremonium proliferatum (C). D. Verticillium-like (Clonostachys palmae). E. Stilbella-like (Mycocitrus synnematus). F, G. Penicilliumlike: Clonostachys cylindrica (F), Sesquicillium candelabrum (G). H. Gliocladium-like conidiophores (Roumegueriella echinulata). Scale bars: E = 100 μm; A–D, F–H = 10 μm. The apex is either simple or features an inconspicuous or distinct apical ring. Ascospores are ellipsoid to broadly ellipsoid, globose, fusoid, aseptate, 1to multi-septate, hyaline, smooth, striate, spinulose, verrucose, tuberculate, or ornamented with wings or flanges (Rossman et al. 1999) (Fig. 3). Asexual morphs are hyphomycetous, or less commonly coelomycetous, with morphologies acremonium-, verticillium-, stilbella-, penicillium-, or gliocladium-like (Fig. 4). In the Bionectriaceae, most genera have an acremonium-like asexual morph. These fungi are similar to the genus Acremonium, typically characterised by producing septate, General introduction 17 1 smooth hyphae, having conidiophores that are solitary, straight or irregularly curved, unbranched or basitonically branched, with narrow, tapered, mostly lateral phialides and unicellular conidia arranged in slimy heads or chains (Hou et al. 2023). Some genera with stilbella-like conidiophores forming synnemata are bundles of conidiophores (sporebearing structures) arranged together in a vertical or upright position (Gams 1971). Genera with verticillium-like conidiophores emerging from either submerged hyphae or aerial mycelium are upright, predominantly arranged in verticillate whorls, septate, hyaline, with conidiogenous cells that are enteroblastic, monophialidic, terminal or lateral, flaskshaped or aculeate (Giraldo & Crous 2019). Additionally, some genera with penicilliumlike conidiophores are mononematous, with stipes typically arising from submerged or aerial hyphae or from aerial hyphal fascicles, ropes, or strands, bearing penicilli that are monoverticillate or bito multi-verticillate, and conidiogenous cells that are phialidic, narrowly flask-shaped, widest in the lower third or middle, and slightly and continuously tapering toward the tip (Visagie et al. 2014, Zhao et al. 2023). Furthermore, genera with gliocladium-like conidiophores are monomorphic, narrowly penicillate, adpressed, ranging from monoto terverticillate, emerging from the agar surface or aerial mycelium, with phialides either cylindrical or flask-shaped (Rossman et al. 1999). It is worth noting that in Clonostachys, which is the type genus of Bionectriaceae, the asexual morph is frequently characterised by dimorphic conidiophores, with primary conidiophores forming early and having a verticillium-like or narrowly penicillate structure, while secondary conidiophores develop later, showing a penicillium-like morphology and often aggregating into cushionshaped, more or less distinct sporodochia (Schroers 2001, Zhao et al. 2023). Ecological niches and industrial relevance of Bionectriaceae and acremonium-like fungi Fungi of the Bionectriaceae and acremonium-like species are distributed worldwide, occupying diverse ecological niches and belonging to various ecological functional groups. These fungi can be bryophilous, growing on mosses; coprophilous, thriving on dung; corticolous, inhabiting tree bark; fungicolous, living on or parasitizing other fungi; foliicolous, found on leaves; herbicolous, growing on herbaceous plants; and lichenicolous, forming associations with or parasitizing lichens (Hyde et al. 2020, Perera et al. 2023, Zhao et al. 2023). In terms of ecological roles, many species are saprotrophic, decomposing complex organic materials, such as plant litter and dead wood to acquire carbon. Their filamentous growth facilitates nutrient translocation, and they produce a variety of enzymes that break down compounds like cellulose, hemicellulose, chitin, pectin, and lignin. Through these processes, saprotrophs play a crucial role in nutrient cycling and maintaining ecosystem sustainability (Baldrian 2017). Additionally, some species act as plant pathogens infecting and causing diseases in plants, forming close associations to access host resources and overcome plant defences, often causing significant agricultural and ecological damage Chapter 1 18 (Bosland & Barchenger 2024). Furthermore, some species also play a role in infectious diseases in humans and animals, with some causing opportunistic infections, which can be particularly harmful to immunocompromised individuals (Summerbell & Scott 2015, Summerbell et al. 2018). Another important ecological role is mycoparasitism, in which fungi parasitize other fungi, either as obligate or facultative parasites, obtaining nutrients from their host to sustain growth, secreting cell wall degrading enzymes, including chitinases, glucanases, and proteases, to degrade the cell wall of the host fungus, and often releasing toxins or antibiotics to weaken or kill the host (Reino et al. 2008, Seidl 2008, Vinale et al. 2008, Ajith & Lakshmidevi 2010, Qualhato et al. 2013, Karlsson et al. 2018, Sun et al. 2020a, b). Additionally, endophytes are fungi that colonise plant tissues internally without causing visible disease symptoms, sometimes conferring benefits to the plant, such as enhanced stress tolerance or pathogen resistance (Gouda et al. 2016). Acremonium-like fungi and Bionectriaceae species can play a significant role in industrial, agricultural, pharmaceutical, and commercial applications, contributing to enzyme production, biological control, and bioremediation, with several genera, especially Clonostachys and Acremonium, garnering significant attention due to their ecological significance and commercial value (Goswami et al. 2008, Bischoff et al. 2009, Wicklow & Poling 2009, Sun et al. 2020a). Clonostachys species are particularly noteworthy for their ability to act as biocontrol agents against numerous fungal plant pathogens, with C. rosea being the most studied species in the genus. It has shown promising results in managing fungal plant diseases through the production of antifungal metabolites and mycoparasitism, and is also capable of biodegradation and biotransformation. To date, C. rosea is effective against many fungal plant pathogens. Jensen et al. (2004) investigated the biopriming of infected carrot seeds using C. rosea, an antagonist, to help control seedborne Alternaria species. Yohalem et al. (2004) demonstrated that C. rosea, as a biocontrol agent, effectively suppresses the sporulation of Botrytis aclada on necrotic leaf tips. However, it does not prevent the pathogen from spreading to adjacent living tissue. Schoneberg et al. (2015) revealed that C. rosea has the potential to control Fusarium head blight pathogens, including Fusarium graminearum and F. cerealis (formerly F. crookwellense), by significantly suppressing perithecia and ascospore production. When applied to wheat straw before pathogen inoculation, it reduced perithecia and ascospore production by 88–100 %. When applied after inoculation, it reduced these by 73 % and 100 %, respectively (Schoneberg et al. 2015). These results highlight the potential of C. rosea as a biocontrol agent against Fusarium head blight (Schoneberg et al. 2015). Furthermore, the identification of efficient microorganisms, such as C. rosea, which can degrade plastic waste like starch films and poly(ɛ-caprolactone), offers a promising solution to mitigate the environmental burden of plastic pollution (Urbanek et al. 2017). Species of Acremonium s. lat. have proven to be of great importance in agroforestry, industry, the medical and pharmaceutical sectors, and for food storage and preservation, General introduction 19 1 with some species also serving as effective biological control agents against fungal plant pathogens (Goswami et al. 2008, Choi et al. 2009, Auer & Ludwig-Müller 2014, Bobeck & Pearce 2017). Furthermore, Acremonium s. lat. species are utilised in industrial processes to produce enzymes, antibiotics, and other bioactive compounds. For example, Hapsidospora chrysogena (formerly Acremonium chrysogenum) is a source of cephalosporins, a class of β-lactam antibiotics widely used in clinical practice to treat bacterial infections (Burton & Abraham 1951, Gams 1971, Hamilton-Miller 2000). So far, over 600 secondary metabolites have been extracted from Acremonium s. lat. species, and the wide variety of bioactive compounds continues to draw considerable interest from chemists and pharmacologists (Qin et al. 2024). These fungi are highly relevant in the medical field as opportunistic pathogens, causing infections such as onychomycosis, keratomycosis, peritonitis, and fungemia, particularly in immunocompromised individuals (Gams 1971, Novicki et al. 2003, Summerbell & Scott 2015, Summerbell et al. 2018). Additionally, species of Acremonium s. lat. are known to cause food spoilage, contaminating cereals, fruits, vegetables, dairy products, beverages, and processed foods, including frozen meat and salami, posing challenges for food storage and safety (Fernández-Trujillo 1997, Fujikawa 1997, Pitt & Hocking 1997, 2009, 2022, Summerbell & Scott 2015, Summerbell et al. 2018, Samson et al. 2019). Aims and outline of this thesis This thesis aims to provide a comprehensive revision of Bionectriaceae, including an indepth study of its type genus Clonostachys, as well as acremonium-like fungi, focusing on the morphological, taxonomic, and phylogenetic aspects of these microfungi with reduced morphology. Through this study, I seek to clarify the classification of these fungi, identify novel species within these groups, and explore their evolutionary relationships using both morphological characteristics and molecular data. The insights gained from this research will not only refine the taxonomy and classification of Bionectriaceae and acremonium-like fungi but also provide a basis and phylogenetic reference for future investigations into their ecological roles and commercial potential. These data will contribute to the more effective utilisation of these fungi in biocontrol, pharmaceutical, and agricultural applications. In Chapter 2, I revise Clonostachys, the type genus of Bionectriaceae, with C. rosea as the type species. Clonostachys rosea is recognised as an excellent biological agent and plays a crucial role in biological control of numerous fungal plant pathogens due to its ability to inhibit pathogen growth through competitive exclusion, production of secondary metabolites, and mycoparasitism (Sun et al. 2020a, Jensen et al. 2022). Its significance extends beyond ecological roles, as C. rosea has garnered attention for its potential in integrated pest management and sustainable agriculture. Given the importance of Clonostachys in both ecological and biotechnological contexts, the aim of this study is Chapter 1 20 to reconstruct the phylogenetic backbone of the genus Clonostachys using specific gene regions to establish a clearer evolutionary framework. A further aim is to distinguish different species based on molecular and morphological data, and to re-evaluate Clonostachys within the Bionectriaceae, thereby clarifying its placement and circumscription within a broader taxonomic context. The Bionectriaceae includes members that play a crucial role in biotechnological and commercial applications, such as biocontrol agents and biodegraders, and are a source of bioactive secondary metabolites. Despite their significant ecological and industrial relevance, Bionectriaceae remains poorly understood, with many genera lacking clear taxonomic definitions. In Chapter 3, I aim to address these gaps by reconstructing a clearer phylogenetic backbone for this family to better delineate species and genera within Bionectriaceae. These results contribute to resolving taxonomic ambiguities within Bionectriaceae and provide a robust phylogenetic framework for future studies on this ecologically and commercially important group of fungi. In Chapter 4, building on the revision of the Bionectriaceae in Chapter 3, I found that many genera within the family exhibit acremonium-like asexual morphs, but do not belong to the Acremonium s. str. This fungal group is not limited to the genus Acremonium but is also present in Bionectriaceae as well as other families within Hypocreales and related orders. Due to the highly reduced micromorphological structures of acremonium-like fungi and the poorly differentiated and overlapping features among different taxa, species classification within this group is particularly challenging. 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Clear lactic acid was used as mounting medium for the observation of micromorphological structures of stromata, perithecia, perithecial walls, asci, ascospores, conidiophores and conidia (Schroers 2001). Observations of micro-morphological characteristics were processed with a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope. Photomicrographs and measurements were taken with a Nikon DS-Ri2 high-definition colour digital camera using the NIS-elements D software v. 4.50 (Nikon, Tokyo, Japan). All descriptions, illustrations and nomenclatural data were deposited in MycoBank (www. MycoBank.org; Crous et al. 2004), and specimens were deposited in the CBS Fungarium. RESULTS Phylogenetic analyses For inferring the phylogeny of the genus Clonostachys within the Bionectriaceae, we analysed aligned DNA sequence data from five concatenated loci (ITS, LSU, RPB2, TEF1, and TUB2) in dataset 1. To obtain a more precise phylogenetic relationship of species within Clonostachys, more inclusive analyses based on DNA sequence data from five loci were carried out for the genus (dataset 2). Dataset 1: Concatenated and aligned ITS, LSU, RPB2, TEF1, and TUB2 sequences from 269 taxa represent several genera belonging to the Bionectriaceae, with Tilachlidium brachiatum (CBS 363.97, CBS 505.67), Flammocladiella anomiae (CLL 16017), F. aceris (CBS 138906) and F. decora (CBS 142776) serving as outgroups (Hypocreales, Tilachlidiaceae & Flammocladiellaceae; Fig. 1). The final alignment consisted of 4245 characters, including alignment gaps (gene boundaries ITS: 1–691, 691 bp; LSU: 692–1495, 804 bp; RPB2: 1496– 2288, 793 bp; TEF1: 2289–3103, 815 bp, TUB2: 3104–4245, 1142 bp). Among these, 2315 character sites were conserved (ITS: 257, LSU: 605, RPB2: 297, TEF1: 484, TUB2: 672), 1872 were variable (ITS: 407, LSU: 198, RPB2: 477, TEF1: 330, TUB2: 460), and 1646 were parsimony informative characters (ITS: 338, LSU: 167, RPB2: 453, TEF1: 281, TUB2: 407). The phylogenetic trees based on dataset 1 were generated with Maximum-likelihood and Bayesian analyses. According to the result of MrModelTest, the GTR+I+G model was proposed for all loci investigated. The Bayesian analysis of the concatenated five-locus alignment lasted for 10,075000 generations and 20152 trees were generated after the average standard deviation of split frequencies value was below 0.01 in the BI analysis. A Chapter 2 32 total of 15114 trees were used for calculating the posterior probabilities (PP) after the first 25 % of trees were discarded as the burn-in phase. The three phylogenetic analyses (RAxML, IQ-TREE, and MrBayes) overall displayed the same species clades and mainly differed with regards to the backbone relationships between species clades/ lineages. The best RAxML tree based on the combined dataset is presented here with bootstrap support values of ML analyses (RAxML-BS / IQ-TREE-BS) and relevant Bayesian posterior probabilities (PP) shown at the nodes (RAxML-BS > 50 % / IQ-TREE-BS > 90 % / PP > 0.90) (Fig. 1). RAxML Emericellopsis donezkii CBS 489.71_T Stilbocrea macrostoma CBS 114375 Stanjemonium fuscescens CBS 264.96_T Stilbocrea colubrensis CLLM 16003_T Acremonium egyptiacum CBS 114785_T Stanjemonium ochroroseum CBS 656.79_T Ovicillium oosporum CBS 110151_T Acremonium alternatum CBS 407.66_T Acremonium stroudii CBS 138820_T Ovicillium subglobosum CBS 101963_T Emericellopsis mirabilis CBS 176.53 Geosmithia lavendula CBS 344.48_T Emericellopsis stolkiae CBS 159.71_T Flammocladiella anomiae CLL 16017 Emericellopsis salmosynnemata CBS 182.56_T Emericellopsis fimetaria CBS 176.60 Acremonium charticola CBS 547.86 Emericellopsis pallida CBS 624.73 Ovicillium attenuatum CBS 399.86_T Tilachlidium brachiatum CBS 363.97 Emericellopsis glabra CBS 119.40_T Emericellopsis pallida CBS 490.71_T Emericellopsis humicola CBS 180.56_T Emericellopsis maritima CBS 491.71_T Emericellopsis maritima CBS 379.70F Stanjemonium grisellum CBS 655.79_T Acremonium egyptiacum CBS 124.42 Stilbocrea macrostoma CBS 141849 Emericellopsis pusilla CBS 226.62_T Emericellopsis alkalina CBS 120049 Acremonium psychrophilum CBS 139.93 Emericellopsis robusta CBS 105.70_T Acremonium charticola CBS 117.25 Acremonium brachypenium CBS 866.73_T Emericellopsis terricola CBS 120.40_T Emericellopsis alkalina CBS 127350_T Emericellopsis minima CBS 190.55_T Geosmithia pallidum CBS 260.33_T Acremonium sordidulum CBS 385.73_T Stilbocrea walteri CBS 144627_T Emericellopsis microspora CBS 380.62_T Flammocladiella aceris CBS 138906_T Emericellopsis stolkiae CBS 139531 Tilachlidium brachiatum CBS 505.67 Flammocladiella decora CBS 142776 Acremonium sordidulum CBS 102413 Emericellopsis mirabilis CBS 177.53_T 85/95/- 100/100/1 92/99/1 100/100/1 96/100/1 100/100/1 92/99/1 100/100/1 69/100/1 100/100/1 100/100/1 100/100/1 88/98/1 50/-/0.94 100/100/1 100/100/1 58/95/0.99 68/93/1 100/100/1 51/-/- 100/100/1 100/100/1 100/100/1 57/94/1 100/100/1 67/99/0.95 88/100/1 100/100/1 97/100/1 100/100/1 100/100/1 100/100/1 99/100/1 100/100/1 100/100/1 84/100/1 97/100/1 100/100/1 97/100/1 81/100/0.99 100/100/1 68/100/1 100/100/1 100/100/1 2X 2X Stilbocrea Ovicillium Geosmithia Acremonium Stanjemonium Emericellopsis Fig. 1. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on aligned and concatenated ITS, LSU, RPB2, TEF1 and TUB2 sequences of 269 strains representing Bionectriaceae and outgroups. Numbers at branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP) above 50 % / 90 % / 0.9. New species are printed in red font, new combinations in blue font and coloured boxes highlight genera. Roman numerals indicate subgenera as coded in the legend. “T” indicates ex-type strains. The tree is rooted to Flammocladiella aceris CBS 138906, F. decora CBS 142776, F. anomiae CLL 16017, Tilachlidium brachiatum CBS 363.97, and T. brachiatum CBS 505.67 (Hypocreales, Flammocladiellaceae & Tilachlidiaceae). Scale bar represents expected number of changes per site. Revising Clonostachys and allied genera in Bionectriaceae 33 2 trees targeting each of the used partitions individually (ITS, LSU, RPB2, TEF1) are presented as Supplementary Figs S1–S4; TUB2 is not shown as those sequences were mainly available only for Clonostachys. The resulting phylogenetic tree (Fig. 1) resolved 24 well-supported clades, representing 24 genera in the Bionectriaceae. In our study, one clade has isolates preliminarily identified as Sesquicillium microsporum that are here reassigned to Nectriopsis (100 % /100 % / 1), with one new species, Nectriopsis didymii. Two clades have isolates preliminarily identified as Clonostachys that are here assigned to Mycocitrus (96 % / 98 % / 0.99) and Stephanonectria (100 % / 100 % / 1). The genus Sesquicillium (97 % / 100 % / 1) is resurrected to accommodate the subgenera Epiphloea (except C. setosa) and Uniparietina, with three new species and seven new combinations. The genus Clonostachys (99 % / 100 % / 1) includes the subgenera Astromata, Bionectria, Myronectria and Zebrinella with 49 known and 19 new species (Figs 1, 2). However, the here presented dataset also supports Lasionectria antillana CBS 122797_T Hydropisphaera fungicola CBS 122304_T Gliomastix roseogrisea CBS 134.56_T Geonectria subalpina CBS 143540_T Gliomastix masseei CBS 794.69_T Lasionectria mantuana CBS 114291 Protocreopsis phormiicola CBS 567.76_T Lasionectria mantuana A.R. 4029 Fusariella atrovirens CBS 311.73 Paracylindrocarpon pandanicola KUMCC 17-0272_T Synnemellisia aurantia COAD 2070_T Fusariella arenula CBS 330.77 Lasionectriopsis germanica CBS 143538_T Lasionectria sylvana CBS 566.76 Fusariella concinna CBS 312.73 Fusariella hughesii CBS 435.70 Gliomastix masseei CBS 557.75 Fusariella sp. CBS 128364 Lasionectria antillana CBS 114748 Hydropisphaera peziza CBS 296.65 Hydropisphaera peziza CBS 139487 Gliomastix roseogrisea CBS 380.70A Lasionectriopsis pteridii CBS 783.69 Gliomastix polychroma CBS 181.27_T Protocreopsis caricicola CBS 140572_T Hydropisphaera cyatheae CBS 575.76 Paracylindrocarpon nabanheensis KUMCC 16-0147_T Fusariella curvata MFLUCC 15-0844_T Ochronectria calami CBS 125.87 Protocreopsis pertusa CBS 568.76 Paracylindrocarpon aloicola CBS 141300_T Paracylindrocarpon aloicola CBS 135907 Ochronectria thailandica MFLUCC 15-0140_T Hydropisphaera suffulta CBS 122.87 Verrucostoma martinicense CBS 138731_T Lasionectria krabiense MFLUCC 15-0673_T Lasionectriella herbicola CBS 140156_T Verrucostoma freycinetiae MAFF 240100_T Gliomastix murorum CBS 154.25_T Ochronectria calami CBS 445.96 Gliomastix murorum CBS 195.70 Gliomastix polychroma CBS 184.30 Fusariella arenula CBS 329.77 Lasionectriopsis pteridii CBS 782.69_T Paracylindrocarpon xishuangbannaensis KUMCC 16-0144_T Roumegueriella rufula CBS 346.85 Lasionectriella rubioi CBS 140157_T 98/100/1 100/100/1 100/100/1 100/100/1 100/100/1 97/100/1 83/99/0.98 100/100/1 98/100/1 100/100/1 100/100/1 76/99/0.99 84/98/1 100/100/1 90/100/1 100/100/1 57/-/0.98 100/100/1 100/100/1 100/100/1 100/100/1 100/100 100/100/1 100/100/1 100/100/1 61/-/0.98 95/100/1 90/100/1 100/100/1 79/100/1 100/100/1 100/100/1 100/100/1 100/100/1 95/99/1 100/100/1 100/100/1 59/-/- 100/100/1 100/100/1 100/100/1 62/100/- 100/100/1 86/100/1 52/-/0.98 100/100/1 Protocreopsis Lasionectriopsis Lasionectriella Ochronectria Lasionectria Geonectria Hydropisphaera Gliomastix Synnemellisia Roumegueriella Verrucostoma Paracylindrocarpon Fusariella Fig. 1. (Continued). Chapter 2 34 Mycocitrus aurantium BAFC 3843 Nectriopsis didymii CBS 326.79 Sesquicillium neerlandicum CBS 148201 Nectriopsis fuliginicola CBS 400.82_T Sesquicillium symmetricum CBS 124.79_T Sesquicillium neerlandicum CBS 148202 Sesquicillium symmetricum CBS 485.78 Nectriopsis microspora CBS 102560 Nectriopsis microspora CBS 933.69_T Sesquicillium saulense BRFM 2782_T Sesquicillium buxi JW6017 Stephanonectria keithii CBS 943.72 Sesquicillium neerlandicum CBS 148214 Sesquicillium spinulosisporum CLLG12001_T Nectriopsis candicans CBS 440.65 Mycocitrus coxeniae BRIP 49599a_T Nectriopsis lindauiana CBS 897.70_T Nectriopsis didymii CBS 395.82 Stephanonectria keithii CBS 100007 Nectriopsis didymii CBS 355.70 Sesquicillium rossmaniae CBS 210.93 Mycocitrus coccicola BUcCo Nectriopsis rexiana CBS 305.70A Sesquicillium neerlandicum CBS 148213 Sesquicillium sesquicillii CBS 180.88_T Nectriopsis violacea CBS 914.70_T Stephanonectria chromolaenae MFLUCC 18-0589_T Nectriopsis candicans CBS 701.79_T Stephanonectria chromolaenae CBS 476.91 Stephanonectria chromolaenae CBS 475.91 Sesquicillium buxi JW94008 Sesquicillium neerlandicum CBS 148210 Mycocitrus phyllostachydis CBS 330.69 Nectriopsis microspora CBS 954.72 Stephanonectria keithii CBS 434.70 Nectriopsis microspora CBS 582.77 Sesquicillium neerlandicum CBS 148212 Sesquicillium essexcoheniae CBS 918.97 Nectriopsis didymii CBS 852.70A_T Sesquicillium phyllophilum CBS 662.83 Sesquicillium buxi CBS 288.62 Sesquicillium phyllophilum CBS 921.97_T Mycocitrus odorus CBS 120610 Nectriopsis microspora CBS 354.70 Mycocitrus aurantium BAFC 51693 Sesquicillium buxi JW58015 Nectriopsis rexiana CBS 305.70C Nectriopsis didymii CBS 852.70B Sesquicillium rossmaniae CBS 211.93_T Sesquicillium buxi JW182006 Nectriopsis violacea CBS 849.70 Nectriopsis rexiana CBS 542.92 Mycocitrus coccicola BUcS Sesquicillium rossmaniae CBS 221.93 Nectriopsis didymii CBS 788.85 Sesquicillium buxi JW259005 Mycocitrus synnematus CBS 126677_T Sesquicillium intermediophialidicum CBS 685.96_T Sesquicillium buxi JW199009 Sesquicillium neerlandicum CBS 148215 Sesquicillium buxi CBS 696.93 Sesquicillium neerlandicum CBS 148209 Sesquicillium neerlandicum CBS 148203_T Mycocitrus odorus CBS 100104_T Sesquicillium buxi CBS 202.69 Nectriopsis didymii CBS 652.70 Nectriopsis sporangiicola CBS 166.74_T Sesquicillium essexcoheniae BRIP 75170a_T 61/94/0.99 52/95/1 100/100/1 90/100/1 78/99/1 97/100/1 86/100/1 87/100/0.97 98/99/1 100/100/1 95/99/1 100/100/1 89/100/1 87/99/1 94/98/1 100/100/1 52/-/- 95/98/1 76/100/0.95 99/100/1 100/100/1 100/100/1 57/98/- 99/100/1 96/98/0.99 98/100/1 100/100/1 100/100/1 100/100/1 100/100/1 63/-/0.92 94/100/1 97/100/1 100/100/1 61/98/- 99/100/1 100/100/1 90/100/1 100/99/1 100/100/1 100/100/1 72/98/- 93/98/0.94 78/99/1 55/96/0.98 59/96/0.97 100/100/1 100/100/1 99/100/1 Nectriopsis Mycocitrus Stephanonectria Sesquicillium II I I Fig. 1. (Continued). that Sesquicillium and Clonostachys derive from the same ancestor, as their phylogenetic sister group relatedness is highly supported (95 % / 99 % / 1). Revising Clonostachys and allied genera in Bionectriaceae 35 2 Sesquicillium candelabrum JW31018 Clonostachys australiana CBS 102423 Sesquicillium lasiacidis NL19-086015 Clonostachys australiana CBS 102421_T Clonostachys pityrodes CBS 102033_T Sesquicillium lasiacidis CBS 147133 Clonostachys chlorina CBS 287.90_T Sesquicillium lasiacidis CBS 179.88_T Clonostachys fusca CBS 101925 Clonostachys fusca CBS 207.93_T Clonostachys vesiculosa HMAS 183151_T Sesquicillium candelabrum JW79008 Clonostachys setosa CBS 917.97_T Sesquicillium candelabrum YFCC 896 Sesquicillium lasiacidis CBS 190.38 Clonostachys leucaenae MFLUCC 20-0008_T Sesquicillium candelabrum YFCC 895 Clonostachys venezuelae CBS 107.87_T Sesquicillium lasiacidis NL19-089008 Clonostachys lucifer CBS 100008_T Sesquicillium candelabrum CBS 513.67 Clonostachys pilosella CLLG19028_T Sesquicillium lasiacidis CBS 504.67 Sesquicillium lasiacidis NL19-085006 Clonostachys buxicola CBS 102419_T Sesquicillium candelabrum CBS 205.69 Sesquicillium lasiacidis JW235005 Sesquicillium lasiacidis NL19-085003 Clonostachys grammicosporopsis CBS 114.87_T Clonostachys longiphialidica CBS 112.87_T Sesquicillium candelabrum YHH 896 Sesquicillium candelabrum CBS 119045_T Clonostachys levigata CBS 101916_T Sesquicillium candelabrum CBS 194.53 Clonostachys grammicospora CBS 209.93_T Clonostachys fusca CBS 996.97 Sesquicillium candelabrum CBS 512.67 Clonostachys lucifer CBS 126.87 Clonostachys ellipsoidea CBS 175.76_T Clonostachys intermedia CBS 508.82_T Clonostachys subquaternata CBS 100003_T Clonostachys aurantiaca CBS 124757_T Clonostachys ellipsoidea CBS 102566 Clonostachys vacuolata CBS 191.93_T Sesquicillium candelabrum JW1015 Clonostachys flava CBS 915.97_T Sesquicillium candelabrum CBS 204.69 Clonostachys pallens PAD S00004_T 97/99/1 100/100/1 100/100/1 97/100/1 100/100/1 99/100/1 81/98/1 95/99/1 100/100/1 97/100/1 98/100/1 100/100/1 99/100/1 100/100/1 96/99/1 93/98/1 80/97/- 81/97/0.98 99/100/1 100/100/1 86/97/1 78/96/1 97/100/1 71/100/- 83/98/1 100/100/1 98/99/1 95/99/1 Clonostachys III IV I Sesquicillium Fig. 1. (Continued). The individual gene trees had variable success in resolving genus clades (Supplementary Figs S1–S4). Although they generally resolved the same genus clades, the order and association between the clades were not always the same due to low support in the backbones of the trees. The ITS phylogeny (Supplementary Fig. S1) recovered all the genus clades presented in Fig. 1, with the exception that it intermixed species of Emericellopsis and Stanjemonium. The LSU phylogeny (Supplementary Fig. S2) recovered all genera presented in Fig. 1, but did not cluster species of Lasionectria in a monophyletic clade and intermixed several subclades of Sesquicillium and Clonostachys intermixed in a polytomy. The RPB2 phylogeny (Supplementary Fig. S3) recovered all genera presented in Fig. 1, with the exception that it intermixed species of Emericellopsis and Stanjemonium. The TEF1 phylogeny (Supplementary Fig. S4) recovered all genera presented in Fig. 1, but did not cluster all species of Nectriopsis in a monophyletic clade (Nectriopsis lindauiana clustered Chapter 2 36 0.08 Clonostachys rhizophaga CBS 202.37_T Clonostachys compactiuscula CBS 913.97_T Clonostachys rogersoniana CBS 920.97_T Clonostachys sporodochialis CBS 101921_T Clonostachys pnagiana CLLG19041_T Clonostachys eriocamporesii CBS 148221 Clonostachys ambigua PAD S00003_T Clonostachys krabiensis MFLUCC 16-0254_T Clonostachys fujianensis CBS 127474_T Clonostachys oligospora HMAS 290895_T Clonostachys farinosa IBP2 Clonostachys hongkongensis CBS 116542 Clonostachys rosea CBS 710.86_T Clonostachys garysamuelsii CBS 123964_T Clonostachys apocyni CBS 130.87 Clonostachys kunmingensis CBS 101920 Clonostachys swieteniae MFLUCC 18-0572_T Clonostachys farinosa CBS 124067 Clonostachys kunmingensis YFCC 898_T Clonostachys rosea f. catenulata CBS 154.27_T Clonostachys chongqingensis HMAS 290894_T Clonostachys samuelsii CBS 699.97_T Clonostachys leptoderma HMAS 255834_T Clonostachys ralfsii CBS 703.97_T Clonostachys epichloe CBS 101037_T Clonostachys agarwalii CBS 533.81_T Clonostachys penicillata CBS 653.70 Clonostachys pseudostriatopsis MAFF 239827_T Clonostachys reniformis CBS 695.86_T Clonostachys aureofulvella CBS 100980_T Clonostachys palmae CBS 119.87_T Clonostachys penicillata CBS 729.87_T Clonostachys hongkongensis CBS 115291_T Clonostachys moreaui CLL19024_T Clonostachys eriocamporesii MFLUCC 19-0486_T Clonostachys pseudochroleuca CBS 187.94_T Clonostachys cylindrica CBS 101113_T Clonostachys farinosa CBS 364.78_T Clonostachys oblongispora CBS 100285_T Clonostachys chloroleuca CBS 141588_T Clonostachys farinosa MFLUCC 17-2620 Clonostachys viticola CAA 944_T Clonostachys obovatispora CBS 118752_T Clonostachys farinosa MFLUCC 17-0131 Clonostachys divergens CBS 967.73B_T Clonostachys farinosa PAD S00020 Clonostachys capitata CBS 218.93_T Clonostachys solani CBS 697.88_T Clonostachys parasporodochialis CBS 192.93_T Clonostachys zelandiaenovae CBS 100979_T Clonostachys eriocamporesii CBS 647.91 Clonostachys bambusae CBS 139411_T Clonostachys farinosa PAD S00011 Clonostachys penicillata CBS 148211 Clonostachys solani f. nigrovirens CBS 183.30_T Clonostachys pseudostriata CBS 120.87_T Clonostachys sp. CBS 496.90 Clonostachys miodochialis CBS 997.69_T Clonostachys kowhai CBS 461.95_T 100/100/1 53/92/- 52/97/0.93 51/-/- 54/97/- 97/100/1 100/100/1 91/100/1 95/100/1 51/98/- 55/-/- 82/100/1 81/100/0.98 100/100/1 96/100/1 92/99/1 94/100/1 98/99/1 84/100/0.99 100/100/1 97/100/1 85/100/0.99 63/99/0.97 98/100/1 90/99/1 54/99/1 99/100/1 56/-/0.93 66/-/- 75/91/- 87/98/- 55/98/- Clonostachys subgenera: I: Epiphloea II: Uniparietina III: Myronectria IV: Zebrinella V: Astromata VI: Bionectria -/99/- -/92/- V VI Clonostachys basal to Stilbocrea without any support) while Clonostachys buxicola and Clonostachys pityrodes formed a clade sister to the monophyletic Stephanonectria clade but with an unsupported connecting node. Dataset 2: This dataset consisted of 394 ingroup isolates that formed a fully supported clade representing Clonostachys, with the bionectriaceous Acremonium alternatum Fig. 1. (Continued). Revising Clonostachys and allied genera in Bionectriaceae 37 2 Clonostachys subquaternata CBS 133487 Clonostachys australiana CBS 102421_T Clonostachys leucaenae MFLUCC 20-0008_T Clonostachys chlorina CBS 287.90_T Clonostachys levigata CBS 124005 Clonostachys setosa CBS 834.91 Clonostachys fusca CBS 996.97 Clonostachys buxicola CBS 102419_T Clonostachys grammicosporopsis CBS 114.87_T Clonostachys aurantiaca CBS 124757_T Clonostachys australiana CBS 102423 Clonostachys lucifer CBS 126.87 Clonostachys setosa CBS 917.97_T Clonostachys pityrodes CBS 322.78 Clonostachys lucifer CBS 100008_T Clonostachys intermedia CBS 508.82_T Clonostachys grammicosporopsis CBS 102834 Clonostachys pilosella CLLG19028_T Clonostachys grammicosporopsis CBS 111.87 Clonostachys subquaternata CBS 108.87 Clonostachys pityrodes CBS 102035 Clonostachys levigata CBS 203.69 Clonostachys ellipsoidea CBS 102566 Clonostachys pityrodes CBS 246.78 Clonostachys levigata JW182010 Clonostachys grammicosporopsis CBS 102835 Clonostachys fusca CBS 101925 Clonostachys pityrodes CBS 249.78 Clonostachys vacuolata CBS 191.93_T Clonostachys grammicosporopsis CBS 115.87 Clonostachys pallens PAD S00004_T Clonostachys fusca CBS 207.93_T Clonostachys vesiculosa HMAS 183151_T Acremonium alternatum CBS 407.66_T Clonostachys setosa CBS 112025 Clonostachys levigata JW199015 Clonostachys subquaternata CBS 100003_T Clonostachys flava CBS 915.97_T Clonostachys venezuelae CBS 107.87_T Clonostachys grammicosporopsis CBS 102843 Clonostachys levigata JW259015 Clonostachys grammicospora CBS 209.93_T Clonostachys levigata CBS 948.97 Clonostachys ellipsoidea CBS 175.76_T Clonostachys levigata CBS 101916_T Acremonium stroudii CBS 138820_T Clonostachys longiphialidica CBS 112.87_T Clonostachys pityrodes CBS 126394 Clonostachys pityrodes CBS 102033_T 98/100/1 100/100/1 100/100/1 96/98/1 100/100/1 94/98/1 65/98/0.93 94/98/1 100/100/1 97/100/1 99/100/1 62/96/- 60/100/0.99 94/100/0.99 97/98/1 100/100/1 73/99/- 91/100/0.96 100/100/1 61/95/0.96 100/100/1 100/100/1 97/100/1 86/97/0.96 86/100/1 79/100/0.98 100/100/1 94/100/1 98/100/1 96//98/1 65/95/0.96 4X 4X 100/100/1 100/100/1 100/100/1 100/100/1 100/100/1 -/91/- Fig. 2. Phylogenetic tree inferred from a Maximum Likelihood (RAxML-ML) analysis based on aligned and concatenated ITS, LSU, RPB2, TEF1 and TUB2 sequences of 394 strains representing Clonostachys and outgroups. Numbers at branches indicate support values (RAxML-BS / IQ-TREE-BS / BI-PP) above 50 % / 90 % / 0.9. New species are printed in red font and coloured boxes highlight species clades / lineages. “T” indicates ex-type strains. A detailed view of the collapsed clade at the bottom of the phylogenetic tree can be found in Fig. S9. The tree is rooted to Acremonium alternatum CBS 407.66 and A. stroudii CBS 138820 (Hypocreales, Bionectriaceae). Scale bar represents expected number of changes per site. Chapter 2 38 Clonostachys compactiuscula CBS 123781 Clonostachys penicillata CBS 653.70 Clonostachys hongkongensis CBS 115291_T Clonostachys ralfsii CBS 141088 Clonostachys compactiuscula CBS 592.93 Clonostachys penicillata CBS 729.87_T Clonostachys compactiuscula CBS 919.97 Clonostachys compactiuscula CBS 122571 Clonostachys ralfsii CBS 129.87 Clonostachys oligospora HMAS 290895_T Clonostachys compactiuscula CBS 913.97_T Clonostachys eriocamporesii CBS 647.91 Clonostachys ralfsii CBS 102852 Clonostachys ralfsii CBS 102849 Clonostachys compactiuscula CBS 101923 Clonostachys samuelsii CBS 700.97 Clonostachys miodochialis CBS 997.69_T Clonostachys divergens CBS 532.69 Clonostachys divergens CBS 229.80 Clonostachys ralfsii CBS 703.97_T Clonostachys samuelsii CBS 198.93 Clonostachys ralfsii CBS 267.36 Clonostachys divergens JW190011 Clonostachys samuelsii CBS 188.94 Clonostachys divergens CBS 102426 Clonostachys ralfsii CBS 102850 Clonostachys divergens CBS 967.73B_T Clonostachys eriocamporesii CBS 148221 Clonostachys ralfsii CBS 127880 Clonostachys divergens JW183010 Clonostachys obovatispora CBS 118752_T Clonostachys ralfsii CBS 102845 Clonostachys samuelsii CBS 196.93 Clonostachys ralfsii CBS 141089 Clonostachys hongkongensis CBS 116542 Clonostachys compactiuscula CBS 123.79 Clonostachys epichloe CBS 101037_T Clonostachys penicillata CBS 148211 Clonostachys compactiuscula CBS 123759 Clonostachys cylindrica CBS 101113_T Clonostachys samuelsii CBS 100976 Clonostachys compactiuscula CBS 123786 Clonostachys compactiuscula CBS 122580 Clonostachys eriocamporesii MFLUCC 19-0486_T Clonostachys divergens JW158008 Clonostachys samuelsii CBS 699.97_T Clonostachys compactiuscula CBS 102563 Clonostachys divergens CBS 381.77 Clonostachys compactiuscula CBS 119318 Clonostachys samuelsii CBS 199.93 Clonostachys fujianensis CBS 127474_T Clonostachys divergens CBS 120975 Clonostachys samuelsii CBS 701.97 Clonostachys ralfsii CBS 102851 Clonostachys samuelsii CBS 201.93 100/100/1 69/99/1 100/100/1 52/-/- 99/100/1 85/100/1 96/98/0.96 100/100/1 92/98/0.99 100/100/1 100/100/1 100/100/1 100/100/1 97/100/1 99/100/1 67/100/- 100/100/1 98/100/- 63/98/1 -/98/- -/-/0.94 Fig. 2. (Continued). (CBS 407.66) and A. stroudii (CBS 138820) used as outgroups (Fig. 2). The final alignment consisted of 4055 characters, including alignment gaps (gene boundaries ITS: 1–565, 565 bp; LSU: 566–1366, 801 bp; RPB2: 1367–2133, 767 bp; TEF1: 2134–2941, 808 bp; TUB2: 2942–4055, 1114 bp). Among these, 2654 character sites were conserved (ITS: 330, LSU: Revising Clonostachys and allied genera in Bionectriaceae 39 2 Clonostachys aureofulvella CBS 102839 Clonostachys zelandiaenovae CBS 217.93 Clonostachys ambigua PAD S00003_T Clonostachys zelandiaenovae CBS 234.80B Clonostachys zelandiaenovae CBS 124343 Clonostachys palmae CBS 119.87_T Clonostachys rogersoniana CBS 668.70 Clonostachys zelandiaenovae CBS 100978 Clonostachys rogersoniana CBS 102572 Clonostachys zelandiaenovae CBS 123949 Clonostachys rogersoniana CBS 394.85 Clonostachys zelandiaenovae CBS 102846 Clonostachys pseudostriata CBS 309.96 Clonostachys zelandiaenovae CBS 233.80 Clonostachys sp. CBS 496.90 Clonostachys viticola CAA 944_T Clonostachys zelandiaenovae CBS 102844 Clonostachys zelandiaenovae CBS 234.80A Clonostachys pseudostriatopsis MAFF 239829 Clonostachys aureofulvella CBS 195.93 Clonostachys agarwalii CBS 533.81_T Clonostachys rogersoniana CBS 582.89 Clonostachys pseudostriatopsis MAFF 239841 Clonostachys pseudostriatopsis MAFF 239827_T Clonostachys rogersoniana CBS 102564 Clonostachys pseudostriata CBS 120.87_T Clonostachys zelandiaenovae CBS 197.93 Clonostachys rogersoniana CBS 377.65 Clonostachys zelandiaenovae CBS 234.80C Clonostachys aureofulvella CBS 102837 Clonostachys rogersoniana CBS 139551 Clonostachys aureofulvella CBS 102836 Clonostachys krabiensis MFLUCC 16-0254_T Clonostachys viticola CAA 946 Clonostachys zelandiaenovae CBS 102422 Clonostachys zelandiaenovae CBS 100977 Clonostachys krabiensis CBS 192.96 Clonostachys bambusae CBS 139411_T Clonostachys reniformis CBS 695.86_T Clonostachys aureofulvella CBS 235.80 Clonostachys rogersoniana CBS 121653 Clonostachys zelandiaenovae CBS 232.80 Clonostachys chongqingensis HMAS 290894_T Clonostachys aureofulvella CBS 100980_T Clonostachys rogersoniana CBS 920.97_T Clonostachys aureofulvella CBS 200.93 Clonostachys aureofulvella CBS 102838 Clonostachys capitata CBS 218.93_T Clonostachys rogersoniana CBS 139287 Clonostachys viticola CAA 945 Clonostachys leptoderma HMAS 255834_T Clonostachys zelandiaenovae CBS 100979_T Clonostachys swieteniae MFLUCC 18-0572_T 91/100/1 95/99/1 90/100/1 100/100/1 93/98/- 57/90/- 98/100/- 92/98/0.96 94/98/- 75/-/- 98/100/1 81/98/- 67/-/- 96/100/1 99/100/1 96/100/1 100/100/- 90/100/1 98/100/1 99/100/1 94/100/1 Fig. 2. (Continued). 694, RPB2: 378, TEF1: 576, TUB2: 676), 1366 were variable (ITS: 220, LSU: 106, RPB2: 382, TEF1: 232, TUB2: 426), and 1185 were parsimony informative (ITS: 175, LSU: 90, RPB2: 353, TEF1: 194, TUB2: 373). The phylogenetic trees based on dataset 2 were generated with Maximum-likelihood analyses and Bayesian analyses. According to the result of MrModelTest, the SYM+I+G Chapter 2 40 Clonostachys solani f. nigrovirens CBS 223.72A Clonostachys solani f. nigrovirens CBS 229.74 Clonostachys solani CBS 142837 Clonostachys pseudochroleuca CBS 192.94 Clonostachys solani f. nigrovirens CBS 144943 Clonostachys solani f. nigrovirens JW96008 Clonostachys pseudochroleuca CBS 187.94_T Clonostachys solani f. nigrovirens CBS 147.65 Clonostachys solani JW11016 Clonostachys solani JW191026 Clonostachys solani CBS 697.88_T Clonostachys solani CBS 142845 Clonostachys solani f. nigrovirens CBS 325.78 Clonostachys pseudochroleuca CBS 194.93 Clonostachys solani f. nigrovirens CBS 728.69 Clonostachys solani f. nigrovirens CBS 101924 Clonostachys solani CBS 102418 Clonostachys solani f. nigrovirens CBS 101919 Clonostachys solani CBS 708.86 Clonostachys solani f. nigrovirens NL19-049009 Clonostachys solani f. nigrovirens CBS 191.31 Clonostachys solani f. nigrovirens CBS 223.72B Clonostachys solani f. nigrovirens CBS 223.72C Clonostachys solani f. nigrovirens JW10009 Clonostachys solani f. nigrovirens CBS 125111 Clonostachys solani CBS 144941 Clonostachys solani CBS 224.72A Clonostachys pseudochroleuca CBS 185.94 Clonostachys solani f. nigrovirens CBS 183.30_T Clonostachys solani NL19-22014 Clonostachys solani CBS 144338 Clonostachys solani CBS 702.97 Clonostachys pseudochroleuca CBS 220.93 Clonostachys solani CBS 144944 Clonostachys solani f. nigrovirens NL19-008005 Clonostachys solani f. nigrovirens JW211002 Clonostachys solani f. nigrovirens CBS 142.91 Clonostachys solani f. nigrovirens CBS 101926 Clonostachys solani f. nigrovirens CBS 350.76 Clonostachys pseudochroleuca CBS 124610 Clonostachys solani CBS 224.72F Clonostachys solani f. nigrovirens JW187017 Clonostachys solani BE19-005010 Clonostachys pseudochroleuca CBS 219.93 Clonostachys pseudochroleuca CBS 447.96 Clonostachys solani f. nigrovirens CBS 144349 Clonostachys solani f. nigrovirens JW1072 Clonostachys solani CBS 752.68 Clonostachys pseudochroleuca CBS 124754 Clonostachys solani CBS 102425 Clonostachys pseudochroleuca CBS 191.94 Clonostachys solani CBS 906.72D Clonostachys solani f. nigrovirens CBS 228.74 Clonostachys pseudochroleuca CBS 186.94 98/100/1 92/96/1 93/100/1 100/100/1 97/100/1 87/99/1 78/100/0.96 Fig. 2. (Continued). model was proposed for ITS, GTR+I+G model was proposed for LSU, RPB2, and TEF1, and HKY+I+G model was proposed for TUB2. The Bayesian analysis of the concatenated fivelocus alignment lasted for 67,905000 generations and 135812 trees were generated after the average standard deviation of split frequencies value was below 0.013 in the BI analysis. Revising Clonostachys and allied genera in Bionectriaceae 47 2 Sesquicillium, while noting that S. microsporum differed from other Sesquicillium species in size ranges and shapes of its phialides and conidia, rather dry conidial chains, and more irregularly branched and poorly developed penicilli. Relatedness of S. microsporum and Nectriopsis was hypothesised earlier on the basis of the myxomyceticolous lifestyle and analyses of LSU sequences (Rogerson & Stephenson 1993, Schroers 2001). In the present study, according to our phylogenetic analyses, strains identified as S. microsporum clustered in a lineage (Fig. 1; 61 % / 98 % / -) of the genus Nectriopsis and is closely related to N. didymii (Fig. 1). For morphological comparison with the other species of this genus, see notes under N. didymii. Mycocitrus Möller, Bot. Mitt. Tropen. 9: 297. 1901. Synonym: Shiraiella Hara, Bot. Mag. (Tokyo) 28: 274. 1914. Type: Mycocitrus aurantium Möller Sexual morph on the natural substratum. Stroma well-developed, surface buff to rufous or light orange, KOH-, clasping and surrounding the substratum. Ascomata perithecial, Fig. 4. Nectriopsis microspora (ex-type CBS 933.69). A–C. Colonies on OA, PDA, SNA after 7 d at 25 °C. D, E. Conidiophores. F. Conidia. Black arrows indicate intercalary phialides. Scale bars = 10 μm. Chapter 2 48 surface partially to fully immersed, with apices barely visible, densely gregarious, forming a single layer. Asci cylindrical, ascal apex simple, 8-spored. Ascospores 1-septate, ellipsoid, hyaline, spinulose. Asexual morphs commonly acremonium-like (adapted from Rossman et al. 1999). Notes: Mycocitrus was first described from culms of living bamboo and Microstachys in southern Brazil (Möller 1901). The type species, M. aurantium, is characterised by its large, fleshy, orange stromata that clasp and surround bamboo culms, with perithecial ascomata partially to fully immersed in the upper region of the stromata. Later, a second species, M. phyllostachydis (bas. Ustilaginoidea phyllostachydis), was collected in Japan on Phyllostachys and added to the genus (Doi 1967). Mycocitrus was originally placed in the “Hypocreaceen, Didymosporae” (Möller 1901), and later in Hypocreaceae (Doi 1967). The proposal by Rossman et al. (1999) to include Mycocitrus in the Bionectriaceae was based on morphology. Leite et al. (2018) obtained two bamboo-inhabiting M. aurantium cultures in South America. Their phylogeny was based on ITS sequences and suggested prematurely that Mycocitrus forms an independent lineage within Hypocreales, distinct from Bionectriaceae, Nectriaceae, Cordycipitaceae, Clavicipitaceae, and Hypocreaceae. However, Hou et al. (2023) revealed that Mycocitrus clusters within the Bionectriaceae based on a multi-locus phylogenetic analysis, what is supported in the present analysis (Fig. 1). Mycocitrus coccicola (J.A. Stev.) Lin Zhao & Crous, comb. nov. MycoBank MB 848434. Basionym: Tubercularia coccicola J.A. Stev., Rep. (Annual) Puerto Rico Insular Exp. Sta., 1916–1917: 91. 1917. Synonyms: Clonostachys coccicola (J.A. Stev.) H.T. Dao, Mycol. Prog. 15: 6. 2016. Nectria tuberculariae Petch, Trans. Brit. Mycol. Soc. 7: 157. 1921. Material examined: Australia, New South Wales, Cornwallis, armoured scale insects, date and collector unknown, culture BucCo; New South Wales, Somersby, armoured scale insects, date and collector unknown, culture BucS. Notes: Mycocitrus coccicola was originally described as Tubercularia coccicola (Stevenson 1917) before Dao et al. (2016) placed it in Clonostachys as C. coccicola. In our study, the phylogenetic analysis of the combined ITS, LSU, TEF1, RPB2, and TUB2 dataset revealed that C. coccicola clusters within the genus Mycocitrus (Fig. 1) and a new combination is proposed here. Mycocitrus coxeniae (Y.P. Tan et al.) Lin Zhao & Crous, comb. nov. MycoBank MB 848911. Basionym: Clonostachys coxeniae Y.P. Tan et al., Index of Australian Fungi 5: 3. 2023. Revising Clonostachys and allied genera in Bionectriaceae 49 2 Description: Tan & Shivas (2023). Notes: Tan & Shivas (2023) recently coined Clonostachys coxeniae, but without any proper description, illustration or discussion of morphological characters. Their deposited sequence identifies this species as a member of genus Mycocitrus (Fig. 1). Mycocitrus odorus L.W. Hou et al., Stud. Mycol. 105: 111. 2023. Fig. 5. Typus: Netherlands, Amsterdam, Slotervaart Hospital, onychomycosis (human), unknown date, W.C. van Dijk & W. Pauw (holotype specimen CBS H-24690, ex-type living culture CBS 100104). Description and illustration: Hou et al. (2023). Additional materials examined: Netherlands, human skin, unknown collection date and collector, culture CBS 120610. Sweden, Stockholm, human skin, unknown collection date and collector, culture CBS 232.75B. Note: Mycocitrus odorus was recently described by Hou et al. (2023) as a novel species in Mycocitrus (Fig. 1). Mycocitrus phyllostachydis (Syd. & P. Syd.) Yoshim. Doi, Bull. Natl. Sci. Mus., Tokyo, N.S. 10: 31. 1967. Basionym: Ustilaginoidea phyllostachydis Syd. & P. Syd., Mém. Herb. Boissier 4: 5. 1900. Synonyms: Hypocreopsis phyllostachydis (Syd. & P. Syd.) I. Miyake & Hara, Bot. Mag., Tokyo 24: 333. 1910. Shiraiella phyllostachydis (Syd. & P. Syd.) Hara, Bot. Mag., Tokyo 28: 402. 1914. Description and illustration: Doi (1967). Material examined: Japan, on Phyllostachys sp. (Poaceae), unknown date, W. Gams, specimen CBS H-14839, culture CBS 330.69 = IFO 8912. Notes: Gams (1971) examined the culture of M. phyllostachydis (CBS 330.69) isolated from Phyllostachys sp. in Japan. Based on the phylogenetic analyses, classification of U. phyllostachydis in Mycocitrus, as proposed by Doi (1967), is confirmed here (Fig. 1). Mycocitrus synnematus Lin Zhao & Crous, sp. nov. MycoBank MB 848435. Fig. 6. Chapter 2 50 Fig. 5. Mycocitrus odorus (CBS 120610). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–K. Conidiophores. L. Conidia. Scale bars = 10 μm. Etymology: Name refers to the production of synnemata. Typus: Sri Lanka, from wood, date unknown, G.J. Samuels (holotype designated here CBS H-25131, ex-type living culture CBS 126677). Sexual morph unknown. Synnemata erect, golden brown, occurring singly or in groups, abundant in culture. Conidiophores macronematous, branched, asymmetric-biverticillate Revising Clonostachys and allied genera in Bionectriaceae 51 2 Fig. 6. Mycocitrus synnematus (ex-type CBS 126677). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Synnemata. G. Detail of the apical portion of a synnema. H–K. Conidiogenous cells. L. Conidia. Scale bars: F = 100 μm; G = 50 μm; H–L = 10 μm. or monoverticillate. Conidiogenous cells phialidic, cylindrical, straight or slightly curved, slightly tapered at the apex, in terminal whorls of 2–5, (13.2–)16.2–45.4(–59.6) × (1.2– )1.5–2.0(–2.1) μm. Conidia aseptate, hyaline, smooth, ellipsoid, straight to slightly curved, distally broadly rounded, without recognisable hilum, (4.3–)5.5–7.7(–8.0) × (2.7–)3.0– 3.8(–4.0) μm (av. 6.8 × 3.5 μm, n = 150). Chapter 2 52 Culture characteristics: Colonies on OA reaching 30–35 mm diam. after 7 d in darkness at 25 °C, flat, with entire margin, aerial mycelium scanty, finely floccose, whitish, reverse concolourous. Colonies on PDA reaching 27–28 mm diam., flat, with entire margin, aerial mycelium moderately dense, floccose, pale yellow, reverse concolourous. Colonies on SNA reaching 19–22 mm diam., flat, with entire margin, aerial mycelium moderate, floccose white, reverse whitish. Notes: Phylogenetically, M. synnematus is closely related to M. coccicola, M. coxeniae, M. odorus, and M. phyllostachydis (Fig. 1). Morphologically, M. synnematus can be distinguished from M. odorus and M. coccicola by its larger conidia, (4.3–)5.5–7.7(–8.0) × (2.7–)3.0–3.8(–4.0) μm (av. 6.8 × 3.4 μm) in M. synnematus, 3.2–5.5 × 2.1–3.3 (av. 4 × 2 μm) in M. coccicola and (3.4–)3.7–5.0(–6.6) × (1.8–)2.1–2.8(–3.0) μm (av. 4.4 × 2.5 μm) in M. odorus. Furthermore, CBS 126677 (M. synnematus) and CBS 330.69 (M. phyllostachydis) have clearly different ITS (93.9 % identity, with 31 bp differences), LSU (97.6 %, 18 bp), RPB2 (89.1 %, 81 bp), and TEF1 (96.6 %, 27 bp) sequences, while M. synnematus (CBS 126677) has different ITS (95 % identity, with 24 bp differences) sequences when compared with M. coxeniae (BRIP 49559a). Stephanonectria Schroers & Samuels, Sydowia 51: 116. 1999. Type: Stephanonectria keithii (Berk. & Broome) Schroers & Samuels Sexual morph on the natural substratum. Stroma superficial, reduced or erumpent through bark. Perithecia brown, KOH-, smooth to rough, minutely papillate. Ostiolum surrounded by a crown-like arrangement of cells. Perithecial wall consisting of two regions. Cells of the crown merging with the cells of the outer wall region, angular to oblong, outwards toothlike. Ascospores 1-septate, covered with short striae. Asexual morph. Conidiophores monomorphic, sporodochial, towards the margin of the colony solitary, irregularly penicillate, or sparsely aggregated, not showing regular patterns, with 1–5 phialides on each supporting cell; branches overall diverging, sometimes joined by anastomoses. Phialides cylindrical, sometimes widening in the middle or in the upper part, typically becoming narrower just underneath the apex, with apical periclinal thickening visible, without collarette. Conidia aseptate, hyaline, smooth, ellipsoidal, hilum median, slightly laterally displaced, or not visible (adapted from Schroers et al. 1999a). Notes: Stephanonectria was introduced as a new genus because of characters of the ascomatal wall and aggregations of cells forming a crown-like structure around the ostiolum. The ascospores are covered with short striae that are more or less parallel with the long axis of the spore. The asexual morph was identified as being myrothecium-like Revising Clonostachys and allied genera in Bionectriaceae 53 2 (Schroers et al. 1999a) because of details seen in the shape of phialides. Despite the more irregular branching pattern of conidiophores, sporodochia seen in S. keithii are similar to those formed by many Clonostachys species. The use of “myrothecium-like” for the asexual morph in Stephanonectria (Schroers et al. 1999a) is therefore obsolete. Stephanonectria chromolaenae R.H. Perera & K.D. Hyde, Fungal Diversity 118: 134. 2023. Fig. 7. Typus: Thailand, Chiang Mai Province, Mae Rim District, on dead stem of Chromolaena odorata (Asteraceae), 18 Sep. 2017, R.H. Perera (holotype MFLU 19-0972, ex-type living culture MFLUCC 18-0589). Description and illustration: Perera et al. (2023). Additional materials examined: Turkey, isolated from soil, date unknown, G. Turhan, No. 10, culture CBS 475.91; soil, date unknown, G. Turhan, No. 13, culture CBS 476.91. Notes: Stephanonectria chromolaenae was described from dead stems of Chromolaena odorata. Based on our phylogenetic analysis, S. chromolaenae and S. keithii formed a fullysupported clade within the genus Stephanonectria (Fig. 1). Stephanonectria keithii (Berk. & Br.) Schroers & Samuels, Sydowia 51: 116. 1999. Basionym: Nectria keithii Berk. & Br., Ann. Mag. Nat. Hist., Ser. 4, 27: 144. 1876. Synonym: Nectriella keithii (Berk. & Br.) Sacc., Michelia 1: 279. 1879. Typus: UK, Scotland, Forres, on decorticated stems of cabbage, collection date unknown, Rev. J. Keith, specimen IMI 77877. Description and illustration: Schroers et al. (1999a). Additional materials examined: Netherlands, Utrecht, Berenkuil, soil, Sep. 1972, collector unknown, culture CBS 943.72; Oostelijk Flevoland, agricultural soil, under permanent potato, J. W. Veenbaas-Rijks, Oct. 1969, culture CBS 434.70. New Zealand, Gisborne, Lake Waikaremoana, Ngamoko Trail, on Beilschrniedia tawa, 30 May 1983, G. J. Samuels et al., Samuels culture 83-165, CBS 100007 (PDD 46342; BPI 737629). Notes: This species was originally described as Nectria keithii (Berkeley & Broome 1876). Schroers et al. (1999a) introduced a new genus Stephanonectria to accommodate S. keithii. The three strains included in the phylogenetic analysis cluster in a supported clade (Fig. 1; 87 % / 99 % / 1). Chapter 2 54 Fig. 7. Stephanonectria chromolaenae (CBS 475.91). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–I. Conidiophores. J. Conidia. Scale bars: E, F = 50 μm; G–J = 10 μm. Sesquicillium W. Gams, Acta Bot. Neerl. 17. 455. 1968. Type: Sesquicillium buxi (J.C. Schmidt ex Link) W. Gams Sexual morph on the natural substratum. Perithecial stroma typically formed superficially on plant tissue, reduced, supporting solitary perithecia, typically consisting of prosenchymatous cells not integrating with cells of major perithecial wall regions, sometimes erumpent Revising Clonostachys and allied genera in Bionectriaceae 55 2 through bark, supporting several perithecia and consisting of pseudoparenchymatous cells. Perithecia solitary, gregarious or loosely aggregated, crowded if formed on an erumpent stroma, globose to subglobose, 200–400 µm diam., sometimes smaller, up to 200 µm diam., pale yellow, pale orange to light orange, apically or laterally pinched when dry, not papillate, glabrous; ostiolar region somewhat sunken and slightly darker (brownish). Perithecial wall either consisting of two or one major wall region, sometimes with an additional outermost cell-layer that continues into the prosenchymatous stroma. Asci narrowly to broadly clavate, 8-spored, with flat or rounded apex, with or without visible ring. Ascospores typically 1-septate, equally 2-celled, sometimes aseptate, hyaline, spinulose, warted, with short striae or with warts arranged in striae, typically ellipsoidal to fusiform. Asexual morph. Conidiophores macronematous, mononematous, monomorphic penicillate or somewhat dimorphic, penicillate and verticillium-like, mostly arising from the agar surface or from sparsely formed aerial mycelium. Penicillate conidiophores bito quaterverticillate; branches of the penicilli divergent or adpressed; terminal whorls consisting of narrowly flask-shaped phialides and/or typically one or sometimes two successive intercalary phialides, of which the uppermost bears a solitary terminal phialide; conidiogenous pegs of intercalary phialides typically short, formed laterally just below the upper septum. Conidia aseptate, smooth, hyaline, obovoid, ellipsoid, or fusoid, slightly curved or straight, generally with a slightly laterally displaced hilum and arranged in imbricate chains forming columns or, rarely, with a centrally located hilum and arranged in linear chains (adapted from Schroers 2001). Notes: In this study, Bionectria subgenera Epiphloea and Uniparietina clustered in a statistically supported clade (Fig. 1; 97 % / 100 % / 1) that is sister to clades accommodating the other Clonostachys subgenera (Fig. 1). This separate clade includes C. buxi and C. candelabrum classified by Gams (1968) in Sesquicillium. The type species of the subgenus Uniparietina is Bionectria coronata, for which S. buxi is currently in use. Furthermore, the genus Sesquicillium, already used by Samuels (1989) for several below considerd species, is resurrected to accommodate the subgenera Epiphloea and Uniparietina. Sesquicillium buxi (J.C. Schmidt ex Link) W. Gams, Acta Bot. Neerl. 17: 455. 1968. Basionym: Fusidium buxi J.C. Schmidt ex Link, Willdenow, Sp. pl., Edn 4 6(2): 97. 1825. Synonyms: Fusisporium buxi (J.C. Schmidt ex Link) Fr., Syst. Mycol. 3: 447. 1832. Verticillium buxi (J.C. Schmidt ex Link) Auersw. & Fleischh., Hedwigia 6: 9. 1867. Ramularia buxi (J.C. Schmidt ex Link) Fuckel, Symb. Mycol. p. 97. 1870. Paecilomyces buxi (J.C. Schmidt ex Link) Bezerra, Acta Bot. Neerl. 12: 63. 1963. Clonostachys buxi (J.C. Schmidt ex Link) Schroers, Stud. Mycol. 46: 193. 2001. Nectriella coronata Juel, Arkiv før Botanik 19: 4. 1925. Bionectria coronata (Juel) Schroers, Stud. Mycol. 46: 203. 2001. Chapter 2 56 Typus: Germany, Leipzig, Auerswald [neotype for Fusidium buxi: B, designated by Gams (1968)]. Descriptions and illustrations: Juel (1925), Gams (1968), Rossman et al. (1993), Schroers (2001). Additional materials examined: France, Pyrénées Atlantiques, Île de Sauveterre de Béarn; 400 m alt., Buxus sempervirens, leaf litter, 17 Oct 1993, F. Candoussau, BPI 802851, culture CBS 696.93. Netherlands, Buxus sempervirens, collection date unknown, J.L. Bezerra, culture CBS 288.62; Culemborg, soil, collection date unknown, R. Fuld, culture JW182006; Netherlands, Utrecht, soil, collection date unknown, M. Wickham, culture JW199009; Ermelo, soil, collection date unknown, Marit en Mette Elmers, culture JW259005; Deurne, soil, collection date unknown, Martina Hoeben, culture JW58015; Eemnes, soil, collection date unknown, Herman Wim Vos, culture CBS 147861 = JW6017; Rijen, soil, collection date unknown, Gijs & Lotte Schijvenaars, culture JW94008. Sweden, Uppsala, Botanical Garden, on leaves of Buxus sempervirens, 10 Oct. 1924, O. Juel (type of Nectriella coronata: holotype S, isotype BPI). UK, England, Kew Gardens, Buxus sempervirens, leaf litter, Nov 1967, W. Gams, CBS H-18217, culture CBS 202.69. Notes: Sesquicillium buxi was originally described as Fusidium buxi in Link (1825). Later, it was transferred to the genus Sesquicillium (Gams 1968) as S. buxi (type species) with conidiophores having sparsely branched whorls, and intercalary phialides. By accepting a broader genus concept, Schroers (2001) combined S. buxi as Clonostachys buxi. In the present study, the phylogenetic analysis of the combined ITS, LSU, TEF1, RPB2 and TUB2 dataset reveals that taxa of Sesquicillium form the phylogenetic sister of Clonostachys sensu stricto (Fig. 1). Therefore, S. buxi is resurrected here. Sesquicillium candelabrum (Bonord.) W. Gams, Acta Bot. Neerl. 17: 457. 1968. Fig. 8. Basionym: Verticillium candelabrum Bonord., Handb. Allgem. Mykol. (Stuttgart): 97. 1851. Synonyms: Clonostachys candelabrum (Bonord.) Schroers, Stud. Mycol. 46: 192. 2001. Clonostachys chuyangsinensis Hong Yu bis & Yao Wang, Frontiers Microbiol. 14: 3. 2023. Typus: Switzerland, Kt. Bern, Büetigen, Waldhaus Hörnli, Ischlag, strongly decayed Fomitopsis pinicola, 13 Oct. 2005, W. Gams (epitype designed here CBS H-25178, MBT 10012943, ex-epitype culture CBS 119045); Rabenhorst ‘Fungi europaei’ No. 2148, on leaves of Laurus nobilis, leg. P.A. Saccardo, Selva, 1875 (B), designated by Gams (1968) (neotype for Verticillium candelabrum). Sexual morph unknown. Asexual morph. Conidiophores monomorphic, scattered on the agar surface or arising from strands of aerial hyphae, monoto quaterverticillate, divergent Revising Clonostachys and allied genera in Bionectriaceae 63 2 Fig. 11. Sesquicillium lasiacidis (CBS 504.67). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars = 10 μm. Descriptions and illustrations: Samuels (1989), Schroers (2001). Additional materials examined: Germany, root-associated soil using Hordeum vulgare as bait, 22 May 2018, J.G. Maciá-Vicente, culture CBS 147133. Netherlands, Pinus nigra var. austriaca, root collar, unknown date and collector, culture CBS 190.38; wheat field soil, collection date unknown, J.H. van Emden, specimen CBS H-18223, culture CBS 504.67; Eindhoven, soil, 2017, T. Tuinier, culture JW235005; Drenthe, soil, 2019, L. Jurjens, S. Bilstra Chapter 2 64 & J. van Hoorn, culture NL19-085003; Drenthe, soil, 2019, L. Jurjens, S. Bilstra & J. van Hoorn, culture NL19-085006; Drenthe, soil, 15 Nov. 2019, N. Schoon & S. Krol, culture NL19-086015; Drenthe, soil, 2019, S. Schabel & M. Geerisma, culture NL19-089008. Notes: Based on the phylogenetic analyses, S. lasiacidis has a close phylogenetic affinity to S. candelabrum (Fig. 1). Morphologically, S. lasiacidis differs from S. candelabrum in producing longer and narrower conidia, (5.6–)6.4–7.6(–8.2) × (1.8–)2.2–2.8(–3.2) μm vs (3.6–)3.9–4.9(–5.5) × (2.5–)2.6–3.2(–3.7) μm (Schroers 2001). Although phylogenetically closely related, isolate CBS 504.67, which produces shorter conidia, (3.2–)3.6–4.5 µm, may represent another phylogenetic species. Sesquicillium neerlandicum Lin Zhao & Crous, sp. nov. MycoBank MB 848455. Fig. 12. Etymology: Named after the Netherlands where the strains of this species were isolated in the context of a Dutch citizen science project of the Westerdijk Institute and Utrecht University, in collaboration with various schools, sampling garden soils in urban areas. Typus: Netherlands, Gelderland Province, Ravenswaaij, soil, Mar. 2017, L. & N. de Klijne (holotype designated here CBS H-25136, ex-type living culture CBS 148203 = JW 17023). Sexual morph unknown. Asexual morph. Conidiophores monomorphic, scattered on the agar surface or arising from strands of aerial hyphae, penicillate, branches divergent or adpressed, bito quaterverticillate; stipes up to 80 μm long, to 2.4–4.4 μm wide at base, penicilli up to 100 μm high and 100 μm wide; terminal phialides in adpressed whorls of up to five, straight to slightly curved, narrowly flask-shaped, slightly tapering in the upper part, (5.4–)6.5–9.7(–12.7) μm long, 1.9–2.4 μm wide at base, 2.4–3.4 μm at widest point, 0.8–1.2 μm wide near apex (n = 38); intercalary phialides common, solitary or in pairs, subterminally formed lateral pegs up to 7 μm long. Conidia hyaline, aseptate, ellipsoidal to cylindrical, almost straight, without a laterally displaced hilum, with rounded distal end, (4.2–)4.5–5.6(–6.0) × (2.5–)2.7–3.2(–3.5) μm (av. 5.1 × 3.0 μm, n = 150), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 24–27 mm diam. after 7 d at 25 °C in darkness, flat, with crenate margin, aerial mycelium moderate, felty, finely granular, dirty white, reverse concolourous. Colonies on PDA reaching 24–26 mm diam., flat, with crenate margin, aerial mycelium abundant, felty to cottony, finely to coarsely granular, whitish, reverse concolourous. Colonies on SNA reaching 23–25 mm diam., flat, with crenate margin, aerial mycelium sparse, finely to coarsely granular, whitish, reverse concolourous. Revising Clonostachys and allied genera in Bionectriaceae 65 2 Additional materials examined: Netherlands, Gelderland Province, Kapel Avezaath, soil, Mar. 2017, A. Panneman, CBS 148215 = JW79004; Limburg Province, Ell, soil, Mar. 2017, K. Brennand, culture CBS 148213 = JW53028; North Holland Province, Hilversum, soil, Mar. 2017, H., J., A. & J. Bezemer, culture CBS 148201 = JW135005; South Holland Province, Hillegom, soil, Mar. 2017, M. & L. Fleur, culture CBS 148214 = JW71013; Utrecht Province, Nieuwegein, soil, Mar. 2017, J. Schmidt, culture CBS 148212 = JW45022; Utrecht Province, Utrecht, soil, Mar. 2017, G. Bleijlevens, culture CBS 148202 = JW143015; Utrecht, soil, Mar. Fig. 12. Sesquicillium neerlandicum (ex-type CBS 148203). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars: E, F = 50 μm; G–I = 10 μm. Chapter 2 66 2017, R. van den Brink, culture CBS 148209 = JW263008; Utrecht, soil, Mar. 2017, R. van den Brink, culture CBS 148210 = JW263012. Notes: Sesquicillium neerlandicum was isolated from Dutch garden soil. Its species clade and phylogenetic relatedness to S. rossmaniae and S. essexcoheniae is statistically strongly supported (Fig. 1; 100 % / 100 % / 1). It differs from S. rossmaniae in the production of longer stipes (up to 80 μm long in S. neerlandicum vs 40 μm long in S. rossmaniae) and higher and wider penicilli (100 μm high and 100 μm wide in S. neerlandicum vs 50 μm high and 60 μm wide in S. rossmaniae). Sesquicillium neerlandicum differs from S. essexcoheniae in higher and wider penicilli (100 μm high and wide in S. neerlandicum, 30–75 μm high and 70 μm wide in S. essexcoheniae). Sesquicillium phyllophilum (Schroers) Lin Zhao, Crous & Schroers, comb. nov. MycoBank MB 848456. Basionym: Clonostachys phyllophila Schroers, Stud. Mycol. 46: 193. 2001. Typus: France, Forêt des buis, Coudrée (Haute Savoie), Buxus forest, on fallen leaves of Viscum album, Sep. 1996, H.-J. Schroers & T. Gräfenhan (holotype CBS H-7945, ex-type culture CBS 921.97). Description and illustration: Schroers (2001). Additional material examined: Japan, Tokyo, Shinjuku Gyoen Garden, Sep. 1983, W. Gams, culture CBS 662.83. Notes: Sesquicillium phyllophilum was originally described as Clonostachys phyllophila by Schroers (2001) from fallen leaves of Viscum album collected in France. The present study places it phylogenetically in the genus Sesquicillium and therefore a new combination is proposed here (Fig. 1). Sesquicillium rossmaniae (Schroers) Lin Zhao, Crous & Schroers, comb. nov. MycoBank MB 848457. Basionym: Clonostachys rossmaniae Schroers, Stud. Mycol. 46: 177. 2001. Synonym: Bionectria rossmaniae Schroers, Stud. Mycol. 46: 177. 2001. Typus: French Guiana, Piste de Saint-Elie, km 16 on road between Sinnamary and St. Elie, ‘Ecerex’, Orstom research area, 05°20’N, 00°53’W, on twigs of recently dead tree, Feb.– Mar. 1986, G.J. Samuels, G.J.S. 3970, G.J.S. isolate 86-246 (isotype CBS H-7944, ex-type culture CBS 211.93). Revising Clonostachys and allied genera in Bionectriaceae 67 2 Description and illustration: Schroers (2001). Additional materials examined: French Guiana, on bark of living liana, Jan.–Mar. 1986, G.J. Samuels, culture CBS 210.93; ibid. bark of recently dead tree, Jan.–Mar. 1986, G.J. Samuels, culture CBS 221.93. Note: Sesquicillium rossmaniae was originally described as Clonostachys (Bionectria) rossmaniae by Schroers (2001) and is shown here to cluster with other species of Sesquicillium (Fig. 1). Phylogenetically, S. rossmaniae is closely related to S. neerlandicum, but with clearly different ITS (99.4 % identity, with 3 bp differences), RPB2 (97.4 %, 19 bp), and TEF1 (97.5 %, 20 bp) sequences. For morphological comparison, see notes under S. neerlandicum. Sesquicillium saulense (Lechat & J. Fourn.) Lin Zhao & Crous, comb. nov. MycoBank MB 848458. Basionym: Clonostachys saulensis Lechat & J. Fourn., Ascomycete.org 11(3): 65. 2019. Typus: French Guana, Saül, Gros Arbres trail, on dead bark of Bauhinia sp., 22 Aug. 2018, C. Lechat, CLLG18023-A5 (holotype LIP CLLG18023-A5, ex-type culture BRFM 2782). Description and illustration: Lechat et al. (2019). Notes: The culture was isolated from dead bark of Bauhinia sp. collected from Saül in French Guiana, and described as Clonostachys saulensis (Lechat et al. 2019). Phylogenetically, it falls in a well-supported lineage among other species of the genus Sesquicillium (Fig. 1). Sesquicillium sesquicillii (Samuels) Lin Zhao, Crous & Schroers, comb. nov. MycoBank MB 848459. Basionym: Nectria sesquicillii Samuels, Mem. New York Bot. Gard. 49: 268. 1989. Synonyms: Bionectria sesquicillii (Samuels) Schroers, Stud. Mycol. 46: 190. 2001. Clonostachys sesquicillii Schroers, Stud. Mycol. 46: 190. 2001. Typus: Guyana, Cuyuni-Mazaruni Region, No. VII, Mazaruni Subregion, No. VII-2, foothills immediately S of Mt. Ayanganna, ca 1 km W of Pong River, 05°28’N, 60°04’W, 550–600 m alt., on twigs and lichen, 26 Feb. 1987, G.J. Samuels, J. Pipoly & G. Gharbarran, G.J.S. 4825, G.J.S. isolate 87-23 (isotype CBS H-7413, culture ex-type CBS 180.88). Descriptions and illustrations: Samuels (1989), Schroers (2001). Chapter 2 68 Notes: Sesquicillium sesquicillii was originally described as Nectria sesquicillii by Samuels (1989) from twigs and a lichen. It was subsequently transferred to Clonostachys (Schroers 2001). According to our phylogenetic inference, the ex-type of N. sesquicillii falls in the highly supported Sesquicillium clade (Fig. 1; 97 % / 100 % / 1). Perithecia of S. sesquicillii are formed in dense groups on erumpent stromata (Schroers 2001: fig. 87a, b, d), while the perithecia of other Sesquicillium species are formed solitarily on superficial, thus, nonerumpent, typically reduced stromata. Sesquicillium spinulosisporum (Lechat & J. Fourn.) Lin Zhao & Crous, comb. nov. MycoBank MB 848460. Basionym: Clonostachys spinulosispora Lechat & J. Fourn., Ascomycete.org 10(4): 128. 2018. Typus: French Guiana, Régina, nouragues natural Reserve, Inselberg camp, primary rainforest, on aerial, dead palm leaf of Astrocaryum vulgare (Arecaceae), 16 Jun. 2012, C. Lechat [holotype CLLG12001 (LIP), ex-type culture CBS 133762]. Description and illustration: Lechat & Fournier (2018). Notes: The type culture was isolated from a dead palm leaf of Astrocaryum vulgare (Arecaceae) collected from Régina in French Guiana and originally described as C. spinulosispora. The present study places it phylogenetically in the genus Sesquicillium, being closely related to S. phyllophilum (Fig. 1). Sesquicillium spinulosisporum can be distinguished from S. phyllophilum based on the absence of intercalary phialides and its shorter conidia (4.5–6.5 μm in S. spinulosisporum vs (5.4–)5.8–7(–8.8) μm in S. phyllophilum). Sesquicillium symmetricum Lin Zhao & Crous, sp. nov. MycoBank MB 848461. Fig. 13. Etymology: Name refers to the symmetrical conidia produced by this species. Typus: Colombia, Dep. de Meta, Municipio de Villavicencio, 25 km from Villavicencio to Acacías, 550 m alt., agricultural soil, 18 Feb. 1978, O. Rangel (holotype designated here CBS H-25134, ex-type living culture CBS 124.79). Sexual morph unknown. Asexual morph. Conidiophores monomorphic, penicillate, arising from the agar surface or aerial mycelium; stipe 20–130 μm long, 1.9–3.9 μm wide at base; penicilli up to 120 μm high, 90 μm wide; terminal phialides in whorls of up to six, adpressed or divergent at acute angles, narrowly flask-shaped, (8.7–)10.4–21.6(–22.6) μm long, (1.2– )1.6–2.5(–2.8) μm wide at base, (2.0–)2.5–3.6(–4.0) μm at widest point, (0.8–)0.9–1.2(– Revising Clonostachys and allied genera in Bionectriaceae 69 2 Fig. 13. Sesquicillium symmetricum (ex-type CBS 124.79). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I, J. Conidia in linear chains. Scale bars = 10 μm. 1.3) μm wide near aperture (n = 60); intercalary phialides present, subterminally formed lateral pegs 3–5 μm long. Conidia hyaline, ellipsoidal to obovoid to somewhat clavate, symmetrical, distally broadly rounded, without laterally displaced hilum, (5.5–)5.8–8.9(– 9.8) × (2.2–)2.3–3.3(–3.9) μm (av. 7.1 × 2.7 μm, n = 100), arranged in linear chains. Culture characteristics: Colony on OA attaining 18–20 mm diam. after 7 d at 25 °C in Chapter 2 70 darkness, flat, with entire margin, aerial mycelium sparsely developed, finely felty, dirty white, reverse whitish. Colony on PDA attaining 20–21 mm diam., flat, with entire margin, aerial mycelium moderate, felty, whitish, reverse concolourous. Colony on SNA attaining 20–21 mm diam., flat, with entire margin, aerial mycelium moderate, felty, dirty white, reverse concolourous. Additional material examined: Colombia, Dep. de Meta, Municipio de Villavicencio, 25 km from Villavicencio to Acacías, 550 m alt., maize-field soil, collection and isolation date unknown, O. Rangel, culture CBS 485.78. Notes: Sesquicillium symmetricum is closely related to S. candelabrum, S. essexcoheniae, S. lasiacidis, S. neerlandicum, and S. rossmaniae (Fig. 1). Sesquicillium symmetricum can be morphologically distinguished from S. candelabrum, S. essexcoheniae, S. rossmaniae and S. neerlandicum based on its longer conidia [(5.5–)5.8–8.9(–9.8) μm long, av. 7.1 μm, in S. symmetricum; (3.6–)3.9–4.9(–5.5) μm av. 4.3 μm in S. candelabrum; (3.9–)4.3–5.6(–5.8) μm, av. 4.8 μm in S. essexcoheniae; (4.2–)4.6–5.4(–6.6) μm, av. 5.0 μm in S. rossmaniae; [(4.2–)4.5–5.6(–6.0) μm, av. 5.1 μm in S. neerlandicum]. Sesquicillium symmetricum differs from S. lasiacidis in having longer phialides [(8.7–)10.4–21.6(–22.6) μm in S. symmetricum, 8.4–13.8 μm in S. lasiacidis]. Due to the symmetrical shape of conidia and central placement of their hila, S. symmetricum produces not imbricate but linear conidial chains. Sesquicillium tornatum (Höhn.) Schroers, comb. nov. MycoBank MB 848462. Basionym: Pseudonectria tornata Höhn., Sitzungsber. Akad. Wiss. Wien, Math.-Naturwiss, Kl., Abt. 1. 118: 1470. 1909. Synonyms: Bionectria tornata (Höhn.) Schroers, Stud. Mycol. 46: 184. 2001. Clonostachys tornata (Höhn.) Rossman et al., Stud. Mycol. 80: 242. 2015. Sesquicillium asymmetricum Samuels, Mem. New York Bot. Gard. 49: 276. 1989. Clonostachys asymmetrica (Samuels) Schroers, Stud. Mycol. 46: 184. 2001. Nectria sesquiphialis Samuels, Mem. New York Bot. Gard. 49: 276. 1989. Typus: Type for Pseudonectria tornata: Indonesia, Java, Tjibodas, on decaying leaves of Pandanus sp. (no holotype specimen recorded). Type for Nectria sesquiphialis and Sesquicillium asymmetricum: Venezuela, Edo, Bolivar, 110–111 km S of El Dorado on road between El Dorado and Sta Elena, on leaf of Zingiberaceae, 6 Aug. 1972, R.F. Cain, G.J. Samuels & C. Blanco [holotype Dumont-VE 7184 (VEN), isotype NY, dried culture of C.T.R. isolate 72-193 (= ATCC 66892), derived from ascospores of Dumont-VE 7184, type of N. sesquiphialis, and filed with it (NY)]. Descriptions and illustrations: Von Höhnel (1909), Samuels (1989), Schroers (2001). Revising Clonostachys and allied genera in Bionectriaceae 71 2 Notes: Pseudonectria tornata and Nectria sesquiphialis, including the asexual morphtypified S. asymmetricum, were synonymised based on sexual and asexual morph characters encountered in the type of N. sesquiphialis (Dumont-VE 7184) and P. tornata (FH no. 2899) (Schroers 2001: figs 81 vs 82) and sexual morph characters described by von Höhnel (1909). Asexual morph characters consisted of conidiophores showing intercalary phialides below solitary terminal phialides and size range and shape of conidia; sexual morph characters, the habit of superficially formed perithecia on decaying leaves of Pandanus sp., perithecial walls composed of two regions, and 1-septate ascospores (9–12.8 × 2–3 μm for P. tornata; 10.8–20.8 × 2–4 μm for N. sesquiphialis) (Schroers 2001). Accordingly, morphological character interpretations were used for linking P. tornata and N. sesquiphialis to Bionectria subgenus Epiphloe and combining P. tornata into Bionectria and the same arguments are here adopted for combining the species into Sesquicillium. Obtaining and analysing phylogenetic marker genes from strain ATCC 66892 (= C.T.R. isolate 72-193, ex-type strain of N. sesquiphialis) is therefore required to further support the here suggested taxonomy. Clonostachys Corda, Pracht-Fl. Eur. Schimmelbild.: 31. 1839. Type: Clonostachys araucaria Corda Sexual morph. Stromata typically present, well developed if erumpent through bark, reduced if formed on associated fungus hosts, typically made of pseudoparenchymatous cells, rarely entirely superficial. Perithecia orange, yellowish orange, brownish orange, rarely brownish, KOH-, sometimes becoming paler in lactic acid, crowded in often large groups on stromata, rarely solitary, globose or somewhat higher than wide, sometimes obovoid, smooth or warted. Perithecial wall of two or three regions, rarely a single region, texture prosenchymatous or pseudoparenchymatous. Perithecial surface smooth, rough, or warted. Perithecial warts, if present, strongest developed in the upper part of the perithecia, irregularly scattered or radiating from the perithecial apex downwards. Asci 8-spored, apically rounded or flat, sometimes with prominent edges, with subapically thickened walls and apical ring. Ascospores typically 1-septate, coarsely or finely warted or striate, hyaline, ellipsoidal, tapering slightly towards their ends, sometimes with warts arranged in striae, rarely rough or smooth. Asexual morph. Sporodochia often on erumpent stroma when formed on the natural substratum or cushion-shaped in culture and without stroma; synnemata present in some species. Conidiophores frequently dimorphic (primary and secondary conidiophores), sometimes monomorphic. Primary conidiophores mononematous, early-formed, verticillum-like or narrowly penicillate. Secondary conidiophores mononematous, later-formed, penicillate, often aggregating into cushinshaped, more or less distinct sporodochia, less commonly synnematous; stipes typically arising from submerged or aerial hypha or from aerial hyphal fascicles, ropes or strands; penicilli monoverticillate or bito more-level verticillate. Conidiogenous cells phialidic. Chapter 2 72 Phialides on primary conidiophores almost cylindrical, slightly and gradually tapering; phialides on secondary conidiophores narrowly flask-shaped, widest in the lower third or middle, and slightly and continuously tapering toward tip or, in some species, intercalarly formed below terminal phialides and with subapically formed conidiogenous, lateral pegs. Conidiophores rarely with setae. Conidia aseptate, ellipsoid to subfusoid, typically with one more flattened side and a laterally displaced hilum resulting in a somewhat kidney-like appearance, hyaline or greenish hyaline, held in watery droplets or heads when formed on primary conidiophores or imbricate chains when formed on secondary conidiophores or sporodochia, conidial masses collapsing in either off-white, orange or greenish slimy masses. Notes: Clonostachys araucaria, the type species of Clonostachys, was described as producing white colonies “on forest soil”, incubated at around 17 °C, and oblongellipsoidal, obviously hyaline, and imbricately arranged conidia that adhere in columns and are formed by a penicillate conidiophore (Corda 1839). Although type material is not available, it is clear that Corda’s drawing illustrates a secondary conidiophore formed by many Clonostachys species. Samuels (1988a) linked members of the Nectria ochroleuca group to Clonostachys, and Schroers et al. (1999b) classified Clonostachys as the asexual morph of Bionectria. Thereafter, Clonostachys binominals were also provided for sexual morph-typified Bionectria names to allow usage of Clonostachys binomials for all species considered at that time (Schroers 2001), and Clonostachys became the name selected in the single name system (Rossman et al. 2013). Dimorphic conidiophores, described for Clonostachys rosea (as Gliocladidum roseum) for the first time by Bainier (1905, 1907), i.e., primary and secondary conidiophores, slightly curved conidia showing laterally displaced hila (i.e., a scar at the base of a conidium), perithecial walls consisting of three regions, warted ascospores and stromata erumpent through bark, characterise a core group of species of subgenus Bionectria (Fig. 1 and 2, terminal clade including C. reniformis). The group is further subdivided morphologically based on characteristics of both types of conidiophores, bearing either diverging or adpressed branches or phialides, while further differentiations of species based on morphological characters are difficult (Moreira et al. 2016). Species of this core group show rather homogenous character patterns, however, two of its species do not produce dimorphic conidiophores but sporodochia only. This core group also includes C. rosea and C. solani, for which two forms were accepted based on either light or (partly dark) green conidial masses (Schroers 2001) and named by using the reduced species epithets from Gliocladium catenulata Gilman & Abbott or, respectively, G. nigrovirens van Beyma. We recommend using the form-based system in situations where users of names wish to emphasise the green phenotype trait of their strains. However, the use of the binominals C. rosea or C. solani is equally correct (Fig. 2, Supplementary Fig. S9). Revising Clonostachys and allied genera in Bionectriaceae 79 2 hilum, broadly rounded at the end, (5.6–)6.4–9.2(–10.3) × 1.6–2.0(–2.4) μm (av. 7.7 × 1.9, n = 150), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 32–36 mm diam. after 7 d at 25 °C in darkness, flat, with crenate margin, aerial mycelium moderate, felty, dirty white to pale yellow, with concentric rings, reverse pale yellow. Colonies on PDA reaching 34–36 mm diam., flat, with crenate margin, aerial mycelium moderate, felty to cottony, dirty white, with concentric rings, reverse concolourous. Colonies on SNA reaching 28–31 mm diam., flat, with entire margin, aerial mycelium sparse, dirty white, reverse concolourous. Notes: Phylogenetically, C. bambusae (CBS 139411) and Clonostachys sp. CBS 496.90 (Figs 1, 2) form a sister clade to the core group of subgenus Bionectria characterised by regularly formed dimorphic conidiophores and slightly curved conidia having a laterally displaced hilum. Monomorphic, penicillate conidiophores, with somewhat divergent to adpressed branches and frequent occurrence of intercalary phialides characterise C. bambusae. The two strains differ genetically in ITS (99.2 % identity, with 4 bp differences), LSU (99.6 %, 3 bp), RPB2 (94 %, 43 bp), TEF1 (98.5 %, 12 bp) and TUB2 (98 %, 16 bp) sequences. Schroers (2001: fig. 93a) illustrated CBS 496.90 to directly compare conidiophores and intercalary phialides with those of C. setosa, which also is characterised by a sesquicillium-like asexual morph. Due to their phylogenetic affinity, Clonostachys sp., CBS 496.90 and C. setosa are to be classified in Clonostachys and not Sesquicillium. Clonostachys buxicola Lin Zhao & Crous, sp. nov. MycoBank MB 848480. Fig. 17. Etymology: Name refers to the host genus, from which this fungus was isolated, Buxus. Typus: France, Pyrénées Atlantiques, Isle de Sauveterre de Bearn, on bark of dead Buxus sempervirens, 25 Oct. 1998, G.J. Samuels & F. Candoussau (holotype designed here CBS H-25137; ex-type culture CBS 102419). Sexual morph unknown. Asexual morph. Conidiophores variable, somewhat penicillate or typically with short conidiophores gradually integrating into indistinct sporodochia. Sporodochia arising from the aerial mycelium or from agar surface, appearing at first as distinct white pustules, with time coalescing, arranged in tufts throughout the colony, covered with greenish grey to dark green conidial masses. Penicillate mononematous conidiophores arising from agar surface or aerial mycelium, with irregularly branched penicilli. Sporodochial conidiophores irregularly penicillate, terto quaterverticillate or more frequently branched; cells supporting phialides frequently widening distally; terminal phialides in whorls of 2–5, also singly, adpressed or divergent at acute angles, straight Chapter 2 80 Fig. 17. Clonostachys buxicola (ex-type CBS 102419). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D. Sporodochia producing green conidial masses on OA. E, F. Sporodochia. G. Phialides. H. Conidiophores. I, J. Conidia. Scale bars = 10 μm. or curved, cylindrical or somewhat narrowly flask-shaped, generally slightly tapering in the upper part, without a visible collarette, (7.4–)8.7–16.9(–21.0) μm long, (1.4–)1.6– 2.4 μm wide at base, 0.9–1.2 μm wide near aperture; intercalary phialides rare, solitary, mostly below a single terminal phialide, sometimes arising from ± square-shaped cells, the conidiogenous peg of intercalary phialides sometimes as long as terminal phialides. Revising Clonostachys and allied genera in Bionectriaceae 81 2 Conidia aseptate, greenish hyaline, smooth, ovoid to ellipsoid, straight or minutely curved, sometimes widest in the lower part, with a median or slightly laterally displaced, not protruding, distinctly flat or almost invisible hilum, (4.6–)5.2–8.0(–8.6) × (2.3–)2.7–3.5(– 3.8) μm (av. 6.4 × 3.1 μm, n = 200), arranged in linear chains. Culture characteristics: Colonies on OA reaching 24–26 mm diam. after 7 d at 25 °C in darkness, flat, with entire margin, and aerial mycelium sparsely developed, felty, dirty white, reverse concolourous. Colonies on PDA reaching 25–28 mm diam., flat, with entire margin, aerial mycelium moderate, felty, white, reverse concolourous. Colonies on SNA reaching 16–17 mm diam., flat, with irregular margin, finely granulose, felty, white, reverse concolourous. Notes: Based on phylogenetic analysis, C. buxicola and C. pityrodes, both forming distinctly greenish pigmented conidial masses, were included in a fully-supported clade (Figs 1, 2). Clonostachys buxicola and C. pityrodes are morphologically similar, but have clearly differing ITS (96.3 % identity, with 18 bp differences), LSU (98.7 %, 10 bp), RPB2 (84.2 %, 119 bp), TEF1 (95.3 %, 38 bp), and TUB2 (90.3 %, 104 bp) sequences. Both species form a unique lineage near the root of the Clonostachys clade (Figs 1, 2). Clonostachys pityrodes was placed in its own subgenus, subgenus Myronectria, because of morphological characters deviating from those of the other subgenera (Schroers 2001). Clonostachys cylindrica Lin Zhao & Crous, sp. nov. MycoBank MB 848481. Fig. 18. Etymology: Name refers to the cylindrical conidia produced by this species. Typus: Venezuela, Estación Biológica de Rancho Grande, Parque Nacional Henry Pittie, Estado Aragua, 1 200 m alt., Nov. 1997, R.F. Castañeda (holotype designated here CBS H-25150, ex-type living culture CBS 101113). Sexual morph unknown. Asexual morph. Conidiophores dimorphic. Primary conidiophores verticillium-like, or acremonium-like, arising from the agar surface or from aerial mycelium, with monoverticillate or more-level-verticillate divergent phialides, sometimes with sidebranches; stipe up to 165 μm long, 2.3–4.5 μm wide at base; penicilli up to 134 μm high; terminal phialides in whorls of 4, in lower levels also solitary, straight, cylindrical, but slightly and continuously tapering towards the tip, with or without a minute collarette, (10.4–)18.4–39.0(–41.7) μm long, (1.7–)1.8–2.6(–3.1) μm wide at base, (1.0–)1.2–1.6(– 1.7) μm wide near aperture (n = 52), each producing a small, hyaline drop of conidia. Secondary conidiophores penicillate, scattered on the agar surface or arising from strands of aerial hyphae, up to terverticillate, branches adpressed or somewhat divergent; stipes Chapter 2 82 Fig. 18. Clonostachys cylindrica (ex-type CBS 101113). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G–I. Secondary conidiophores. J. Conidia. Scale bars: E = 50 μm; D, F–J = 10 μm. 41–152 μm long, 3.2–5.0 μm wide at base; penicilli up to 93 μm high, to 71 μm diam. at widest point; terminal phialides adpressed, in whorls of up to seven, straight to slightly curved, narrowly flask-shaped, slightly tapering in the upper part, with or without a visible collarette, (7.7–)8.2–12.8(–15.7) μm long, (1.6–)1.8–2.9(–3.3) μm wide at base, (2.4–)3.0– 4.0 μm at widest point, (0.9–)1.0–1.3(–1.4) μm wide near aperture (n = 100); intercalary phialides (5.4–)6.7–10.2(–12.3) × (2.8–)3.0–4.0(–4.3) with to 4.5 μm long lateral pegs, Revising Clonostachys and allied genera in Bionectriaceae 83 2 below a whorl of terminal phialides. Conidia aseptate, hyaline, smooth, cylindrical, straight or slightly curved, with a laterally displaced or sometime central hilum, (7.7–)8.6–11.9(– 13.5) × (2.3–)2.5–3.3(–4.3) μm (av. 10.1 × 2.9 μm, n = 150), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 32–34 mm diam. after 7 d at 25 °C in darkness, with entire margin, aerial mycelium moderate, felty, whitish, reverse concolourous. Colonies on PDA reaching 35–38 mm diam., with entire margin, aerial mycelium moderate, felty to cottony, finely to coarsely granular, whitish, reverse concolourous. Colonies on SNA reaching 31–36 mm diam., with entire margin, aerial mycelium sparse, granular, whitish, reverse concolourous. Notes: Clonostachys cylindrica resembles C. compactiuscula and C. penicillata, but these species can be distinguished based on conidial size [(7.7–)8.6–11.9(–13.5) × (2.3–)2.5–3.3(– 4.3) μm in C. cylindrica) vs (3.9–)5.4–6.6–7.5(–12.4) × (1.5–)1.9–2.2–2.5(–3.2) μm in C. compactiuscula and (4.5–)5.0–6.8(–7.6) × (1.6–)1.7–2.2(–2.4) μm in C. penicillata. However, the morphologically differing C. divergens and C. samuelsii (sporodochial conidiophores) and C. hongkongensis and C. rogersoniana (broadly ellipsoidal to oval conidia) are also members of the two clades. Clonostachys cylindrica produces mononematous, dimorphic conidiophores, pale conidial masses, and clearly longer conidia (av. 10.1 μm), while C. divergens (clostest phylogenetic sister lineage in Fig. 2) forms greenish pigmented conidial masses, and less than 5 μm long conidia. Also the molecular sequences distinguish both species clearly (ITS: 96.3 % identity, with 18 bp differences; LSU: 99.4 %, 5 bp; and TEF1: 97.9 %, 17 bp). Clonostachys ellipsoidea Lin Zhao & Crous, sp. nov. MycoBank MB 848482. Fig. 19. Etymology: Name refers to the broadly ellipsoidal conidia produced by this fungus. Typus: Indonesia, Java, Jogyakarta, date unknown, F.J.J. Jongeleen (holotype designated here CBS H-25144, ex-type living culture CBS 175.76). Sexual morph unknown. Asexual morph. Conidiophores monomorphic, penicillate, arising from the sparse aerial mycelium, branches divergent or somewhat divergent, monoto biverticillate; stipes 10–60 to μm long, to 4.3 μm wide at base; terminal phialides in loose apical whorls of 2–6, adpressed or divergent at acute angles, straight or curved, cylindrical and slightly tapering towards the tip or narrowly flask-shaped and slightly widening in the middle, without visible collarette, (7.9–)8.5–18.9(–21.5) μm long, (1.4–)1.5–2.4(–2.7) μm wide at base, (2.2–)2.4–3.5(–3.9) μm at widest point, 1.0–1.4 μm wide near aperture (n = 100); intercalary phialides rare, conidiogenous pegs to 5 μm long, formed solitarily Chapter 2 84 Fig. 19. Clonostachys ellipsoidea (ex-type CBS 175.76). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–H. Conidiophores. I, J. Conidia. Scale bars = 10 μm. below whorls of terminal phialides. Conidia aseptate, hyaline, smooth, broadly ellipsoidal, rarely minutely curved, ends broadly rounded, hilum laterally displaced, almost median or invisible, (4.2–)4.8–5.9(–6.3) × (3.0–)3.2–3.7(–4.0) (av. 5.3 × 3.5, n = 150), arranged in imbricate chains that may collapse into slimy masses. Culture characteristics: Colonies on OA reaching 40–42 mm diam. after 7 d at 25 °C in darkness, flat, with entire margin, aerial mycelium sparse, floccose, whitish, reverse Revising Clonostachys and allied genera in Bionectriaceae 85 2 concolourous. Colonies on PDA reaching 39–41 mm diam., with entire margin, aerial mycelium moderate, felty to cottony, whitish, reverse concolourous. Colonies on SNA reaching 38–40 mm diam., flat, with entire margin, aerial mycelium sparse, felty, finely granular, whitish, reverse concolourous. Additional material examined: India, Amarkantak, Shahdol, soil, Jan. 1993, T. Okuda, No. TC 1304, culture CBS 102566. Notes: Based on the phylogenetic analyses (Figs 1, 2), C. ellipsoidea is closely related to C. leucaenae (MFLUCC 20-0008), C. pallens (PAD S00004), C. vacuolata (CBS 191.93) and C. venezuelae (CBS 107.87). Clonostachys ellipsoidea differs from C. leucaenae in ITS (95.3 %, with 23 bp differences), LSU (98.3 %, 14 bp), from C. pallens in the ITS (92.5 % identity, with 14 bp differences) sequence, and from C. venezuelae (CBS 107.87) in ITS (95.5 % identity, with 23 bp differences), LSU (98.6 %, 11 bp), RPB2 (96 %, 30 bp), TEF1 (96.2 %, 30 bp), and TUB2 (97.1 %, 30 bp) sequences. In addition, C. ellipsoidea differs from C. vacuolata in producing smaller and wider conidia (av. 5.3 × 3.5 μm vs av. 6.0 × 2.9 μm). Morphology of conidiophores and conidia supports classification of C. ellipsoidea in subgen. Zebrinella. Clonostachys eriocamporesii R.H. Perera & K.D. Hyde, Fungal Diversity 100: 199. 2020. Fig. 20. Typus: Thailand, Southern Thailand, on dead stems of Pennisetum polystachion (Poaceae), 11 Nov. 2017, A. Karunarathne, Bion 78 (holotype MFLU 18-2718, ex-type culture MFLUCC 19-0486). Description based on CBS 647.91: Sexual morph unknown. Asexual morph. Sporodochia formed as distinct white pustules, with time coalescing to support dark greenish conidial masses. Conidiophores monomorphic, sporodochial, phialides straight or curved, cylindrical and slightly tapering towards the tip or narrowly flask-shaped and slightly widening in the middle, with or without a visible collarette, (7.6–)9.5–17.5(–19.6) μm long, (2.0–)2.2–3.2(– 3.5) μm at base, (2.3–)2.5–3.5(–3.7) at widest point, (0.8–)0.9–1.2(–1.3) μm wide near aperture (n = 60). Conidia aseptate, greenish hyaline, ellipsoidal to narrowly clavate, straight or slightly curved, with or without a slightly protruding or slightly laterally displaced hilum and a rounded distal end, (4.4–)5.9–8.6(–12.6) × (2.4–)2.7–3.8(–4.2) μm (av. 7.4 × 3.2 μm, n = 150), arranged in linear chains. Culture characteristics: Colonies on OA reaching 30–45 mm diam. after 7 d at 25 °C in darkness, with entire margin, aerial mycelium moderate, felty to cottony, whitish, reverse concolourous. Colonies on PDA reaching 32–41 mm diam., with entire margin, aerial Chapter 2 86 Fig. 20. Clonostachys eriocamporesii (CBS 647.91). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D. Sporodochia after 1 wk. E. Sporodochia after 2 wk. F, G. Sporodochia. H, I. Conidia. Scale bars = 10 μm. mycelium moderate, felty to cottony, whitish, reverse concolourous. Colonies on SNA reaching 27–32 mm diam., with entire margin, aerial mycelium moderate in the centre, sparsely at periphery, floccose, whitish, reverse concolourous. Additional material studied: Germany, Berlin, unknown date and collector, culture CBS 647.91. Netherlands, soil, Oct. 27, 2019, T. Vercruisse, culture CBS 148221= NL19-060010. Revising Clonostachys and allied genera in Bionectriaceae 87 2 Notes: Clonostachys eriocamporesii was first described from dead stems of Pennisetum polystachion (Hyde et al. 2020a). According to our phylogenetic analyses (Figs 1, 2), C. eriocamporesii is closely related to C. epichloe, C. fujianensis, C. miodochialis and C. obovatispora. Morphologically, C. eriocamporesii differs from C. epichloe, C. miodochialis and C. obovatispora, in producing larger conidia [(4.4–)5.9–8.6(–12.6) × (2.4–)2.7–3.8(– 4.2) av. 7.4 × 3.2 μm in C. eriocamporesii, (4.8–)6.0–7.0(–9.6) × (1.6–)2.2–2.8(–3.6) av. 6.6 × 2.6 μm in C. epichloe, (5.2–)5.8–7.2(–8.0) × (1.8–)2.6–3.0(–3.4) av. 6.6 × 2.8 μm in C. miodochialis, and (4.7–)5.8–7.6(–8.2) × (1.7–)2.0–2.5(–2.9) av. 6.7 × 2.3 μm in C. obovatispora]. Clonostachys eriocamporesii (CBS 647.91) and C. fujianensis (CBS 127474) are clearly different by ITS (98.2 % identity, with 9 bp differences), LSU (99.6 %, 3 bp), TEF1 (95.8 %, 34 bp), and TUB2 (91.5 %, 90 bp) sequences. Clonostachys farinosa (Henn.) Rossman, IMA Fungus 5: 86. 2014. Basionym: Nectriella farinosa Henn., Hedwigia 36: 219. 1897. Synonyms: Nectria farinosa (Henn.) Möller, in Schimper, Bot. Mitt. Tropen 9: 296. 1901. Nectria byssicola Berk. & Broome, J. Linn. Soc. Bot. 14: 116. 1873. Bionectria byssicola (Berk. & Broome) Schroers & Samuels, Z. Mykol. 63: 152. 1997. Clonostachys byssicola Schroers, Stud. Mycol. 46: 80. 2001. Clonostachys eriocamporesiana R.H. Perera & K.D. Hyde, Fungal Diversity 100: 197. 2020. Clonostachys indica Prasher & R. Chauhan [as ‘indicus’], Kavaka 48: 22. 2017. Clonostachys wenpingii (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang, Mycol. Progr. 13: 969. 2014. Clonostachys granuligera (Starbäck) Forin & Vizzini, Persoonia 45: 240. 2020. Clonostachys squamuligera (Sacc.) Forin & Vizzini, Persoonia 45: 245. 2020. Clonostachys spinulosa R.H. Perera, E.B.G. Jones & K.D. Hyde, Fungal Diversity 118: 109. 2023. Description and illustration: Schroers (2001). Typus: Venezuela, Edo Sucre, between Los Pocitos and Santa Isabel, on unidentified wood, 11 Jul. 1972, G.J. Samuels & K.P. Dumont (isotype of C. byssicola, CBS H-7918, ex-type living culture CBS 364.78 = C.T.R. 72-123-ss7 = VE 4681). Notes: Nectria byssicola was described by Berkeley & Broome (1873) from the portion ‘173d’ (type specimen) as having pale orange, scurfy perithecia and ascospores. It was transferred to Bionectria as B. byssicola (Schroers & Samuels 1997). Subsequently, Clonostachys byssicola was newly described by Schroers (2001). However, with the introduction of the One Fungus–One Name concept, B. byssicola became a synonym for C. byssicola. Therefore, the oldest available name, N. byssicola (1873), cannot be Chapter 2 88 recombined in Clonostachys. Accordingly, the next available epithet, Nectriella farinosa (1897), was placed in Clonostachys and published as a new combination, C. farinosa (Rossman 2014). According to the multi-gene phylogenetic inferences, the ex-type strains of C. eriocamporesiana (MFLUCC 17-2620), C. granuligera (PAD S00011), C. indica (IBP2), C. spinulosa (MFLUCC 17-0131), C. squamuligera (PAD S00020) and C. wenpingii (CBS 124067) cluster near the ex-type culture of C. byssicola (CBS 364.78; Figs 1, 2) and together with several other strains identified by Schroers (2001) as C. byssicola (Fig. 2). Judged on the basis of gatherings of perithecia on recently dead trees, C. farinosa is one of the more frequently occurring species in tropical regions (Samuels 1976, Schroers 2001). A couple of asexual morph characters were discussed that could help identifying C. farinosa in cultures (Schroers 2001, as C. byssicola); however, distinguishing this species from others forming dimorphic conidiophores is difficult. It is therefore not surprising that rather numerous synonymous species were described since 2001 on the basis of asexual morph-typified material. Clonostachys flava Lin Zhao, Crous, & Schroers, sp. nov. MycoBank MB 848483. Fig. 21. Etymology: Name refers to the yellow colonies of this species (OA, PDA, and SNA). Typus: French Guiana, St. Laurent-Du-Marouni, Canton de Maripasoula, S. along Route De belizon, decaying bark, 200 m alt., 14 Sep. 1994, S.M. Huhndorf, BPI 737845, culture G.J.S. 94-75 (holotype designated here CBS H-25139, ex-type culture CBS 915.97). Sexual morph known from natural specimen (not shown). Asci 49–70 × 8.5–10 μm. Ascospores 1-septate, hyaline, striate, ellipsoid, 9.7–10.8–12.8 × 3.3–4.0–4.9 μm. Asexual morph. Conidiophores dimorphic, mononematous, scattered on the agar surface or arising from strands of aerial hyphae. Primary conidiophores narrowly penicillate, adpressed, monoto terverticillate; stipe 20–120 μm long, 2.2–4.0 μm wide at base; penicilli 40–100 μm high; phialides straight to slightly curved, almost cylindrical, slightly tapering towards the tip, sometimes with a short collarette, (22.4–)25.9–43.2(–46.9) μm long, (1.4–)1.6– 2.4(–2.6) μm wide at base, (1.1–)1.2–1.6(–1.7) μm wide near aperture (n = 50). Secondary conidiophores penicillate, terto quaterverticillate, branches adpressed or divergent, phialides adpressed or somewhat divergent; phialides in loose whorls of 2–5, straight or slightly curved, flask-shaped or somewhat cylindrical, widest in the lower third, slightly tapering in the upper part towards the tip, without a collarette, (11.4–)12.6–18.8(–20.7) μm long, (1.2–)1.5–2.3(–2.8) μm wide at base, (1.8–)2.0–2.7(–2.8) μm at widest point, (0.8–)0.9–1.3(–1.4) μm wide near aperture (n = 50). Conidia aseptate, hyaline, smooth, ellipsoid to obovoid, distally broadly rounded, apex minutely tapering, with a median, invisible, or rarely somewhat laterally displaced hilum, (4.7–)4.9–6.5(–7.8) × (2.4–)2.6– Revising Clonostachys and allied genera in Bionectriaceae 95 2 concolourous. Colonies on PDA reaching 28–31 mm diam., flat, with entire margin, aerial mycelium moderate, felty to cottony, whitish, reverse concolourous. Colonies on SNA reaching 28–30 mm diam., flat, with entire margin, aerial mycelium sparse, felty, whitish, reverse concolourous. Notes: According to the multi-gene phylogenetic inference, C. garysamuelsii groups together with C. moreaui and C. oblongispora (Fig. 2; support below threshold values) or C. parasporodochialis and C. sporodochialis (Fig. 1, poorly to well-supported). Morphologically, C. garysamuelsii differs from C. moreaui and C. oblongispora in producing smaller conidia [(3.5–)4.5–7.1(–8.2) × (2.0–)2.3–3.3(–3.7) μm vs 5.0–10.0(–12.0) × 3.5–5.0 μm and (9.0– )12.6–14.0(–19.8) × (2.6–)3.2–3.8(–4.2) μm]. However, C. garysamuelsii differs from C. sporodochialis in producing larger conidia [(3.5–)4.5–7.1(–8.2) × (2.0–)2.3–3.3(–3.7) μm vs (3.2–)4.4–5.4(–6.8) × (1.6–)2.0–2.2(–2.6) μm]. Clonostachys garysamuelsii can also be distinguished from C. moreaui and C. parasporodochialis by having dimorphic conidiophores, while conidiophores of C. moreaui and C. parasporodochialis are monomorphic (Lechat et al. 2020). Clonostachys hongkongensis Lin Zhao & Crous, sp. nov. MycoBank MB 848487. Fig. 24. Etymology: Named after the location where the fungus was collected, Hong Kong, China. Typus: China, Hong Kong, Pokfulam Reservoir, wood, 14 Jun. 2001, unknown collector (holotype designated here CBS H-25149, ex-type living culture CBS 115291). Sexual morph unknown. Asexual morph. Conidiophores dimorphic, mononematous. Primary conidiophores verticillium-like, arising from the agar surface or from aerial mycelium, with monoverticillate or more-level-verticillate divergent phialides, sometimes with side-branches; stipe up to 225 μm long, 2.3–4.6 μm wide at base; penicilli 40–130 μm high; phialides in whorls of 2–4, in lower levels also solitary, straight, cylindrical, slightly and continuously tapering towards the tip, with or without a minute collarette, (19.9–)20.4–39.1(–43.4) μm long, (1.6–)1.9–2.3(–2.6) μm wide at base, 1.1–1.5(–1.7) μm wide near aperture (n = 50), each producing a small, hyaline drop of conidia. Secondary conidiophores penicillate, scattered on the agar surface or arising from strands of aerial hyphae, bito quaterverticillate; branches adpressed or somewhat divergent; stipes 93– 135 μm long, to 5 μm wide at base; penicilli 30–58 μm, to 55 μm diam. at widest point; terminal phialides adpressed, in whorls of up to 4–5, straight to slightly curved, narrowly flask-shaped, slightly tapering in the upper part, with or without a visible collarette, (9.0– )10.3–14.5(–15.9) μm long, (1.5–)1.6–2.2(–3.0) μm wide at base, (2.2–)2.4–2.9(–3.4) μm at widest point, (0.8–)0.9–1.3 μm wide near aperture (n = 50); intercalary phialides rare, Chapter 2 96 Fig. 24. Clonostachys hongkongensis (ex-type CBS 115291). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Primary conidiophores. F, G. Secondary conidiophores. H, I. Conidia. Scale bars: E, F = 50 μm; D, G–I = 10 μm. with to 3 μm long lateral pegs, below a whorl of terminal phialides (not shown). Conidia aseptate, hyaline, smooth, ellipsoid to obovoid, straight or slightly curved, with a laterally displaced or sometimes median hilum, (4.1–)4.3–5.9(–6.3) × (2.8–)2.9–3.4 μm (av. 4.8 × Revising Clonostachys and allied genera in Bionectriaceae 97 2 3.1 μm, n = 70), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 36–40 mm diam. after 7 d at 25 °C in darkness, flat, with crenate margin, aerial mycelium moderate, finely to coarsely granular, felty to cottony, whitish to pale yellow, reverse yellowish. Colonies on PDA reaching 37– 43 mm diam., flat, with crenate margin, aerial mycelium moderate, finely to coarsely granular, felty to cottony, whitish, reverse concolourous. Colonies on SNA reaching 39–40 mm diam., flat, with crenate margin, aerial mycelium sparse, finely to coarsely granular, whitish, reverse concolourous. Additional material examined: China, Hong Kong, Pokfulam Reservoir, wood, 14 Jun. 2001, unknown collector, culture CBS 116542. Notes: Based on the multi-locus phylogenetic analyses (Figs 1, 2), C. hongkongensis has a close phylogenetic affinity to C. cylindrica, C. divergens, C. rogersoniana and C. samuelsii. Morphologically, C. hongkongensis differs from C. cylindrica, C. divergens, C. rogersoniana and C. samuelsii in producing shorter conidia [(4.1–)4.3–5.9(–6.3) μm vs (7.7–)8.6–11.9(– 13.5) μm in C. cylindrica, (4.8–)5.8–6.4(–7.4) μm in C. divergens, (4.8–)5.8–7.2(–9.6) μm in C. rogersoniana and (4.4–)5.8–7.0(–11.6) μm in C. samuelsii]. Loosely branched sporodochia showing diverging branches and greenish conidial masses distinguish C. divergens from C. hongkongensis. Clonostachys kunmingensis Hong Yu bis & Yao Wang, Frontiers Microbiol. 14: 8. 2023. Fig. 25. Typus: China, Yunnan Province, Kunming City, Wild Duck Forest Park (25°13’N, 102°87′E, 2 100 m alt.), from soil on the forest floor, 10 Aug. 2019, Yao Wang (holotype YHH 898 dried specimen, ex-type culture YFCC 898). Description based on CBS 101920: Sexual morph from natural specimen (not shown). Asci 49.1–59.1–66.9 × 6.7–8.4–10.0 μm (n = 16). Ascospores 1-septate, hyaline, smooth or finely spinulose, ellipsoid, 10.1–12.7–16.6 × 3.6–4.2–4.9 μm (n = 24). Asexual morph. Conidiophores dimorphic, mononematous. Primary conidiophores arising from the agar surface or from strands of aerial mycelium, branches adpressed or somewhat divergent, phialides adpressed; stipe 30–140 μm long, 2.8–4.8 μm wide at base; penicilli 30–80 μm high; phialides in apical whorls of 2–5, also solitary arising from lower levels, straight, almost cylindrical, slightly tapering towards the tip, with a somewhat visible collarette, (15.4–)17.6–31.2(–32.5) μm long, (1.6–)1.9–2.5(–2.7) μm wide at base, 1.1–1.5(–1.6) μm wide near aperture (n = 80). Secondary conidiophores penicillate, bito quaterverticillate, Chapter 2 98 primary branches typically divergent, higher level branches and phialides somewhat divergent to almost adpressed; stipe 20–120 μm long, 2.5–4.5 μm wide; penicilli 30–70 μm long; 25–65 μm wide; terminal phialides in loose whorls of up to 6, straight or slightly curved, flask-shaped, widest in the lower third, slightly tapering in the upper part towards the tip, without or with a collarette, (9.0–)10.0–16.7(–28.1) μm long, (1.2–)1.6–2.5(–2.8) μm wide at base, (1.9–)2.1–2.7(–2.8) μm at widest point, (0.9–)1.0–1.3 μm wide near Fig. 25. Clonostachys kunmingensis (CBS 101920). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G–I. Secondary conidiophores. J, K. Conidia. Scale bars: G = 50 μm; D–F, H–K = 10 μm. Revising Clonostachys and allied genera in Bionectriaceae 99 2 aperture (n = 100); intercalary phialides sometimes observed, bearing one or several terminal phialides, conidiogenous pegs to 5 μm long. Conidia aseptate, hyaline, smooth, ellipsoid, commonly asymmetric with one more flattened sides, distally broadly rounded, with laterally displaced hilum, (3.7–)4.5–6.3(–7.1) × (2.1–)2.4–3.1(–3.3) μm (av. 5.4 × 2.7 μm, n = 150), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 51–53 mm diam. after 7 d at 25 °C in darkness, flat, with fimbriate margin, aerial mycelium abundant, felty, whitish, reverse concolourous. Colonies on PDA reaching 46–50 mm diam., flat, with entire margin, aerial mycelium abundant, felty, pale yellow, with greenish tinge diffusing into the medium outside the colony, reverse yellowish to light orange. Colonies on SNA reaching 38–45 mm diam., flat, with crenate margin, floccose, aerial mycelium scanty, whitish, reverse concolourous. Material examined: China, Yunnan Province, Kunming City, Songming County, Dashao Village (25°24’N, 102°55′E, 2750 m alt.), from soil on the forest floor, 24 Aug. 2019, Y. Wang, culture YFCC 892. Jamaica, Hanover Parish, Dolphin Head Mt., vic. Askenish, wood, collection and isolation date unknown, collected by D. Korf et al., isolated by C.T. Rogerson & G.J. Samuels MJ 946, C.T.R. 71-116, culture CBS 101920. Notes: Clonostachys kunmingensis was described from soil in Kunming City, China, but its holomorph was encountered in the neotropics. Schroers (2001) filed CBS 101920 under C. solani due to overall branching patterns of the primary and secondary conidiophores. Based on our phylogenetic analysis, C. kunmingensis is closely related to C. rhizophaga (Figs 1, 2). Length of phialides in primary conidiophores distinguish both species [(15.4– )17.6–31.2(–32.5) μm in C. kunmingensis, (15.6–)22.0–34.2(–48.2) μm in C. rhizophaga]. Clonostachys kunmingensis (CBS 101920) has different ITS (99.2 % identity, with 4 bp differences), LSU (99.9 %, 1 bp), RPB2 (97.5 %, 19 bp), TEF1 (99.6 %, 3 bp), and TUB2 (98.3 %, 18 bp) sequences when compared with C. rhizophaga (CBS 202.37). Clonostachys longiphialidica Lin Zhao, Crous, & Schroers, sp. nov. MycoBank MB 848488. Fig. 26. Etymology: Names refers to the long conidiogenous peg of intercalary phialides produced by this species. Typus: Venezuela, Dept. Rio Negro, Cerro de la Neblina, summit camp 5, valley at N base of Pico Phelps, 1000–1250 m alt., bark, cloud forest, 12 Apr. 1984, G.J. Samuels, G.J.S. 1301 (holotype designated here CBS H-25142, ex-type living culture CBS 112.87 = G.J.S. 84-330). Chapter 2 100 Sexual morph from natural specimen (not shown). Asci 46.1–66.3 × 6.6–10.3 μm (n = 11). Ascospores 1-septate, hyaline, striate, ellipsoid, 10.4–12.7–15.8 × 3.4–4.2–5.3 μm (n = 27). Asexual morph. Conidiophores monomorphic, aggregated in pustules, arising from the agar surface or sparse aerial mycelium, adpressed or with more or less divergent branches, phialides adpressed or somewhat divergent, bito quaterverticillate; stipe 10–50 μm long, 1.9–3.4 μm wide at base; penicilli up to 75 μm long, 60 μm wide; terminal phialides in whorls of 2–6, cylindrical or slightly tapering toward the tip, or slightly widening in lower third, Fig. 26. Clonostachys longiphialidica (ex-type CBS 112.87). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars = 10 μm. Revising Clonostachys and allied genera in Bionectriaceae 101 2 frequently with a small collarette, (7.6–)9.2–16.7(–24.2) μm long, (1.5–)1.7–2.8(–2.9) μm wide at base, (2.0–)2.3–3.3(–3.5) μm at widest point, (1.1–)1.2–1.5(–1.6) μm wide near aperture (n = 80); intercalary phialides rare, formed below whorls of terminal phialides, with to 8 μm long conidiogenous pegs. Conidia aseptate, hyaline, smooth, narrowly ellipsoid to cylindrical, straight or nearly straight, distally broadly rounded, (6.3–)6.7–8.5(– 9.3) × (2.2–)2.4–3.0(–3.4), (av. 7.5 × 2.7 μm, n = 150), arranged in imbricate chains that may collapse into slimy masses. Culture characteristics: Colonies on OA reaching 40–42 mm diam. after 7 d at 25 °C in darkness, with entire margin, aerial mycelium sparse, felty, whitish, reverse concolourous. Colonies on PDA reaching 37–43 mm diam., with entire margin, aerial mycelium moderate, felty to cottony, finely to coarsely granular, white yellowish, reverse pale yellow. Colonies on SNA reaching 39–44 mm diam., with entire margin, aerial mycelium moderate, felty to cottony, whitish, reverse concolourous. Notes: Clonostachys longiphialidica is represented by a single strain isolated from bark in Venezuela. It is phylogenetically different from the closely related C. vacuolata (ITS: 96.0 % sequence similarity; LSU: 96.0 %, RPB2: 93.6 %, and TEF1: 97.6 %). Their morphological differences are discussed under C. vacuolata. Specimen G.J.S. 1301 was identified as C. grammicospora on the basis of ascospore morphology and characters of the perithecia (Schroers 2001: fig. 62a, c, e, f). However, its ascospores cover a slightly larger length range as Raunkiaer 3103 (isotype of Nectria grammicospora, ascospores 10.7–13.1 × 3.8–4.9 µm) and specimen Samuels 3285 (type of C. grammicospora, ascospores 9–13.5 × 3.6–5.4 μm) (Samuels 1988b, Schroers 2001). Sexual and asexual morphology suggest classification of C. longiphialidica in subgen. Zebrinella. Clonostachys obovatispora Lin Zhao & Crous, sp. nov. MycoBank MB 848489. Fig. 27. Etymology: Names refers to the obovate conidial shown by this species. Typus: Germany, Frankfurt, Bergen-Enkheim, on living leaves with Epichloe typhina, 10 Jun. 2005, R. Kirschner (holotype designated here CBS H-25148, ex-type living culture CBS 118752). Sexual morph unknown. Asexual morph. Conidiophores monomorphic, sporodochial. Sporodochia appearing at first as distinct white pustules, with time coalescing and with green coloured conidial masses; phialides in whorls of 2–4, flask-shaped to cylindrical, widest near the middle, narrowing in the uppermost part, without a visible collarette, (9.2– )9.8–16.4(–17.0) μm long, (1.6–)1.8–2.7(–2.9) μm at base, (2.3–)2.4–3.2(–3.5) at widest point, (0.7–)0.8–1.1(–1.2) μm wide near aperture (n = 60). Conidia aseptate, greenish Chapter 2 102 Fig. 27. Clonostachys obovatispora (ex-type CBS 118752). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D, E. Sporodochia. F, G. Phialides. H, I. Conidia. Scale bars = 10 μm. hyaline to pale green, smooth, oblong-ellipsoid or clavate to obovoid, (4.7–)5.8–7.6(–8.2) × (1.7–)2.0–2.5(–2.9) (av. 6.7 × 2.3 μm, n = 100), arranged in linear chains. Culture characteristics: Colonies on OA reaching 27–30 mm diam. after 7 d at 25 °C in darkness, with slightly lobate margin, aerial mycelium moderate, felty, finely to coarsely granular, whitish, reverse concolourous. Colonies on PDA reaching 26–27 mm diam., with slightly lobate margin, aerial mycelium moderate, felty to cottony, whitish, reverse Revising Clonostachys and allied genera in Bionectriaceae 103 2 concolourous. Colonies on SNA reaching 19–22 mm diam., flat, with entire margin, membranous without aerial mycelia, colourless, reverse colourless. Notes: According to phylogenetic inferences in the present study (Figs 1, 2), C. obovatispora is closely related to C. epichloe, and C. miodochialis. However, C. obovatispora differs from C. epichloe (CBS 101037) in ITS (98.8 % identity, with 6 bp differences), LSU (99.3 %, 5 bp), RPB2 (91.3 %, 66 bp), TEF1 (95.8 %, 34 bp), and TUB2 (95.1 %, 50 bp) sequences; C. obovatispora differs from C. miodochialis (CBS 997.69) in ITS (98.4 % identity, with 8 bp differences), LSU (99.5 %, 4 bp), RPB2 (91.7 %, 62 bp), TEF1 (96.1 %, 32 bp), and TUB2 (95.3 %, 47 bp) sequences. Morphologically, C. obovatispora differs from C. epichloe and C. miodochialis in producing shorter phialides [(9.2–)9.8–16.4(–17.0) μm in C. obovatispora vs (7–)12–17(–29) μm in C. epichloe], and narrower conidia [(1.6–)2.2–2.6–2.8 (–3.6) μm in C. obovatispora vs (1.8–)2.6–3(–3.4) μm in C. miodochialis)]. However, overall nature of morphological characters, i.e., occurrence of sporodochia, greenish pigmentation of conidial masses and shape of conidia, is similar in these closely related species and aligns C. obovatispora well into the morphological concept of subgenus Astramata. Clonostachys palmae Lin Zhao, Crous & Schroers, sp. nov. MycoBank MB 848490. Fig. 28. Etymology: Name refers to palm, the host from which the ex-type culture of this fungus was isolated. Typus: Indonesia, Sulavesi, Eastern Dumoga-Bone Nat. Park, between Maddison’s Camp, from palm leaves, 5 Oct. 1985, G.J. Samuels, G.J.S 2156, culture G.J.S. 85-155 (holotype designated here CBS H-25153, ex-type culture CBS 119.87). Sexual morph known from natural specimen (not shown). Asci 45–67.5 × 7.0–10.8 μm. Ascospores striate, ellipsoid with gently tapering ends, 1-septate, 10.0–14.5 × 3.0–5.4 μm. Asexual morph. Conidiophores dimorphic. Primary conidiophores mononematous, arising from either the aerial mycelium or the agar surface, either unbranched, acremonium-like or verticillium-like, monoto terverticillate, with divergent branches and phialides diverging at more or less acute angles; stipe 40–180 μm long, 2.4–4.6 μm wide at base; penicilli 50–100 μm high; phialides in apical whorls of 2–4, straight, cylindrical, slightly tapering towards the tip, with a minute visible collarette, (21.0–)22.4–42.2(–48.8) μm long, (1.6– )1.8–2.8(–3.0) μm wide at base, 1.2–1.8(–2.0) μm wide near aperture (n = 70). Secondary conidiophores broadly penicillate, arising from the agar surface or aerial mycelium terto quinquiesverticillate, aggregated in pustules or sporodochia; phialides slightly divergent or adpressed, in whorls of 2–4, flask-shaped, widest in the lower third or almost cylindrical, slightly tapering in the upper part towards the tip, without a visible collarette, (9.2–)11.0– Chapter 2 104 Fig. 28. Clonostachys palmae (ex-type CBS 119.87). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–F. Primary conidiophores. G, H. Secondary conidiophores. I, J. Conidia. Scale bars = 10 μm. 19.0(–21.6) μm long, (1.5–)1.6–2.6(–2.9) μm wide at base, (2.1–)2.2–2.9(–3.1) at widest point, (1.0–)1.1–1.7(–1.8) μm wide near aperture (n = 80). Conidia aseptate, hyaline, smooth, ellipsoid, some slightly curved or asymmetric with one more flattened side, with a laterally displaced hilum, (4.5–)5.3–8.2(–10.2) × (2.4–)2.7–3.2(–3.6) (av. 6.5 × 2.9 μm, n = 150), arranged in imbricate chains. Culture characteristics: Colonies on OA reaching 49–53 mm diam. after 7 d at 25 °C in Revising Clonostachys and allied genera in Bionectriaceae 111 2 reniformis differs from C. viticola in producing longer primary conidiophore (stipe 40–150 μm, penicilli 40–120 μm in C. reniformis vs 70.5 ± 17.9 μm in C. viticola). Clonostachys reniformis can be distinguished from C. swieteniae by its shorter and narrower stipes of secondary conidiophores (20–70 × 2.0–4.5 vs 130–200 × 5.0–8.0 μm), and curved conidia with clearly laterally displaced hila, while hila are typically central in C. swieteniae (Perera et al. 2020). Clonostachys rosea (Link) Schroers et al., Mycologia 91: 369. 1999. Basionym: Penicillium roseum Link, Mag. Gesell. Naturf. Freunde, Berlin 7: 37. 1816. Synonyms: Gliocladium roseum Bainier, Bull. Soc. Mycol. Fr. 23: 111. 1907. Clonostachys araucaria var. confusa Pinkerton, Ann. Mo. Bot. Gdn 23: 44. 1936. Gliocladium aureum Rader, Phytopathology 38: 450. 1948. Gliocladium verticillioides (G.A. Newton) Pidoplitschka, Sugar Ind. Sci. Notes Kieff 10: 365. 1930. Nectria aureofulva Cooke & Ellis, Grevillea 7: 8. 1878. Bionectria aureofulva (Cooke & Ellis) Schroers & Samuels, Z. Mykol. 63: 153. 1997. Nectria gliocladioides Smalley & H.N. Hansen, Mycologia 49: 533. 1957. Verticillium intertextum I. Isaac & R.R. Davies, Trans. Br. Mycol. Soc. 38: 155. 1955. Clonostachys araucaria Corda, Pracht-Fl. Eur. Schimmelbild.: 31. 1839. Stachylidium araucaria (Corda) Bonord., Handb. Allgem. Mykol. (Stuttgart): 110. 1851. Verticillium pulverulentum Gouw., Meded. Phytopath. Labor. Willie Commelin Scholten Baarn 8: 55. 1924. Verticillium foexii J.F.H. Beyma, Meded. Phytopath. Labor. Willie Commelin Scholten Baarn 12: 31. 1928. Clonostachys gneti Oudem., Verslag. Meded. K. Akad. Wetensch., Afd. Natuurk., ser. 3 7: 321. 1890. Clonostachys populi Harz, Bull. Soc. Imp. Nat. Moscou 44: 116. 1871. Clonostachyopsis populi (Harz) Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 116: 149. 1907. Nectria phyllostachydis Hara [as ‘Nectoria’], Bot. Mag., Tokyo 27: 247. 1913. Gliocladium cholodnyi Pidopl., Fungus flora of coarse fodder: 196. 1953 Dendrodochium strictum D. Sacc., Atti Soc. Veneto-Trent. Sci. Nat. 2: 29. 1896. Verticillium epimyces Berk. & Broome, Ann. Mag. Nat. Hist., Ser. 27: 102. 1851. Torula rosea Preuss, in Sturm, Deutschl. Fl., 3 Abt. (Pilze Deutschl.) 6: 25. 1848. Oospora rosea (Preuss) Sacc. & Voglino, in Saccardo, Syll. Fung. (Abellini) 4: 18. 1886. Alysidium roseum (Preuss) Kuntze, Revis. gen. pl. (Leipzig) 3: 442.1898. Clonostachys populi var. aesculi Oudem., Ned. kruidk. Archf, 3 sér. 2: 1121.1904. Clonostachys araucaria var. compacta Preuss, Linnaea 25: 727. 1853. Verticillium stigmatellum Berk. & M.A. Curtis, in Berkeley, Grevillea 3: 110. 1875. Chapter 2 112 Dendrodochium densipes Sacc. & Ellis, in Ellis & Everhart, J. Mycol. 4: 117. 1888. Nectria congesta Sacc., Michelia 2: 256. 1881. Dialonectria congesta (Sacc.) Cooke, Grevillea 12: 110. 1884. Sphaeria ochroleuca Schwein., Trans. Am. phil. Soc., New Series 4: 204. 1832 (1834). Nectria ochroleuca (Schwein.) Berk., Grevillea 4: 16. 1875. Bionectria ochroleuca (Schwein.) Schroers & Samuels, Z. Mykol. 63: 151. 1997. Clonostachys aranearum Wan H. Chen, et al., Mycosystema 35: 1063. 2016. Description and illustration: Schroers (2001). Typus: Netherlands, soil, on buried sclerotia of Sclerotinia minor, date unknown, A. van Zaayen & W. Gams (neotype CBS H-7917, ex-type culture CBS 710.86). Additional material studied: China, Guizhou, Qianlingshan Park, spider, Jun. 2016, W.H. Chen, culture QLS0625clo (type of Clonostachys aranearum). France, rotten cardboard, date unknown, G. Bainier, culture CBS 100502 (type of Gliocladium roseum Bainier). USA, on decaying bulb of Lilium auratum, Jan. 1955, E.B. Smalley, culture CBS 194.57 (ex-type strains of Nectria gliocladioides Smalley & Hansen); New York, causing lesions on stored carrot roots, date unknown, W.E. Rader, culture CBS 226.48 (ex-type strain of Gliocladium aureum). Notes: Clonostachys rosea is the most commonly isolated species in the genus, with complex taxonomical and nomenclatural history (Schroers 2001). Based on our phylogenetic analyses, the recently described C. aranearum (Chen et al. 2016) is conspecific with C. rosea and included as synonym of C. rosea. Clonostachys vacuolata Lin Zhao, Crous & Schroers, sp. nov. MycoBank MB 848494. Fig. 32. Etymology: Name refers to vacuoles formed by this species inside phialides. Typus: Venezuela, Edo. Miranda: Parque Nacional Guatopo, trail between Agua Blanca and La Cruceta, 10°03’N, 66°26’W N, 500–600 m alt., bark, 27–30 Nov. 1990, G.J. Samuels, B. Hein & S.M. Huhndorf, VE-7664 (holotype designated here CBS H-25143, ex-type living culture CBS 191.93). Sexual morph from natural specimen (not shown). Asci 48–74.4 × 9.5–16.6 μm. Ascospores 1-septate, hyaline, striate, ellipsoid, 13.2–14.7–17.6 × 4.4–5.4–6.3 μm (n=23). Asexual morph. Conidiophores monomorphic, densely aggregated, confluent, formed in pustules, not sporodochial, arising from the agar surface or sparse aerial mycelium, branches Revising Clonostachys and allied genera in Bionectriaceae 113 2 divergent, bito quaterverticillate, phialides somewhat divergent; stipe 10–55 μm long, 1.8–3.8 μm wide at base; penicilli 20–65 μm high, up to 70 μm wide, frequently higher than the length of the stipe; terminal phialides generally in whorls of 2–5, straight or slightly curved, cylindrical, or flask-shaped, with vacuoles, widest in the lower third or middle, slightly tapering in the upper part towards the tip, collarette absent, (7.0–)9.4–21.8(–25.7) μm long, (1.5–)1.7–2.4(–2.6) μm wide at base, (2.1–)2.2–2.9(–3.0) μm at widest point, Fig. 32. Clonostachys vacuolata (ex-type CBS 191.93). A–C. Colonies on OA, PDA and SNA after 7 d at 25 °C. D–G. Conidiophores. H, I. Conidia. Scale bars = 10 μm. Chapter 2 114 (1.0–)1.1–1.5(–1.6) μm wide near aperture (n = 100); intercalary phialides rare, formed below whorls of terminal phialides. Conidia aseptate, hyaline, smooth, ellipsoid to obovoid, straight, both ends broadly rounded, without visible hilum, (5.1–)5.4–6.8(–7.7) × (2.5– )2.7–3.1(–3.3) (av. 6.0 × 2.9 μm, n = 100), arranged in imbricate chains that may collapse into slimy masses. Culture characteristics: Colonies on OA reaching 37–40 mm diam. after 7 d at 25 °C in darkness, with entire margin, aerial mycelium sparsely developed, felty, finely to coarsely granular, yellowish white, reverse concolourous. Colonies on PDA reaching 38–45 mm diam., with entire margin, and aerial mycelium moderate, felty, finely to coarsely granular, pale yellow, reverse concolourous. Colonies on SNA reaching 35–37 mm diam., with entire margin, aerial mycelium sparse, felty, whitish, reverse concolourous. Notes: Clonostachys vacuolata clusters together with C. longiphialidica, C. pallens, C. venezuelae and C. ellipsoidea (Figs 1, 2). It can be morphologically distinguished from C. longiphialidica by producing shorter conidia [(5.1–)5.4–6.8(–7.7) μm vs (6.3–)6.7–8.5(–9.3) μm]. Clonostachys vacuolata differs from C. ellipsoidea in producing longer and narrower conidia (av. 6.0 × 2.9 μm vs av. 5.3 × 3.5 μm). A morphological comparison with C. vacuolata and C. pallens is difficult, because C. palllens was only described based on a sexual morph (Forin et al. 2020), while we observed only asexual morph characters in culture. Schroers (2001) filed the specimen incorrectly under C. grammicospora, as the longer ascospores distinguish VE-7664 from this species. Sexual and asexual morphology suggest classification of C. vacuolata in subgen. Zebrinella. Clonostachys venezuelae Lin Zhao, Crous & Schroers, sp. nov. MycoBank MB 848495. Etymology: Named after the country where the fungus was collected, Venezuela. Typus: Venezuela, Edo Merida, 7 km NE of Merida, ca 4 km inside San Javier del Valle resort, 24 Jul. 1971, K.P. Dumont & G.J. Samuels (holotype designated here CBS H-25240, ex-type living culture CBS 107.87); specimen VE 2865 (NY), culture C.T.R. 71-349. Sexual morph known from natural specimen (not shown). Asci 63.0–90.0 × 10.0–16.0 μm (n = 14). Ascospores 1-septate, hyaline, striate, ellipsoid, (16.0–)17.9(–21.0) × 5.0–6.1– 7.0 μm (n = 22). Asexual morph (not shown), from dried CMD culture, obtained from isolated ascospores. Conidia (8.3–)13.1(–18.8) × (3.8–)5.5(–6.8) μm (fide Schroers) or (5.0– )10.3(–18.0) × (3.0–)5.0(–7.0) μm (fide Samuels). Deposited CBS cultures not sporulating. Clonostachys venezuelae differs from its closest phylogenetic neighbours C. pallens and C. ellipsoidea by unique nucleotide substitutions and indels in the five investigated loci Revising Clonostachys and allied genera in Bionectriaceae 115 2 (see direct sequence comparisons deposited at doi: 10.6084/m9.figshare.22894592): C. venezuelae and C. pallens: ITS position 24 (C), 43 (C), 44 (T), 45 (A, insertion), 52 (C), 53 (gap), 67 (gap), 111 (C), 142 (G), 143 (A), 149(C), 153 (C), 157 (C), 158 (C), 162 (gap), 171 (C), 192 (C), 207 (A, insertion). C. venezuelae and C. ellipsoidea: ITS position 44 (T, insertion), 52 (C), 67 (gap), 69 (A), 111 (C), 143 (A), 150 (T), 153 (C), 158 (C), 162 (gap), 171 (C), 172 (T), 191 (C), 200 (A), 207 (A, insertion), 277 (T), 359 (C), 403 (T), 429 (A), 430 (G), 431 (C), 494 (C), 496 (A), 516 (G), 519 (C), 522 (C), 548 (G), 552 (C, insertion); LSU position 643 (A), 704 (C), 987 (A), 990 (A), 991 (G), 1005 (T), 1007 (T), 1008 (C), 1042 (T), 1045 (T), 1065 (C); RPB2 position 1398 (T), 1407 (C), 1422 (T), 1493 (G), 1599 (A), 1614 (T), 1620 (T), 1650 (C), 1701 (C), 1710 (G), 1719 (A), 1743 (G), 1779 (A), 1782 (C), 1788 (T), 1803 (A), 1804 (C), 1809 (C), 1812 (A), 1833 (C), 1862 (C), 1870 (C), 1900 (G), 1916 (T), 2002 (C), 2011 (G), 2047 (T), 2105 (C), 2126 (C); TEF1 position 2156 (G), 2165 (T), 2174(C), 2180 (C), 2186 (T), 2198 (C), 2255 (T), 2375 (T), 2391 (A), 2392 (A), 2393 (G), 2414 (C), 2416 (G), 2426 (T), 2432 (C), 2447 (T), 2454 (T), 2468 (T), 2469 (C), 2591 (C), 2528 (C), 2585 (T), 2645 (C), 2652 (C), 2748 (T), 2749 (T), 2752 (C), 2840 (C), 2846 (T), 2914(A); TUB2 position 3196 (A), 3199 (C), 3205–3206 (gap), 3207 (A), 3209 (gap), 3219 (G), 3223 (C), 3225 (C), 3236 (A), 3241 (G), 3246 (G), 3249 (T), 3250 (T), 3252 (A), 3260 (T), 3268 (A), 3282 (T), 3356 (C), 3416 (C), 3620 (C), 3684 (A), 3693 (T), 3705 (C), 3744 (C), 3816 (C). Notes: The CBS culture of C. venezuelae does not sporulate anymore, but the phylogenetic analyses show no similarity of this culture with other sequenced cultures (Figs 1, 2). However, the asexual morph is known from a dried culture filed together with specimen VE 2865 studied by G.J. Samuels (see Samuels 1988b) and Schroers (2001). Both filed the specimen under C. (Nectria) subquaternata. Sexual and asexual morphology suggest classification of C. venezuelae in subgen. Zebrinella. DISCUSSION In this study, we investigated 420 strains identified as Clonostachys and allied genera based on morphological characters and phylogenetic analyses. The current study presents the largest sampling of Clonostachys ever subjected to multi-locus sequencing analyses and provides a comprehensive phylogenetic backbone and framework for future studies of Clonostachys. Most cultures examined previously by Schroers et al. (1999b) and Schroers (2000, 2001) were incorporated and newly compared with so far unstudied isolates. Based on phylogenetic analyses of five loci, ITS, LSU, RPB2, TEF1, TUB2, and morphological characters, a rich set of taxa were identified as new species of Clonostachys, Mycocitrus, Nectriopsis, and Sesquicillium (Fig. 1) and our data support that they all belong to the Bionectriaceae. Sesquicillium microsporum differs from other Sesquicillium species in size Chapter 2 116 ranges of phialides and conidia (Samuels 1989) and a myxomyceticolous lifestyle (Rogerson & Stephenson 1993). Our phylogenetic analyses (Fig. 1) support an earlier hypothesis that S. microsporum is a species of Nectriopsis (Schroers 2001), which accommodates also other myxomyceticolous species (Samuels 1988a). We clarified that Clonostachys accommodates the subgenera Astromata, Bionectria, Myronectria and Zebrinella with 19 new and 49 known species, while the genus Sesquicillium, with three new species and eight new combinations was resurrected for subgenera Epiphloea and Uniparietina. Although the purpose of describing subgenera in Clonostachys/Bionectria was to delineate groups of species with similar morphological characters, Schroers (2001) reported that some but not all subgenera are monoor paraphyletic based on ITS and TUB2 sequences. In the present study, the subgenus Astromata (Clade V in Fig. 1) is comprised of six species, including known species C. epichloe, C. eriocamporesii, C. miodochialis and C. oligospora, which agrees with the suppositions in the previous study of Schroers (2001), and new species C. fujianensis and C. obovatispora. Most species of this group form perithecia either directly on their substrata such as fruiting bodies of other fungi or on clearly reduced stromata, sporodochial asexual morphs and greenish pigmented conidial masses, and conidia with a somewhat laterally protruding hilum, that results in a somewhat clavate spore shape (Schroers 2001). Within subgenus Bionectria (Clade VI in Fig. 1), the known species C. compactiuscula, C. divergens, C. ralfsii, C. rogersoniana and C. samuelsii and the new species C. bambusae, C. cylindrica, C. hongkongensis and C. penicillata accumulate numerous nucleotide differences in sequences (Figs 1, 2) when compared to others. All these species cluster outside a wellsupported clade comprising the above discussed core group with homogeneous species of the subgenus Bionectria characterised by dimorphic conidiophores (Fig. 1: 90 % / 99 % / 1; Fig. 2: 92 % / 98 % / 0.96). Also see the discussion under the taxonomic treatment of Clonostachys above. The subgenus Myronectria (Clade III in Fig. 1), comprising two species, C. pityrodes and C. buxicola, is fully supported (Fig. 1: 100 % / 100 % / 1; Fig. 2: 100 % / 100 % / 1). It accommodates an individual branch within the supported Clonostachys clade and is unrelated to the other subgenera. Schroers (2001) erected subgenera Myronectria and Astromata due to the circumstance that they produce synnematous conidiophores and rather dark greenish pigmented conidial masses. Clonostachys pityrodes is also the only Clonostachys species forming somewhat curved, broadly rounded, and comparatively large ascospores. The subgenus Zebrinella forms a monophyletic lineage in Clonostachys (Clade IV in Fig. 1: 93 % / 98 % / 1). Our phylogenetic analyses agree well with the previous study of Schroers (2001), who placed C. chlorina, C. grammicospora, C. grammicosporopsis, C. intermedia, Revising Clonostachys and allied genera in Bionectriaceae 117 2 C. levigata, C. lucifer and C. subquaternata into the subgenus Zebrinella. The subgenus, is, however, also well-supported on the basis of morphological characters. Clearly striate ascospores and perithecial walls showing two regions distinguish it from species of the subgenus Bionectria typically forming warted ascospores and perithecial walls consisting of three regions. The subgenus Epiphloea is shown to be polyphyletic in the present study (Clades labelled I in Fig. 1), with the majority of species now accepted in Sesquicillium, while the sesquicillium-like Clonostachys setosa is accepted in Clonostachys sensu stricto. The subgenus Uniparietina (Clade II in Fig. 1) is represented by Sesquicillium buxi in the present study. Species of Sesquicillium are characterised by macronematous conidiophores that form typically one, rarely two, intercalary phialides just below a single terminal phialide in their penicilli. They should be distinguished from intercalary phialides typically in micronematous conidiophores that look like hyphal cells possessing a lateral conidiogenous peg, for example, in some Acremonium (Gams 1971) and Nectria sensu stricto species (Seifert 1985). This revision, however, shows that high numbers of intercalary phialides are not only formed by species of Sesquicillium but also by C. setosa and Clonostachys sp. CBS 496.90, both classified therefore in subgenus Epiphloea by Schroers (2001). In addition, there are also quite many Clonostachys species producing intercalary phialides at least sporadically [see C. australiana, C. longiphialidica, C. vacuolata, C. ellipsoidea, C. penicillata (this study), C. agarwalii, C. verrucispora, C. compactiuscula, C. rogersoniana, C. grammicospora, C. levigata, and C. chlorina (Schroers 2001)]. It is thus the occurrence of intercalary phialides that link the genera Clonostachys and Sesquicillium morphologically (see also Schroers 2000: fig. 5). Because Clonostachys and Sesquicillium are phylogenetically closely related sister genera (Fig. 1) it is not far-fetched assuming that their intercalary phialides are of homologous nature and perhaps plesiomorphic. The ancestor of these two genera may have possessed conidiophores forming solitary intercalary phialides below terminal phialides. Recognition of Sesquicillium distinguishes species typically forming a reduced perithecial stroma superficially on plant tissue from species in Clonostachys often forming well-developed, through bark erumpent stromata. Also, the asexual morphs, when observed on the natural substratum, are typically mononematous and formed superficially in Sesquicillium, while they are often sporodochial and formed on stromata in Clonostachys. Sesquicillium accommodates a set of species that inhabit leaves, while this lifestyle is rarely seen in Clonostachys, where species may occur as endophytes in woody hosts and subcortical colonisers in recently dead trees, or on fungal hosts associating woody plants. It is possible that the ability to produce stromata erumpent through bark may have evolved in woody hosts (Clonostachys), while stromata were ecologically not required for Chapter 2 118 the superficial lifestyle on plant tissues including leaves (Sesquicillium). However, Bionectria vesiculosa (Luo & Zhuang 2010), here confirmed on the basis of only available nrDNA sequences as a species of Clonostachys, clearly contradicts the concept mentioned above. The sexual morph of this species is astromatous, formed superficially on leaves, and, remarkably, its perithecial wall region consists of a single region only. A similar situation is encountered in Sesquicillium buxi, whose sexual morph consists of astromatous perithecia and a single perithecial wall region (Schroers 2001: fig. 96), nearly indistinguishable from C. vesiculosa (Luo & Zhuang 2010: fig. 1). While it was the asexual morph linking Nectriella coronata first to Sesquicillium (Gams 1968) and then to Clonostachys (Schroers 2000: fig. 5; Schroers 2001), ITS sequences supported classification of C. vesiculosa in Clonostachys (Luo & Zhuang 2010; Supplementary Fig. S1), while LSU places it in a unresolved polytomy among Sesquicillium and Clonostachys (Supplementary Fig. S2). Occurrence of superficially formed perithecia with a simple perithecial wall in both Sesquicillium and Clonostachys may allow hypothesising that these character patterns are (i) plesiomorphic and that (ii) also the common ancestor of Sesquicillium and Clonostachys formed perithecia superficially on plant tissue, perhaps on leaves. Most importantly, however, this concept allows hypothesising that (iii) a diversification of morphs, including perithecial wall anatomies and perithecial wall and stroma interfaces occurred then independently within Sesquicillium and Clonostachys and (iv) this morphological diversification allowed occupation of woody host-related ecological niches and perhaps even mycoparasitism, especially in Clonostachys. Species of Clonostachys are widely distributed all over the world, with the highest known species diversity occurring in tropical regions. These species are commonly found in soils, litter, and dead plant substrata as saprotrophs. They have also been reported as endophytes and epiphytes of living plants (Torcato et al. 2020). Destructive mycoparasitism is especially well documented for C. rosea and a mycophilic habit was inferred for several Clonostachys species that formed their (a)sexual morph structures just on top of other wood associating fungi (Samuels 1976, Schroers et al. 1999b, Schroers 2001). However, while C. rosea has been exhaustively studied (see below), hardly any data are available for the many other species of Clonostachys and Sesquicillium. Sporadic reports have also emphasised parasitic interactions of C. rosea with myxomycetes, nematodes, ticks, molluscs, and leafhoppers (reviewed by Schroers 2001, Toledo et al. 2006, Zhang et al. 2008). However, these reports may rather illustrate the opportunistic nature of this species. Clonostachys rosea was reported as an aggressive mycoparasite in the late 1950s (Barnett & Lilly 1962), and initial attempts to use it for biological control of plant diseases soon followed (Shigo 1958). Since then, there has been a wealth of new knowledge emerging concerning the ecology, physiology and genetics of C. rosea, and its applied use as a biological control agent (BCA) including its formulation, application strategy, efficiency Revising Clonostachys and allied genera in Bionectriaceae 119 2 and safety (Jensen et al. 2022). The biocontrol mechanisms of C. rosea against plant pathogenic fungi are primarily attributed to direct parasitism, secretion of fungal cell wall degrading enzymes, production of secondary metabolites such as antibiotics and toxins, and induction of plant resistance (Sun et al. 2020). Although most reports of biological control of plant diseases involves the species C. rosea, there is evidence to suggest that certain strains from other, closely related species, also possess biocontrol properties, such as C. byssicola, C. chloroleuca, C. rhizophaga and C. solani (García et al. 2003, Krauss et al. 2013, Sun et al. 2017, Broberg et al. 2021). Biological control of plant pathogens via microbial antagonists is one promising component in future disease control strategies (Karlsson et al. 2015). As large-scale genomic sequencing becomes economically viable, the impact of single nucleotide polymorphisms (SNPs) on biocontrol-associated phenotypes can be easily studied across entire genomes of fungal populations. Recently, the genome assemblies of four C. rosea strains have been published (Karlsson et al. 2015, Sun et al. 2015, Liu et al. 2016, Broberg et al. 2018, Wang et al. 2021). The available genome resources are valuable for identifying biocontrol-related genes what will improve our understanding of the biological control ability of C. rosea and related species. The present study should serve as phylogenetic backbone for future taxonomic studies of Clonostachys. Further studies are presently underway to generate full genome sequences of the species studied here in an attempt to identify additional taxa that have biocontrol properties of potential interest to industry. ACKNOWLEDGEMENTS We would like to thank the China Scholarship Council (CSC) for financial support to L.Z. (CSC student number: 202006510014) and the Slovenian Research Agency (ARRS) for financial support to H.-J.S. (grant no. P4-0072; Agrobiodiversity program). The authors are grateful to Dr Gary J. Samuels, who collected most of the holomorphs included in this study. 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The genus Pelonectriella, with a note on bambusicolous Hypocreales with large persistent stroma. Bulletin of the National Science Museum, Tokyo 11: 179–184. Forin N, Vizzini A, Nigris S, et al. (2020). Illuminating type collections of nectriaceous fungi in Saccardo’s fungarium. Persoonia 45: 221–249. Gams W (1968). Die systematische Stellung der Schimmelpilze Fusidium buxi und Verticillium candelabrum. Acta Botanica Neerlandica 17: 455–460. Gams W (1971). Cephalosporium-Artige Schimmelpilze (Hyphomycetes). Stuttgart: Gustav Fischer Verlag. Garcıa RAM, Ten Hoopen GM, Kass DCJ, et al. (2003). Evaluation of mycoparasites as biocontrol agents of Rosellinia root rot in cocoa. Biological Control 27: 210–227. Giraldo A, Hernández-Restrepo M, Crous PW (2019). New plectosphaerellaceous species from Dutch garden soil. Mycological Progress 18: 1135–1154. Bionectriaceae: a poorly known family of hypocrealean fungi with major commercial potential This chapter was published in Studies in Mycology Zhao L, Groenewald JZ, Hou LW, Summerbell RC, Crous PW (2025). Bionectriaceae: a poorly known family of hypocrealean fungi with major commercial potential. Studies in Mycology 111: 115–198. DOI: https://doi.org/10.3114/sim.2025.111.04 Chapter 3 128 Abstract: The ascomycete family Bionectriaceae (Hypocreales) contains cosmopolitan species distributed throughout a broad range of environments, mainly occurring in terrestrial and freshwater ecosystems, with a less frequent occurrence in marine habitats. Members of the family are commonly used in industrial, pharmaceutical, and commercial applications. Applications utilise biodegraders and biocontrol agents, while certain taxa serve as a rich source of bioactive secondary metabolites. In recent years, several studies have proposed new taxonomic concepts within Bionectriaceae based on multigene phylogenetic inference. However, the status of several genera remains controversial or unclear, and many need to be re-collected and subjected to molecular analysis. The present study aims to improve our understanding of Bionectriaceae by re-examining CBS culture collection strains preliminarily identified as taxa within this family. Morphological and molecular phylogenetic analyses are based on alignments of the nuclear ribosomal subunits consisting of the internal transcribed spacer regions and intervening 5.8S nrDNA (ITS), as well as partial sequences for the 28S large subunit (LSU) nrDNA. Additional regions within protein-encoding genes were used, including the DNA-directed RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-alpha (TEF1) regions. The sequences generated were used to reconstruct a phylogenetic backbone of the family Bionectriaceae, and to delineate lineages and generic boundaries within it. Based on these results, seven new genera, 35 new species, and nine new combinations are proposed. A robustly supported phylogenetic framework is provided for Bionectriaceae, resolving 352 species and 50 well-supported genera. This study provides a solid foundation for more indepth future studies on taxa in the family. Key words: Bionectriaceae, industry, multi-locus phylogeny, new taxa, pharmaceutics, taxonomy. Taxonomic novelties: New genera: Clavatomyces Lin Zhao & Crous, Collarimyces Lin Zhao & Crous, Vitreipilata Lin Zhao & Crous, Parageonectria Lin Zhao & Crous, Physaromyces Lin Zhao & Crous, Smyrniomyces Lin Zhao & Crous, Urticomyces Lin Zhao & Crous. New species: Acremonium paramultiramosum Lin Zhao & Crous, Clavatomyces prestoeae Lin Zhao & Crous, Clonostachys novocaledonica Lin Zhao & Crous, Clonostachys tropica Lin Zhao & Crous, Collarimyces guttiformis Lin Zhao & Crous, Emericellopsis mexicana Lin Zhao & Crous, Emericellopsis proliferata Lin Zhao & Crous, Emericellopsis soli Lin Zhao & Crous, Fusariella triangulispora Lin Zhao & Crous, Geonectria alni Lin Zhao & Crous, Geonectria quercus Lin Zhao & Crous, Geosmithia cupressina V. Meshram et al., Geosmithia magnispora Lin Zhao & Crous, Gliomastix olivacea Lin Zhao & Crous, Hydropisphaera armeniaca Lin Zhao & Crous, Hydropisphaera gossypina Lin Zhao & Crous, Hydropisphaera martinicensis Lin Zhao & Crous, Hydropisphaera solani Lin Zhao, L.W. Hou & Crous, Lasionectria chondroidea Lin Zhao & Crous, Lasionectria phormii Lin Zhao, L.W. Hou & Crous, Lasionectriopsis stereicola Bionectriaceae 129 3 Lin Zhao & Crous, Nectriopsis cribrariae Lin Zhao & Crous, Nectriopsis floccosa Lin Zhao & Crous, Ovicillium theobromae Lin Zhao & Crous, Paracylindrocarpon jigongshanense Lin Zhao & Crous, Paracylindrocarpon spartinae Lin Zhao & Crous, Parageonectria arachispora Lin Zhao & Crous, Paragliomastix venezuelana Lin Zhao & Crous, Physaromyces sterilis Lin Zhao & Crous, Protocreopsis chlamydospora Lin Zhao & Crous, Protocreopsis gallica Lin Zhao & Crous, Roumegueriella echinulata Lin Zhao & Crous, Sesquicillium pouteriae Lin Zhao & Crous, Sesquicillium thailandense Lin Zhao & Crous, Smyrniomyces setaceus Lin Zhao & Crous. New combinations: Clavatomyces korfii (Lechat & J. Fourn.) Lin Zhao & Crous, Vitreipilata cirsii (Lechat & J. Fourn) Lin Zhao & Crous, Protocreopsis loweniae (Flakus et al.) Lin Zhao & Crous, Protocreopsis vulpina (Cooke) Lin Zhao & Crous, Proxiovicillium capsici (S.Q. Tong & Y.J. Wu) Lin Zhao & Crous, Sesquicillium shanghaiense (Zhang et al.) Lin Zhao & Crous, Urticomyces pseudoarenulus (Lechat & J. Fourn) Lin Zhao & Crous, Verruciconidia guizhouensis (S.Q. Tong & Y.J. Wu) Lin Zhao & Crous, Verruciconidia saulensis (Lechat & J. Fourn.) Lin Zhao & Crous. New synonyms: Clonostachys aquatica D.F. Bao et al., Emericellopsis microspora Backus & Orpurt. INTRODUCTION The family Bionectriaceae (Hypocreales) was established by Rossman et al. (1999) to accommodate 26 genera, including five cleistothecial genera. Many years later, Wijayawardene et al. (2020, 2022) accepted 47 genera within this family. Subsequently, Trovão et al. (2022) introduced a new genus into the Bionectriaceae, Circumfusicillium, which was isolated from the limestone wall of the Machado de Castro National Museum in Portugal. Perera et al. (2023) introduced three new families based on genera removed from Bionectriaceae, namely Ijuhyaceae, accommodating Ijuhya and Kallichroma, Stromatonectriaceae, accommodating Stromatonectria, and Xanthonectriaceae, accommodating Xanthonectria and Bullanockia. Based on phylogenetic analyses, Globonectria and Septofusidium were transferred to Nectriaceae and Bionectriaceae, respectively, while Trichonectria was left in Hypocreales incertae sedis. Amphichorda, previously placed in Cordycipitaceae, was also transferred to Bionectriaceae as a monophyletic lineage (Guerra-Mateo et al. 2023). Hou et al. (2023) established 10 new genera within Bionectriaceae, namely Alloacremonium, Gossypinidium, Monohydropisphaera, Musananaesporium, Paragliomastix, Proliferophialis, Proxiovicillium, Ramosiphorum, Waltergamsia, and Verruciconidia. The genera Mycoarachis and Nigrosabulum, placed in Bionectriaceae, were considered as synonyms of Hapsidospora, with Heleococcum considered a synonym of Hydropisphaera. Moreover, the genera Bulbithecium, Emericellopsis, Fusariella, and Synnemellisia were reinstated in the Bionectriaceae after having been classified as incertae sedis in Hypocreales (Hyde et al. 2020b, Wijayawardene et al. 2020, 2022, Hou et al. 2023). Chapter 3 130 The Bionectriaceae is characterised by producing globose, subglobose to pyriform perithecial, or rarely cleistothecial ascomata, that are generally superficial or immersed, with or without a stroma. Ascomatal colour ranges from white, yellow, or orange to brown or reddish brown, and sometimes to purple to violet, and does not change in KOH or lactic acid. Ascomatal walls consist of 1–3 cell regions, with each region composed of more or less well-defined, 2–4 cell layers. Periphyses and paraphyses are present or absent. Asci are unitunicate, 8-spored (rarely 4-spored), clavate or globose, and do or do not have an apical ring. Ascospores are aseptate to multi-septate, ellipsoid, broadly ellipsoid, or fusoid; walls are hyaline to brown, and may be smooth, striate, spinulose to tuberculate or ornamented with wings or flanges. Asexual morphs are most commonly hyphomycetous, being acremonium-, clonostachys-, gliocladium-, penicillium-, or verticillium-like. Conidiophores are monoor dimorphic, mononematous, sporodochial or synnematous, smooth to finely echinulate, hyaline, or olivaceous brown to brown. Conidiogenous cells are phialidic, cylindrical to flask-shaped, and have conspicuous or inconspicuous periclinal thickening and cylindrical collarettes at conidiogenous loci. Conidia are ellipsoid, cylindrical or fusoid, aseptate to multi-septate, hyaline to greenish, or olivaceous brown to brown; rarely held together in chains and mostly accumulate in watery or slimy, typically lightcoloured masses; walls are smooth or verrucose, sometimes with papillate or truncate ends. Chlamydospores are present or absent (Rossman et al. 1999, Hyde et al. 2020a, b, Perera et al. 2023, Zhao et al. 2023). Members of the Bionectriaceae are cosmopolitan and distributed across a broad range of environments. They consist of fungi with highly diverse nutritional modes, ranging from saprotrophs and endophytes to parasites of insects, plants, or other fungi, as well as lichenicolous fungi (Hyde et al. 2020b, Perera et al. 2023, Hou et al. 2023, Zhao et al. 2023). They mainly occur in terrestrial and freshwater ecosystems, and less frequently in marine habitats. Members of Bionectriaceae are commonly used in industrial, pharmaceutical, agricultural, and commercial applications, with several genera having significant commercial importance (Goswami et al. 2008, Choi et al. 2009, Wicklow & Poling 2009, Sun et al. 2020). Species of Acremonium sensu lato (s. lat.), including several notable ones, are important in agro-forestry, pharmaceutical industries, and biological control. However, data on such findings are difficult to analyse because many isolates cited in the literature bear outdated or unclear names, are misidentified, or cannot be traced to deposited isolates (Summerbell et al. 2018). Despite these challenges, interpretable data indicate that some species play significant roles in producing valuable enzymes and bioactive secondary metabolites. For example, Sarocladium zeae (formerly Acremonium zeae) produces extracellular hemicellulolytic enzymes that hydrolyse cellulose and hemicellulose (Bischoff et al. 2009, de Almeida et al. 2011), and Acremonium egyptiacum (formerly Acremonium sclerotigenum), isolated from industrially contaminated soil, produces penicillin acylase (Jeevan 2017). The identity of the latter was confirmed with an ITS sequence deposited in Bionectriaceae 131 3 GenBank under KX459421. To date, over 600 secondary metabolites with antimicrobial, antifungal, anticancer, immunosuppressive, and enzyme-inhibitory activities have been characterised from Acremonium s. lat. species (Qin et al. 2024). Additionally, some species of Acremonium s. lat. have been used as biocontrol agents of various plant diseases because of their mycoparasitic and antagonistic activities against plant pathogens. For instance, isolates reported but unverified as Acremonium alternatum and Verruciconidia persicina (formerly A. persicinum) have been used as biological control agents to successfully control coconut tar spot in Brazil (Bettiol 1996). Acremonium alternatum, a historically much-misused name (Summerbell et al. 2018), has repeatedly been assigned to isolates classified as biocontrol agents. Isolates given this name have been shown to control Plasmodiophora brassicae causing clubroot disease of Brassicaceae by reducing symptoms and delaying plant senescence (Pal & Gardener 2006, Doan et al. 2010, Jäschke et al. 2010, Auer & Ludwig-Müller 2014, 2015). Gibellulopsis nigrescens (ATCC 60645; Trichosphaeriales, Trichosphaeriaceae), also wrongly identified as Acremonium alternatum, controls powdery mildew on tomato through induced resistance rather than hyperparasitism (Malathrakis 1985, Kasselaki et al. 2006). To clarify taxonomic identities, any other isolates used in such cases must be DNA barcoded or phylogenetically analysed to determine their actual identities. Notably, the isolate CBS 779.69 (= CMI 49137), originally identified as Cephalosporium acremonium, but now recognised as Hapsidospora chrysogena (= Acremonium chrysogenum), is known as a source of bactericidal β-lactam antibiotics, called cephalosporins and widely used in clinical practice (Burton & Abraham 1951, Gams 1971, Hamilton-Miller 2000). Species of Clonostachys are commonly employed as mycoparasites involved in biocontrol, biodegradation, biotransformation, and fermentation. As an excellent mycoparasite, C. rosea demonstrates robust biocontrol capabilities against a wide range of fungal plant pathogens, nematodes, and insects (Møller et al. 2003, Toledo et al. 2006, Cota et al. 2008, Krauss et al. 2013, Muvea et al. 2014, Rodríguez-Martínez et al. 2018). Clonostachys rosea has been considered as biological control agent against Pythium tracheiphilum in Chinese cabbage (Møller et al. 2003), seedborne Alternaria spp. (Jensen et al. 2004), Botrytis aclada on necrotic leaf tips (Yohalem et al. 2004), Botrytis cinerea causing strawberry grey mould (Cota et al. 2008), fusarium head blight of wheat (Hue et al. 2009, Kosawang et al. 2014, Schoneberg et al. 2015, Samsudin et al. 2017), and Sclerotinia sclerotiorum (Sun et al. 2015). Furthermore, C. rosea functions as a biodegrader of plastic waste (Urbanek et al. 2017). Urbanek et al. (2017) discovered that the C. rosea strain 16G, isolated from the Arctic region (Spitsbergen), exhibited the capability to decompose 100 % of starch films and 52.91 % of poly(ε-caprolactone) films within 30 d at 28 °C. This suggests that C. rosea holds promise as a valuable biodegradation agent for breaking down plastic waste. Emericellopsis includes fungi that have a worldwide distribution in terrestrial Chapter 3 132 environments but are most strongly represented in marine environments. Species of this genus are well-known for their bioactive metabolites, serving as a treasure trove for biological activity compounds. The bioactive antimicrobial peptides (AMPs) produced by these fungi, especially peptaibols, exhibit a wide range of biological activities, making them valuable molecules in various biomedical applications, including antibacterial, antifungal, antiviral, antitumor, and cytotoxic effects (Agrawal & Saha 2021, Gonçalves et al. 2021, Kuvarina et al. 2021). Antimicrobial peptides are emerging as promising alternatives to traditional antifungal drugs. The Norine database, launched by Flissi et al. (2020), is dedicated to cataloguing and characterising non-ribosomal peptides synthases (NRPS), including some compounds produced by species of Emericellopsis, namely antiamoebins I–XI from E. salmosynnemata and E. synnematicola (Pandey et al. 1978, Krishnaswamy & Pattabhi 1987, Jaworski & Brückner 2000), bergofungins A–D from E. donezkii (Berg et al. 1996, 1999, Gessmann 2017), emerimicins II, III, IV from E. microspora and E. minima (Argoudelis & Johnson 1974, Inostroza et al. 2018), zervamicines from E. salmosynnemata (Rinehart et al. 1981), and emericellipsins A–E as novel peptaibols from the alkalophilic E. alkalina that have strong antifungal and cytotoxic properties against drug-resistant pathogenic fungi (Kuvarina et al. 2021). Species of Geosmithia are common saprotrophs associated with bark beetles and are known for attacking hardwoods and conifers in forests around the world. Geosmithia have been extensively studied as fungal symbionts of bark beetles. These fungi are adept at colonising trees subcortically, displaying a spectrum of symbiotic relationships with their insect vectors (Kolařík et al. 2011, Kolařík & Hulcr 2023). Several tree diseases are caused by bark beetles that carry Geosmithia species, and these fungi may form discoloured areas around the beetle galleries. Geosmithia morbida was the first species of the genus documented as a plant pathogen (Kolařík et al. 2011). It is associated with the disease known as thousand cankers disease in walnut trees, causing widespread branch dieback and canopy loss and substantial black walnut tree mortality in North America, especially in the western United States (Tisserat et al. 2009, 2011, Kolařík et al. 2011, Hadziabdic et al. 2014a, b). Additionally, the pathogen has also been found in Europe (Montecchio et al. 2014). While Geosmithia is primarily recognised as a pathogen, it also possesses beneficial characteristics such as the production of a variety of useful secondary compounds. Species of Geosmithia have been demonstrated to produce potential antibacterial and antifungal compounds (Deka & Jha 2018, Veselská et al. 2019, Kolařík & Hulcr 2023). Several recent studies have addressed taxonomic concepts within Bionectriaceae based on multi-gene phylogenetic inference (Hou et al. 2023, Perera et al. 2023). However, limited sequence data are presently available for the family, and many genera and species need to be recollected and sequenced to confirm their placement within or exclusion from Bionectriaceae. This study aims to improve our understanding of Bionectriaceae by reexamining strains that were preliminarily identified as belonging to “Bionectriaceae” in Bionectriaceae 133 3 the CBS culture collection of the Westerdijk Fungal Biodiversity Institute, supplemented with additional strains from the working collection of Pedro W. Crous (CPC). We used morphological and molecular phylogenetic analyses based on alignments of the nuclear ribosomal internal transcribed spacer regions and intervening 5.8S nrDNA (ITS) and partial sequences for the 28S large subunit (LSU) nrDNA, partial protein-encoding genes including the DNA-directed RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-alpha (TEF1) gene regions to reconstruct and enhance a phylogenetic backbone to delineate lineages and define generic boundaries within the Bionectriaceae. An additional objective of this study is to assign names to currently undescribed taxa through comprehensive morphological observations and to revisit old species circumscriptions and reassess their taxonomy. MATERIALS AND METHODS Isolates Strains included in this study were obtained from the CBS culture collection and the collection of Pedro W. Crous (CPC) of the Westerdijk Fungal Biodiversity Institute (WI; Utrecht, the Netherlands). All isolates included in this study were preliminarily identified as Bionectriaceae based on morphological characteristics or initial DNA sequence data (Supplementary Table S1). DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from 7–14-d-old fungal colonies growing on oatmeal agar (OA; Crous et al. 2019) at room temperature. Fungal mycelium was scraped from the colony surface with a sterile scalpel and the genomic DNA was extracted using the Wizard® Genomic DNA purification Kit (Promega Corporation, Madison, WI, USA), following the manufacturer’s protocol. Four loci were amplified: the ITS region was amplified using the primer pair ITS5/ITS4 (White et al. 1990); the LSU region was amplified using the primer pair LR0R/LR5 (Vilgalys & Hester 1990, Rehner & Samuels 1994); RPB2 gene was amplified using the primer pair RPB2-5F2/RPB2-7CR (Liu et al. 1999, Sung et al. 2007); and TEF1 gene was amplified using the primer pair EF-983F/EF-2218R (Rehner & Buckley 2005). The PCR amplifications were performed in a total volume of 12.5 μL. For ITS, the PCR mixture contained 7.69 μL of ddH2O, 1.25 μL of EasyTaq Buffer (Bioline, Luckenwalde, Germany), 0.7 μL of dNTPs (40 μm), 0.63 μL of MgCl2(2 mM), 0.63 μL of DMSO (1 μg/μL), 0.25 μL of each primer (0.2 μm), 0.1 μL of Taq DNA polymerase (Bioline) and 1 μL of genomic DNA; for LSU the reaction mixture contained 7.06 μL of ddH2O, 1.26 μL of MgCl2 (2 mM), 1.25 μL of EasyTaq Buffer, 0.7 μL of dNTPs (40 μm), 0.63 μL of DMSO (1 μg/μL), 0.25 μL of each primer (0.2 μm), 0.1 μL of Taq DNA polymerase and 1 μL of genomic DNA; for RPB2 Chapter 3 134 the reaction mixture contained 7.90 μL of ddH2O, 1.25 μL of EasyTaq Buffer, 0.75 μL of dNTPs (40 μm), 0.50 μL of MgCl2 (2 mM), 0.50 μL of BSA (1 μg/μL), 0.25 μL of each primer (0.2 μm), 0.1 μL of Taq DNA polymerase and 1 μL of genomic DNA; for TEF1 the reaction mixture contained 8.02 μL of ddH2O, 1.25 μL of EasyTaq Buffer, 0.50 μL of dNTPs (40 μm), 0.50 μL of MgCl2 (2 mM), 0.63 μL of DMSO (1 μg/μL), 0.25 μL of each primer (0.2 μm), 0.1 μL of Taq DNA polymerase and 1 μL of genomic DNA. The amplification conditions of ITS, LSU, RPB2, and TEF1 were set as follows: an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 48 °C for ITS and LSU or 56 °C for RPB2 and TEF1 for 45 s, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 7 min. The PCR products were subsequently purified and sequenced using an Applied Biosystems, Hitachi 3730xl DNA analyser (Applied Biosystems Inc., Foster City, California, USA). The consensus sequences were assembled from forward and reverse sequences using Geneious Prime v. 2022.0.2 (http://www. geneious.com, Kearse et al. 2012). Novel sequences generated in this study were deposited in GenBank (http://www. ncbi.nlm.nih.gov, Table S1). Phylogenetic analyses Alignments for individual loci were generated with the online version of MAFFT v. 7 and by using default settings (https://mafft.cbrc.jp/alignment/server/index.html; Katoh et al. 2019), and then manually checked and corrected in MEGA v. 7.0.21 (Kumar et al. 2016). Individual alignments were concatenated using SequenceMatrix v. 1.9 (Vaidya et al. 2011). Phylogenetic analyses were based on Maximum-likelihood (ML) and Bayesian analyses (BA). The ML analyses were conducted using the CIPRES Science Gateway portal v. 3.3 (https:// www.phylo.org/; Miller et al. 2012) and RAxML-HPC2 on ACCESS v. 8.2.12 (Stamatakis 2014) with default GTRCAT model and 1000 rapid bootstrap replications. Additional ML analyses were constructed with IQ-TREE v. 2.1.3 (Nguyen et al. 2015, Minh et al. 2020) and by applying UFBoot2 bootstrapping (ultra-fast bootstrapping, > 95 % is significant) for estimation of branch support (Hoang et al. 2018). The most suitable evolutionary model for each partition was estimated using ModelFinder (Kalyaanamoorthy et al. 2017, Minh et al. 2020) as implemented in IQ-TREE. The most suitable nucleotide substitution models for each partition under the Bayesian Inference Criterion (BIC) were selected using IQ-TREE. For the Bayesian analysis (BA), the best nucleotide substitution model of evolution for each gene partition (ITS, LSU, RPB2, TEF1) were estimated using MrModeltest v. 2.3 under the Akaike Information Criterion (AIC) before the Bayesian analysis (Nylander 2004). The Bayesian analyses were conducted using MrBayes v. 3.2.6 (Ronquist et al. 2012) as described by Hou et al. (2020, 2023). The Markov Chain Monte Carlo sampling (MCMC) analyses was employed with four chains initiated from random tree topologies. These simultaneous Markov chains ran in parallel for 10 M generations, with tree samples taken every 1000 generations. The run was automatically terminated if the average standard deviation of Bionectriaceae 135 3 split frequencies fell below 0.01. The burn-in fraction was set to 0.25, after which the 50 % majority rule consensus trees and posterior probability (PP) values were calculated. The phylogenetic trees were viewed using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/ figtree). The final alignments used for phylogenetic analyses in this study were uploaded to figshare (doi: 10.6084/m9.figshare.28138430). The term “Clade” is applied for accepted lineages in the phylogenetic tree, accommodating a single or multiple taxa. Morphological observations Macroscopic descriptions were recorded from fungal colonies grown on oatmeal agar (OA), malt extract agar (MEA), potato dextrose agar (PDA) (Crous et al. 2019) and synthetic nutrient-poor agar (SNA; Nirenberg 1976). Colony diameters were measured, and characters were recorded after 14 d in the darkness at 25 °C. Colony colours for upper surface and reverse were rated following the colour charts of Rayner (1970). Micro-morphological characters were primarily recorded from 2–5-wk-old colonies on OA under near-UV light at room temperature, but also studied on MEA, PDA, or SNA with autoclaved pine needles. Slide preparations were mounted in clear lactic acid (85–90 %) for the observation of structures of perithecia/cleistothecia, perithecial/cleistothecial walls, asci, ascospores, mycelium, conidiophores, and conidia (Hou et al. 2023). The terminology for sporulation types, such as phalacrogenous, nematogenous, and plectonematogenous, follows Gams (1971). A Nikon AZ100 dissecting microscope, and Nikon Eclipse 80i compound microscope with Differential Interference Contrast (DIC) optics were used for morphological observations. Photomicrographs and measurements were taken with a Nikon DS-Ri2 highdefinition colour digital camera using the Nikon software NIS-Elements D software v. 4.50 (Nikon, Tokyo, Japan). Sections of perithecia were prepared by a Leica CM 1100 freezing cryostat microtome. Taxonomic novelties were deposited in MycoBank (www.MycoBank. org; Crous et al. 2004), and specimens were deposited in the CBS Fungarium, Utrecht, The Netherlands. Abbreviations used for genera in the text are as follows: A. = Acremonium, B. = Bullanockia, C. = Clonostachys, Cl. = Clavatomyces, Col. = Collarimyces, E. = Emericellopsis, F. = Fusariella, G. = Geosmithia, Gl. = Gliomastix, Gn. = Geonectria, H. = Hydropisphaera, I.= Ijuhya, L. = Lasionectria, La. = Lasionectriopsis, N. = Nectriopsis, O. = Ovicillium, Pg. = Parageonectria, Phy. = Physaromyces, Pn. = Paracylindrocarpon, Pr. = Proxiovicillium, Pt. = Protocreopsis, Px. = Paragliomastix, R. = Roumegueriella, S. = Sesquicillium, Sm. = Smyrniomyces, U. = Urticomyces, V. = Verruciconidia, Vit. = Vitreipilata, X. = Xanthonectria. RESULTS Phylogenetic analyses Phylogenetic analyses were conducted using ITS, LSU, RPB2 and TEF1 sequences to Chapter 3 136 Tilachlidium brachiatum CBS 363.97 Flammocladiella aceris CBS 138906T Amphichorda littoralis FMR19404T Bullanockia australis CBS 142093T Proxiovicillium blochii CBS 324.33 Xanthonectria pseudopeziza CBS 140160 Flammocladiella anomiae CBS 142775 Stilbocrea colubrensis CBS 580.73 Hapsidospora globosa CBS 512.70T Amphichorda guana CGMCC 3.17908T Stilbocrea gracilipes CBS 141849 Alloacremonium ferrugineum CBS 102877T Hapsidospora chrysogena CBS 144.62T Bulbithecium hyalosporum CBS 318.91T Hapsidospora globosa CBS110041 Stilbocrea gracilipes CBS 301.96 Hapsidospora flava CBS 596.70T Proxiovicillium capsici SQT01T Stilbocrea sp. CBS 659.83 Hapsidospora scabella CBS 515.70 Proxiovicillium lepidopterorum CBS 101239T Bulbithecium ammophilae CBS 178.78T Stilbocrea walteri CBS 144627T Bulbithecium arxii CBS 737.84T Stilbocrea sp. CBS 532.85 Stilbocrea gracilipes CBS 657.83 Amphichorda coprophila CBS 247.82T Hapsidospora flava CBS 963.87 Stilbocrea gracilipes CBS 142017 Stromatonectria caraganae CBS 127387 Amphichorda cavernicola CGMCC 3.19571T Bulbithecium spinosum CBS 391.66 Amphichorda littoralis FMR19611 Hapsidospora flava CBS 597.70 Bulbithecium truncatum CBS 113718T Proxiovicillium capsici SQT02 Tilachlidium brachiatum CBS 505.67 Stilbocrea sp. CBS 550.84 Stilbocrea colubrensis CBS 134536 Stilbocrea macrostoma CBS 114375 Hapsidospora globosa CBS 481.82 Hapsidospora variabilis CBS 100549T Bulbithecium pinkertoniae CBS 157.70T Bulbithecium spinosum CBS 136.33T Hapsidospora chrysogena CBS 779.69 Bulbithecium pinkertoniae CBS 158.70 Hapsidospora scabella CBS 217.84 Hapsidospora inversa CBS 517.70T Stilbocrea sp. CBS 658.83 Stilbocrea banihashemiana FS1T Bulbithecium spinosum CBS 915.85 Amphichorda felina CBS 648.66 Stromatonectria caraganae CBS 125579 Hapsidospora globosa CBS 416.73 Physaromyces sterilis CBS 139048T Xanthonectria pseudopeziza CBS 141245 Alloacremonium humicola CBS 613.82T Bulbithecium ellipsoideum CBS 993.69T Hapsidospora irregularis CBS 510.70T Amphichorda felina CBS 250.34 Amphichorda guana CGMCC 3.17909 Hapsidospora globosa CBS 268.91 Flammocladiella decora CBS 142776 Hapsidospora globosa CBS 513.70 Hapsidospora scabella CBS 516.70T Hapsidospora chrysogena CBS 401.65 Proxiovicillium blochii CBS 427.93T Stilbocrea banihashemiana Gh093-1 Bulbithecium borodinense CBS 101148T 79/100/1 94/100/0.99 91/100/1 83/100/1 87/99/1 55/96/- 90/100/1 79/100/1 60/95/0.99 97/100/1 94/98/1 87/100/1 99/100/1 97/100/1 79/99/1 90/100/1 90/100/1 77/100/0.99 99/100/1 74/100/0.9 84/100/1 97/100/1 1. Physaromyces 2. Stilbocrea 3. Alloacremonium 4. Amphichorda 5. Hapsidospora 6. Proxiovicillium 7. Bulbithecium Stilbocrea colubrensis CBS 141857T -/94/- 54/-/- Tilachlidiaceae Stromatonectriaceae Xanthonectriaceae Flammocladiellaceae outgroups Bionectriaceae 2× 2× 2× 2× 2× 2× Fig. 1. See legend on following page. Bionectriaceae 143 3 Clonostachys venezuelae CBS 107.87T Clonostachys australiana CBS 135616 Clonostachys oligospora HMAS 290895T Clonostachys australiana CBS 102421T Clonostachys flava CBS 915.97T Clonostachys fusca CBS 996.97 Clonostachys pityrodes CBS 102033T Clonostachys subquaternata CBS 100003T Clonostachys samuelsii CBS 699.97T Clonostachys eriocamporesii MFLUCC 19-0486T Clonostachys grammicospora CBS 209.93T Clonostachys penicillata CBS 653.70 Clonostachys divergens CBS 967.73BT Clonostachys ellipsoidea CBS 175.76T Clonostachys cylindrica CBS 101113T Clonostachys vesiculosa HMAS 183151T Clonostachys rogersoniana CBS 920.97T Clonostachys australiana CBS 102423 Clonostachys levigata CBS 101916T Clonostachys hongkongensis CBS 116542 Clonostachys fujianensis CBS 127474T Clonostachys fusca CBS 101925 Clonostachys miodochialis CBS 997.69T Clonostachys ellipsoidea CBS 102566 Clonostachys aurantiaca CBS 124757T Clonostachys vacuolata CBS 191.93T Clonostachys lucifer CBS 126.87 Clonostachys penicillata CBS 148211 Clonostachys pilosella CLLG19028T Clonostachys tropica CBS 157.96T Clonostachys obovatispora CBS 118752T Clonostachys leucaenae MFLUCC 20-0008T Clonostachys lucifer CBS 100008T Clonostachys rhinolophicola KUMCC 21-0439T Clonostachys chlorina CBS 287.90T Clonostachys intermedia CBS 508.82T Clonostachys buxicola CBS 102419T Clonostachys grammicosporopsis CBS 114.87T Clonostachys fusca CBS 207.93T Clonostachys penicillata CBS 729.87T Clonostachys compactiuscula CBS 913.97T Clonostachys setosa CBS 917.97T Clonostachys longiphialidica CBS 112.87T Clonostachys epichloe CBS 101037T Clonostachys pallens PAD S00004T Clonostachys hongkongensis CBS 115291T 73/99/1 54/95/- -/98/0.99 97/100/1 97/100/1 53/-/0.96 99/100/0.97 95/98/1 84/99/0.94 73/99/1 99/100/1 99/100/1 96/98/1 87/98/1 99/100/1 76/99/1 59/93/1 73/100/- 65/100/0.97 62/100/- 95/100/1 91/100/1 93/100/1 66/100/1 92/100/1 82/100/0.99 99/100/1 89/100/1 99/100/1 21. Clonostachys 68/93/1 -/93/- 57/-/0.93 53/93/- -/-/0.94 Bionectriaceae Fig. 1. (Continued). known species of the genus Sesquicillium (including the generic type species S. buxi), and another species that was recombined into this genus from Clonostachys, S. shanghaiense (basionym: C. shanghaiensis), as well as two novel species, S. pouteriae and S. thailandense. Clade 21 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Clonostachys, which is represented by 69 previously described species (including the generic type species C. rosea), and two novel species, C. novocaledonica and C. tropica. Clade 22 included seven known species of the genus Protocreopsis, and another two species recombined into this genus, Pt. loweniae (basionym: Pronectria loweniae) and Pt. vulpina (syn. Lasionectria vulpina), as well as two novel species, Pt. chlamydospora and Pt. gallica. Clade 23 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Lasionectriopsis, including three known species, La. dentifera, La. germanica, La. pteridii, and one novel species, La. stereicola. Clade 25 (RAxML-BS = 80 %, IQ-TREE- Chapter 3 144 Clonostachys aureofulvella CBS 100980T Protocreopsis loweniae Rodriguez-Flakus 4000T Clonostachys pnagiana CLLG19041T Clonostachys kunmingensis YFCC 898T Clonostachys sp. CBS 136685 Clonostachys viticola CAA 944T Protocreopsis sp. CBS 134534 Clonostachys apocyni CBS 360.78 Protocreopsis finnmarkica CBS 147428T Clonostachys apocyni CBS 130.87 Clonostachys sp. CBS 100005 Protocreopsis finnmarkica CBS 147429 Clonostachys leptoderma HMAS 255834T Clonostachys rosea f. catenulata CBS 154.27T Clonostachys rosea CBS 143541 Clonostachys swieteniae MFLUCC 18-0572T Clonostachys ralfsii CBS 703.97T Protocreopsis gallica CBS 141243 Clonostachys sporodochialis CBS 101921T Clonostachys sp. CPC 45011 Clonostachys palmae CBS 119.87T Protocreopsis vulpina CBS 565.76 Protocreopsis euphorbiae CPC 38896T Protocreopsis gallica CBS 141242 Protocreopsis gallica CBS 141240 Clonostachys pseudochroleuca CBS 187.94T Clonostachys parasporodochialis CBS 192.93T Clonostachys bambusae CBS 139411T Clonostachys moreaui CLL19024T Protocreopsis freycinetiae CBS 573.76T Clonostachys garysamuelsii CBS 123964T Clonostachys reniformis CBS 695.86T Protocreopsis caricicola CBS 110505 Protocreopsis chlamydospora CBS 144254T Protocreopsis rutila CBS 229.70 Clonostachys chongqingensis HMAS 290894T Protocreopsis chlamydospora CBS 141859 Clonostachys artemisiae ZHKUCC 23-0882T Protocreopsis gallica CBS 135079T Protocreopsis pertusa CBS 568.76 Clonostachys capitata CBS 218.93T Protocreopsis sp. CBS 128021 Clonostachys zelandiaenovae CBS 100979T Protocreopsis sp. CBS 141238 Clonostachys pseudostriata CBS 120.87T Protocreopsis rutila CBS 396.66T Clonostachys solani f. nigrovirens CBS 183.30T Clonostachys oblongispora CBS 100285T Protocreopsis sp. CBS 131868 Protocreopsis sp. CBS 141236 Clonostachys agarwalii CBS 533.81T Protocreopsis caricicola CBS 140572T Clonostachys rosea CBS 710.86T Clonostachys krabiensis MFLUCC 16-0254T Clonostachys ambigua PAD S00003T Protocreopsis finnmarkica CBS 147427 Clonostachys chloroleuca CBS 141588T Protocreopsis sp. CBS 142015 Protocreopsis gallica CBS 141241 Clonostachys farinosa CBS 364.78T Clonostachys rhizophaga CBS 202.37T Clonostachys kowhai CBS 461.95T Clonostachys novocaledonica CBS 496.90T Clonostachys moreaui CBS 127881 Protocreopsis phormiicola CBS 567.76T Clonostachys solani CBS 697.88T 94/100/1 98/100/1 90/99/1 98/100/1 64/-/1 70/98/0.94 97/100/0.99 88/100/1 79/100/- -/97/- -/93/0.96 53/97/0.99 86/100/0.97 -/95/0.93 62/99/0.99 58/100/1 76/-/- 95/100/1 92/100/1 80/99/0.93 62/100/1 62/100/0.92 68/99/1 -/98/0.96 66/99/1 99/100/1 84/100/1 94/100/1 89/100/1 97/100/1 65/97/1 71/99/1 22. Protocreopsis 57/90/- 53/-/- -/90/0.94 -/-/0.97 -/-/0.92 21. Clonostachys 2× 2× 2× Bionectriaceae Fig. 1. (Continued). BS = 100 %, BI-PP = 1) accommodated the new genus Clavatomyces, which included Cl. korfii (basionym: Protocreopsis korfii) and a novel species, Cl. prestoeae. Clade 28 (RAxMLBS = 99 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Verruciconidia, Bionectriaceae 145 3 Clavatomyces korfii CBS 138733T Verruciconidia siccicapita CBS 378.70AT Lasionectria antillana CBS 114748 Verruciconidia quercina CBS 183.78 Verruciconidia quercina CBS 355.77 Lasionectriella rubioi CBS 132543 Lasionectria bisepta CBS 752.69 Lasionectria sp. OTU12 Lasionectriella herbicola CBS 140156T Verruciconidia infuscata CBS 100888T Clavatomyces korfii CBS 138710 Lasionectria phormii CBS 102040T Lasionectriopsis germanica CBS 122125 Ramosiphorum polyporicola CBS 110.87 Lasionectriella rubioi CBS 140157T Verruciconidia erythroxyli CBS 728.87T Ramosiphorum thailandicum CBS 101914T Ochronectria calami CBS 454.96 Lasionectriopsis stereicola CBS 123299T Lasionectriopsis pteridii CBS 782.69T Ramosiphorum polyporicola CBS 109.87 Clavatomyces korfii CBS 138714 Ochronectria calami CBS 125.87 Ramosiphorum polyporicola CBS 123779T Lasionectria castaneicola CBS 122792T Verruciconidia persicina CBS 310.59T Verruciconidia verruculosa CBS 989.69T Verruciconidia saulensis CLLG21159T Ochronectria calami CBS 136682 Ochronectria thailandica MFLUCC 15-0140T Lasionectria bisepta CBS 753.69 Lasionectria antillana CBS 122797T Verruciconidia erythroxyli CBS 378.70D Verruciconidia quercina CBS 469.67T Ochronectria calami CBS 445.96 Lasionectriopsis dentifera CBS 574.76T Lasionectriella arenuloides CBS 576.76T Lasionectria olida CBS 799.69T Ramosiphorum echinophoriae CBS 115288T Lasionectria cerealis CBS 144938 Lasionectria olida CBS 798.69 Lasionectria cerealis CBS 393.66 Lasionectriopsis pteridii CBS 579.90 Lasionectria cerealis CBS 461.88 Lasionectria cerealis CBS 208.70 Lasionectria atrorubra CBS 123502T Lasionectriopsis germanica CBS 143538T Verruciconidia verruculosa CBS 299.81B Verruciconidia persicina CBS 113716 Lasionectria krabiense MFLUCC 15-0673T Lasionectria sansevieriae CBS 146973T Ochronectria calami CBS 123492 Verruciconidia unguis CBS 378.70E Verruciconidia unguis CBS 424.93T Lasionectria chondroidea CBS 565.73T Clavatomyces prestoeae CBS 101691T Verruciconidia verruculosa CBS 990.69 Verruciconidia guizhouensis SQT05 Lasionectria bisepta CBS 751.69 Lasionectria mantuana CBS 142926 Lasionectriopsis germanica CBS 121889 Lasionectriopsis stereicola CBS 101910 Ramosiphorum polyporicola CBS 100282 Lasionectriella marigotensis CBS 131606T Lasionectria sylvana CBS 566.76 Lasionectria bisepta CBS 750.69T Ochronectria calami CBS 134535 Lasionectriopsis dentifera CBS 650.75 Lasionectria mantuana CBS 114291 Lasionectriopsis germanica CBS 113762 Lasionectria boothii CBS 129747 Lasionectria lecanodes CBS 139482 Verruciconidia guizhouensis SQT04T 65/97/- 98/100/1 98/100/0.95 97/100/1 -/97/0.99 98/100/1 76/100/- 60/100/- 99/100/1 89/100/1 80/100/1 81/98/1 99/100/1 97/100/0.99 57/100/- 73/100/1 99/100/1 89/100/1 72/98/0.96 95/100/0.98 78/100/1 74/100/1 99/100/1 94/100/1 99/100/1 99/100/1 97/100/1 71/100/1 59/96/0.98 67/100/0.98 66/99/- 97/100/1 98/100/1 25. Clavatomyces 23. Lasionectriopsis 24. Ramosiphorum 26. Lasionectriella 27. Ochronectria 28. Verruciconidia 29. Lasionectria 59/94/- 52/-/- 54/-/- Bionectriaceae Fig. 1. (Continued). Chapter 3 146 Geonectria muralis CBS 149515T Hydropisphaera peziza CBS 233.95 Gliomastix masseei CBS 794.69T Hydropisphaera cyatheae CBS 575.76 Hydropisphaera armeniaca CBS 135905T Hydropisphaera gossypina CBS 140581T Synnemellisia aurantia COAD 2070T Gliomastix tumulicola CBS 128812 Gliomastix murorum CBS 144331 Geonectria muralis CPC 42405 Hydropisphaera peziza CBS 138712 Pseudoacremonium sacchari CBS 137990T Hydropisphaera heliconiae CBS 138704T Caespitomonium squamicola CBS 701.73 Roumegueriella rufula CBS 346.85 Hydropisphaera peziza CBS 296.65 Hydropisphaera aurantiaca CBS 201.35 Hydropisphaera suffulta CBS 122.87 Monohydropisphaera fusigera CBS 124147T Hydropisphaera fungicola CBS 123912 Hydropisphaera peziza CBS 138713 Hydropisphaera fungicola CBS 141091 Gliomastix olivacea CBS 101736T Hydropisphaera fungicola CBS 139554 Selinia pulchra A.R. 2812 Hydropisphaera fungicola CBS 122304T Hydropisphaera peziza CBS 399.66 Paragliomastix venezuelana CBS 102074T Gliomastix masseei CBS 112060 Geonectria alni CBS 140756T Roumegueriella echinulata CBS 276.59T Gliomastix murorum CBS 148.81 Gliomastix masseei CBS 557.75 Verrucostoma freycinetiae MAFF 240100T Gliomastix murorum CBS 154.25T Paragliomastix chiangraiensis CBS 277.80B Septofusidium berolinense CBS 731.70 Pseudosynnemellisia favida CGMCC 3.22477T Musananaesporium tectonae CBS 725.87T Hydropisphaera martinicensis CBS 136679T Hydropisphaera sp. CBS 137311 Hydropisphaera peziza CBS 139487 Gliomastix tumulicola CBS 127532T Gliomastix masseei CBS 511.63 Paragliomastix luzulae CBS 494.67 Hydropisphaera fungicola CBS 136689 Hydropisphaera solani CBS 147425T Paragliomastix luzulae CBS 495.67 Gliomastix murorum CBS 195.70 Hydropisphaera martinicensis CBS 140578 Paragliomastix luzulae CBS 935.69 Hydropisphaera martinicensis CBS 138697 Gliomastix murorum CBS 304.70C Verrucostoma martinicensis CBS 138731T Synnemellisia acaciae BRIP 71652T Geonectria subalpina CBS 143540T Gliomastix murorum CBS 125913 Caespitomonium squamicola CBS 392.73 Hydropisphaera peziza CBS 135908 Paragliomastix znieffensis CBS 140584T Parageonectria arachispora CBS 118.87T Paragliomastix chiangraiensis MFLUCC 14-0397T Hydropisphaera aurantiaca CBS 397.67 Geonectria quercus CBS 137308T Hydropisphaera fungicola CBS 138698 Gliomastix murorum CBS 380.70B Gliomastix murorum CBS 135023 Pseudosynnemellisia favida CGMCC 3.22483 Gossypinidium sporodochiale CBS 101694T Paragliomastix rosea CBS 277.80AT 90/100/1 73/100/0.99 99/100/1 74/100/1 96/99/1 100/100/1 87/98/1 81/98/1 91/100/1 98/100/1 -/96/0.93 99/100/1 87/100/1 91/100/1 69/100/0.95 71/100/0.97 67/100/0.97 97/100/1 65/98/1 97/100/1 98/100/1 99/100/1 99/100/1 76/100/1 -/97/0.94 75/100/0.98 62/98/1 96/100/1 33. Parageonectria 34. Geonectria 30. Pseudoacremonium 31. Septofusidium 32. Paragliomastix 35. Hydropisphaera 36. Monohydropisphaera 37. Musananaesporium 38. Caespitomonium 39. Verrucostoma 40. Selinia 41. Roumegueriella 42. Gossypinidium 43. Synnemellisia 44. Pseudosynnemellisia 45. Gliomastix 2× 4× Bionectriaceae Fig. 1. (Continued). Bionectriaceae 147 3 0.2 Vitreipilata cirsii CBS 139243 Fusariella aegyptiaca CBS 232.73 Paracylindrocarpon aloicola CBS 810.84 Fusariella triangulispora CBS 144361T Fusariella bizzozeriana CBS 867.85 Gliomastix polychroma CBS 143095 Paracylindrocarpon aloicola CBS 335.77 Paracylindrocarpon multiseptatum CBS 333.77 Paracylindrocarpon multiseptatum CBS 337.77T Paracylindrocarpon spartinae CBS 102161T Fusariella sp. CBS 128364 Paracylindrocarpon multiloculatum CBS 340.77 “Hydropisphaera peziza” CBS 102077 Gliomastix polychroma CBS 181.27T Gliomastix musae CBS 561.93 Gliomastix musae CBS 617.94T Vitreipilata cirsii CBS 135615T Fusariella concinna CBS 695.73 Fusariella bizzozeriana CBS 306.73 Fusariella concinna CBS 302.64 Fusariella hughesii CBS 369.76 Urticomyces pseudoarenulus AG09162 Fusariella hughesii CBS 582.97 Paracylindrocarpon aloicola CBS 141300T Fusariella hughesii CBS 435.70 Gliomastix musae CBS 296.70E Gliomastix roseogrisea CBS 213.69 Fusariella arenula CBS 329.77 Urticomyces pseudoarenulus CLL19022T Gliomastix polychroma CBS 210.69 Fusariella concinna CBS 312.73 Paracylindrocarpon xishuangbannaensis KUMCC 16-0144T Fusariella atrovirens CBS 310.73 Gliomastix roseogrisea CBS 784.83 Paracylindrocarpon aurantiacum CBS 135909T Fusariella concinna CBS 309.73 Fusariella arenula CBS 330.77 Gliomastix roseogrisea CBS 134.56T Paracylindrocarpon jigongshanense CBS 128365T Fusariella curvata MFLUCC 15-0844T Smyrniomyces setaceus CBS 130334T Gliomastix roseogrisea CBS 129173 Gliomastix roseogrisea CBS 380.70A Paracylindrocarpon nabanheensis KUMCC 16-0147T Paracylindrocarpon aloicola CBS 126107 Paracylindrocarpon aloicola CBS 135907 Gliomastix musae CBS 678.94 Paracylindrocarpon foliicola CBS 140758T Paracylindrocarpon multiseptatum CBS 338.77 Fusariella concinna CBS 126195 Gliomastix polychroma CBS 296.70D Urticomyces pseudoarenulus CBS 128931 Fusariella atrovirens CBS 311.73 Paracylindrocarpon pandanicola KUMCC 17-0272T Paracylindrocarpon multiloculatum CBS 339.77T Gliomastix musae CBS 296.70A Fusariella hughesii CBS 118230 Fusariella concinna CBS 367.76 Paracylindrocarpon multiseptatum CBS 336.77 Gliomastix roseogrisea CBS 127140 Fusariella concinna CBS 303.73 Vitreipilata cirsii CBS 140570 77/96/0.99 82/100/0.99 95/100/1 73/100/1 99/100/1 92/100/1 75/100/0.95 53/100/- 88/100/- 98/100/1 99/100/1 99/100/1 73/97/0.99 75/100/1 98/100/1 77/100/1 98/100/1 68/96/0.95 83/100/1 95/100/1 98/100/1 98/100/1 -/98/1 -/-/0.99 46. Paracylindrocarpon 50. Fusariella 57/-/- 47. Smyrniomyces 48. Vitreipilata 49. Urticomyces 45. Gliomastix Bionectriaceae including seven known species, and two species that were recombined to the genus, one as V. guizhouensis (basionym: Acremonium guizhouense), and the other as V. saulensis (basionym: Lasionectria saulensis). The genus Lasionectria (Clade 29, RAxML-BS = 100 Fig. 1. (Continued). Chapter 3 148 %, IQ-TREE-BS = 100 %, BI-PP = 1) comprised 12 known species and two novel species, L. chondroidea and L. phormii. Clade 32 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) comprised four species of the genus Paragliomastix, Px. chiangraiensis, Px. luzulae, Px. rosea, Px. znieffensis, and a novel species, Px. venezuelana. The Parageonectria clade (Clade 33) was recognised as a novel monotypic genus, containing Pg. arachispora. Clade 34 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Geonectria with two known species, Gn. subalpina and Gn. muralis, and two novel species, Gn. alni and Gn. quercus. Clade 35 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Hydropisphaera with six known species and four novel species, H. armeniaca, H. gossypina, H. martinicensis, and H. solani. The clade corresponding to Roumegueriella (Clade 41; RAxML-BS = 96 %, IQ-TREE-BS = 100 %, BI-PP = 1) included the type species R. rufula and a novel species, R. echinulata. Clade 45 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BI-PP = 1) comprised six known species of the genus Gliomastix and one novel species, Gl. olivacea. Clade 46 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %, BIPP = 1) accommodated the genus Paracylindrocarpon with eight known species and two novel species, Pn. jigongshanense and Pn. spartinae. The proposed new monotypic genera Smyrniomyces (Clade 47), Vitreipilata (Clade 48), and Urticomyces (Clade 49), along with their respective type species, namely, Sm. setaceus, Vit. cirsii (basionym: Hydropisphaera cirsii), and U. pseudoarenulus (basionym: Hydropisphaera pseudoarenula). Clade 50 (RAxML-BS = 98 %, IQ-TREE-BS = 100 %, BI-PP = 1) accommodated the genus Fusariella, including seven known species and one novel species, F. triangulispora. Other clades such as Stilbocrea (Clade 2), Alloacremonium (Clade 3), Amphichorda (Clade 4), Hapsidospora (Clade 5), Bulbithecium (Clade 7), Circumfusicillium (Clade 9), Waltergamsia (Clade 11), Proliferophialis (Clade 13), Stanjemonium (Clade 14), Stephanonectria (Clade 18), Mycocitrus (Clade 19), Ramosiphorum (Clade 24), Lasionectriella (Clade 26), Ochronectria (Clade 27), Pseudoacremonium (Clade 30), Septofusidium (Clade 31), Monohydropisphaera (Clade 36), Musananaesporium (Clade 37), Caespitomonium (Clade 38), Verrucostoma (Clade 39), Selinia (Clade 40), Gossypinidium (Clade 42), Synnemellisia (Clade 43), and Pseudosynnemellisia (Clade 44) were accepted as taxa of the Bionectriaceae (Fig. 1). Taxonomy According to the multi-locus phylogenetic analyses, combined with morphological characters, we propose new taxa, including seven new genera, 35 new species, and nine new combinations. Two new, however, not-sporulating species are described based on DNA sequence data, following the approach of Hou et al. (2023). Genera are arranged according to their positions on the phylogenetic tree (Fig. 1) and species are arranged alphabetically. Bionectriaceae 149 3 Clade 1. Physaromyces Lin Zhao & Crous, gen. nov. MycoBank MB 856643. Etymology: Referring to the host, Physarum gyrosum, from which the type species was collected. Asexual morph acremonium-like. Mycelium consisting of branched, hyaline, septate, smooth and thick-walled hyphae. Sporulation abundant, phalacrogenous, nematogenous, plectonematogenous. Conidiophores arising from the agar surface and aerial hyphae, or from ropes formed by mycelium, (sub-)erect, unbranched or irregularly branched at 1–3 levels with 1–3 phialides per node, 1–4-septate, occasionally with percurrent proliferation, hyaline, smooth-walled, often with short or long sterile outgrowths. Conidiogenous cells monophialidic, lateral or terminal, cylindrical or subulate, hyaline, with thick and smooth walls, inconspicuous cylindrical collarettes and periclinal thickening at conidiogenous loci. Conidia ellipsoid, cylindrical or oblong, aseptate, hyaline, with thick and smooth walls, rounded at both ends, or with apices rounded and a slightly truncate hilum at bases, arranged in slimy heads. Chlamydospores and sexual morph not observed. Type species: Physaromyces sterilis Lin Zhao & Crous Notes: The monotypic genus Physaromyces is proposed here to accommodate a single species, Phy. sterilis, clustering as the most basal lineage in Bionectriaceae (Fig. 1, clade 1). The species is represented solely by the strain CBS 139048, isolated from old Physarum gyrosum on Fagus litter in the Netherlands. Physaromyces sterilis Lin Zhao & Crous, sp. nov. MycoBank MB 856645. Fig. 2. Etymology: Referring to the production of conidiophores often with sterile outgrowths. Typus: Netherlands, Overijssel Province, Hengelo, Oosterveld, from old Physarum gyrosum on Fagus litter, 17 Apr. 2014, M. Jagers (holotype designated here CBS H-25551, ex-type living culture CBS 139048). Mycelium consisting of branched, hyaline, septate, smooth and thick-walled hyphae, 1.5–3.0 μm wide. Sporulation abundant, phalacrogenous, nematogenous, plectonematogenous. Conidiophores arising from the agar surface and aerial hyphae, or from ropes formed by mycelium, (sub-)erect, unbranched or branched, bearing 1–3 levels with 1–3 phialides per node, occasionally with percurrent proliferation, up to ca 130 μm long, 1.6–2.8 μm wide at base, 1–4-septate, hyaline, smooth-walled, often with short or long sterile outgrowths. Conidiogenous cells monophialidic, lateral or terminal, cylindrical or subulate, hyaline, with Chapter 3 150 Fig. 2. Physaromyces sterilis (ex-type CBS 139048). A–C. Colonies on OA, MEA and PDA after 14 d at 25 °C in darkness. D–J. Morphological structures from OA culture. D–I. Conidiophores. J. Conidia. Scale bars = 10 μm. thick and smooth walls, (14.3–)16.7–40.1(–45.9) μm long, (1.3–)1.4–2.1(–2.3) μm wide at base, (0.7–)0.8–1.1 μm wide near aperture, with inconspicuous cylindrical collarettes and periclinal thickening at conidiogenous loci. Conidia ellipsoid, cylindrical or oblong, aseptate, Bionectriaceae 151 3 hyaline, with thick and smooth walls, rounded at both ends, or with apices rounded and a slightly truncate hilum at bases, variable in size, (3.0–)3.7–7.2(–9.3) × (1.7–)2.0–3.2(–3.6) μm (av. 5.5 × 2.6 μm, n = 100), arranged in slimy heads. Chlamydospores and sexual morph not observed. Culture characteristics: Colonies on OA reaching 30–34 diam. after 14 d at 25 °C in darkness, flat, felty, dusty, white to yellowish, margin entire, reverse primrose. Colonies on MEA reaching 19–27 diam., raised, floccose or felty, white to dirty white, margin lobate, reverse orange, with white radial lines. Colonies on PDA reaching 32–36 diam., flat, felty, dusty, white to yellowish, margin lobate, reverse pale salmon. Colonies on SNA reaching 25–29 diam., flat, sparse aerial mycelium, membranous, colourless, margin entire, reverse colourless. Notes: Phylogenetic analyses based on multiple loci (ITS-LSU-RPB2-TEF1) show that CBS 139048 forms a distinct basal branch in Bionectriaceae (Fig. 1, clade 1). Based on a blastn search of NCBI GenBank nucleotide database, the closest hits using the ITS sequence is listed as Acremonium dichromosporum isolated from Glycyrrhiza uralensis in China [strain RP4; GenBank KF022040; Identity = 449/499 (90 %), 16 gaps (3 %)] [affinity with authentic Stanjemonium dichromosporum (Hou et al. 2023) not analysed]; the closest hit using the LSU sequence is the type of Acremonium subulatum, in GenBank as Acremonium sordidulum [culture CBS 588.73A; GenBank MH872507.1; Identity = 754/778 (97 %), two gaps (0 %)]; the closest hit using the TEF1 sequence is Amphichorda guana [culture LC5815; GenBank KX855211.1; Identity = 763/808 (94 %), no gaps]; no RPB2 sequence was available to include in a blast search. Clade 6. Proxiovicillium L.W. Hou et al., Stud. Mycol. 105: 152. 2023. Mycelium consisting of branched, septate, hyaline, smooth and thin-walled hyphae. Conidiophores arising from the agar surface and aerial hyphae, sometimes radiating out from sterile coils formed by mycelium, (sub-)erect, unbranched or poorly branched, 1–3-septate. Conidiogenous cells monophialidic, terminal, lateral, acicular, cylindrical or subulate, straight to flexuous, hyaline, smooth and thick-walled, with inconspicuous collarettes and periclinal thickening at conidiogenous loci; adelophialides and polyphialides present in some species. Conidia sub-globose, broadly ovoid or ellipsoid, tapering to apiculate base, or with apiculate bases and obtuse apices, aseptate, hyaline, with thin and smooth walls, arranged in long chains, or later collapsing soon as conidial heads. Chlamydospores and sexual morph unknown (adapted from Hou et al. 2023, Tong et al. 2023). Type species: Proxiovicillium blochii (Matr.) L.W. Hou et al. Chapter 3 152 Notes: Proxiovicillium was introduced by Hou et al. (2023) as a genus that is phylogenetically close to Ovicillium and accommodates two species: Proxiovicillium blochii and Proxiovicillium lepidopterorum. A novel combination is described here as Proxiovicillium capsici. Proxiovicillium capsici (S.Q. Tong & Y.J. Wu) Lin Zhao & Crous, comb. nov. MycoBank MB 856647. Basionym: Acremonium capsici S.Q. Tong & Y.J. Wu, MycoKeys 95: 7. 2023. Typus: China, Guizhou Province, Guiyang City, 26°45’75”N, 106°64’87”E, isolated from the rhizosphere soil of Capsicum annuum, Aug. 2022, S.Q. Tong (dried holotype culture SQT H-01, ex-holotype culture SQT01). Description and illustration: Tong et al. (2023). Notes: Acremonium capsici was isolated from the rhizosphere soil of Capsicum annuum by Tong et al. (2023). In the present study, phylogenetic analysis showed that the ex-type culture of A. capsici clusters within the genus Proxiovicillium (Fig. 1, clade 6). Clade 8. Ovicillium Zare & W. Gams, Mycol. Progr. 15: 1020. 2016. Sporulation abundant, phalacrogenous to nematogenous. Conidiophores erect, mostly branched, rarely simple, mostly with secondary branches, bearing verticillate phialides of 4–6 in a whorl, strongly cyanophilic smooth or rough near base, and gradually tapering towards apex, phialide tip undulate in some species. Conidiogenous cells phialidic, terminal or lateral, straight or slightly bent, acicular, with minute collarettes and distinct periclinal thickening at apex, smoothand thin-walled, hyaline. Conidia subglobose, ovoid, napiform, ellipsoid, or cylindrical, more or less apiculate at base, aseptate, smoothand thick-walled, hyaline to subhyaline. Chlamydospores present in some species, globose to ovoid. Sexual morph not observed (adapted from Giraldo et al. 2012, Zare & Gams 2016, Hou et al. 2023). Type species: Ovicillium attenuatum Zare & W. 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Numbers at branches indicate support values (RAxML-BS) above 50 %. The new species are printed in red font and new combinations in blue font. Strains with a type status are indicated with “T”. The tree is rooted to Bullanockia australis (CBS 142093), Flammocladiella aceris (CBS 138906), F. anomiae (CBS 142775), F. decora (CBS 142776), Stromatonectria caraganae (CBS 125579, CBS 127387), Tilachlidium brachiatum (CBS 505.67, CBS 363.97) and Xanthonectria pseudopeziza (CBS 140160, CBS 141245). The scale bar represents the expected number of changes per site. Fig. S2. Phylogenetic tree inferred from a Maximum Likelihood (RAxML) analysis based on a concatenated alignment of ITS, LSU, RPB2, and TEF1 sequences of 751 strains representing Bionectriaceae and allied families in Hypocreales and outgroups. Numbers at branches indicate support values (RAxMLBS) above 50 %. The new species are printed in red font and new combinations in blue font and and different families are highlighted with coloured boxes. Strains with a type status are indicated with “T”. The tree is rooted to Paracremonium inflatum (CBS 485.77), Sarocladium oryzae (CBS 180.74), S. zeae (CBS 800.69), and Xenoacremonium recifei (CBS 137.35). The scale bar represents the expected number of changes per site. Table S1. Strains used in this study with details of their host, location, and GenBank accessions numbers. Revision of acremonium-like fungi 271 4 were reassigned within this genus. Furthermore, their results indicated that most Acremonium species clustered within the Hypocreales, forming smaller clades such as the pinkertoniae-clade and the fusidioides-clade, which were distant from the sclerotigenumclade that includes A. alternatum. The isolates of nine species (A. alcalophilum, A. antarcticum, A. apii, A. brunnescens, A. cucurbitacearum, A. furcatum, A. stromaticum, A. restrictum, and A. nepalense) were placed within the Plectosphaerellaceae (current name: Trichosphaeriaceae). Additionally, Acremonium alabamense, the only named species in Acremonium section Chaetomioidea, was assigned to the Sordariales, while isolates of A. atrogriseum and A. thermophilum were placed in the family Cephalothecaceae, which is sister to the Coniochaetales. These results demonstrated that Acremonium species are phylogenetically divergent and remain polyphyletic. To better clarify the phylogenetic relationships and classification of acremoniumlike taxa, Hou et al. (2023) conducted a comprehensive analysis of 633 cultures with acremonium-like morphology, including 261 ex-type cultures from 89 countries and diverse substrates, most of which were identified as belonging to species in the genus Acremonium or related genera based on the morphological studies of Gams (1971) and Summerbell et al. (2011). Phylogenetic trees based on multiple loci (ITS, LSU, RPB2, and TEF1) were used to classify species at genus and family levels, identifying 63 genera and 14 families within 4 orders. Species with acremonium-like asexual morphs were shown to belong to Cephalothecales (family: Cephalothecaceae), Coniochaetales (family: Coniochaetaceae), Hypocreales (families: Bionectriaceae, Clavicipitaceae, Cordycipitaceae, Myrotheciomycetaceae, Nectriaceae, Niessliaceae, and Sarocladiaceae, as well as five newly established hypocrealean families), and Trichosphaeriales (family: Plectosphaerellaceae, currently referred to as Trichosphaeriaceae). It is worth noting that Acremonium sensu stricto (s. str.) was restricted to the family Bionectriaceae, and most fungi with acremoniumlike morphologies were classified within the Hypocreales (Glenn et al. 1996, Summerbell et al. 2011, Hou et al. 2023). The latter study provided the most comprehensive and up-to-date molecular phylogenetic framework and detailed morphological data for Acremonium species derived from cultures, refined their circumscription, and elucidated the phylogenetic relationships of species recognised within Acremonium. Hypocrea was first described by Fries (1825) and classified within the order Sphaeriacei, under the suborder Sphaerini. Fries (1849) later refined the concept of Hypocrea, adopting a more restricted definition. Subsequently, Lindau (1897) established the family Hypocreaceae, basing it on Hypocrea, and introduced the order Hypocreales to include Hypocreaceae as its sole family. Rossman et al. (1999) revised the taxonomy of Hypocreales, identifying five families: Clavicipitaceae, Hypocreaceae, Nectriaceae, Niessliaceae, and the newly established Bionectriaceae. This classification was later reinforced by LSU sequencebased phylogenetic analysis conducted by Rossman et al. (2001). The sexual morph for Hypocreales is characterised by ascomata that are transparent, white, pale, or brightly to Chapter 4 272 darkly coloured in shades of yellow, orange, red, brown, green, blue, purple, or black, with textures ranging from soft and fleshy to tough, occurring superficially on the substratum, embedded in it, or seated in a weakly to well-developed stroma, and containing unitunicate asci with 2–8 spores and ascospores that are 0–multiseptate, sometimes muriform, and either disarticulating or not; the asexual morphs are typically hyphomycetous and less frequently coelomycetous, often featuring phialidic conidiogenous cells (Rossman et al. 1999, Maharachchikumbura et al. 2015, 2016, Hyde et al. 2020, Perera et al. 2023). The most recent systematic studies of Hypocreales were conducted by Wijayawardene et al. (2022), Perera et al. (2023), and Hou et al. (2023). The outline by Wijayawardene et al. (2022) recognised 15 families and 320 genera within Hypocreales. Perera et al. (2023) conducted a comprehensive revision of Bionectriaceae, Calcarisporiaceae, Hypocreaceae, Nectriaceae, and Tilachlidiaceae using both morphological data and combined gene analyses of the ITS, LSU, rpb2, tef1, and tub2 regions, recognising 17 families within Hypocreales, including the newly established families Ijuhyaceae, Stromatonectriaceae, and Xanthonectriaceae, while excluding Cylindriaceae from Hypocreales. Hou et al. (2023) conducted the most extensive sampling of acremonium-like fungi to date, using multi-locus DNA sequencing analyses, resulting in the establishment of five new families within Hypocreales: Chrysonectriaceae, Neoacremoniaceae, Nothoacremoniaceae, Pseudoniessliaceae, and Valsonectriaceae. Additionally, the family Pseudodiploosporeaceae, introduced by Sun et al. (2023) to accommodate two novel genera, Pseudodiploospora and Zelopaecilomyces, was revisited by Yu et al. (2024), who determined that it contains only a single genus, Pseudodiploospora. Yu et al. (2024) also proposed a new family, Albomorchellophilaceae, with isolates derived from infected sporocarps of cultivated morels. Furthermore, Acremoniopsiaceae comprises isolates mainly derived from mangrove sediments and includes Acremoniopsis (previously placed in Hypocreales incertae sedis), Collarina (previously in Clavicipitaceae), and two newly established genera, Nothoacremoniopsis and Phaeocollarina. The newly proposed family Sedecimiellaceae includes Sedecimiella and Heteroacremonium (Li et al. 2023: additional file 1). Xiao et al. (2023) recently segregated the genera Polycephalomyces, Perennicordyceps, and Pleurocordyceps from the family Ophiocordycipitaceae based on morphological and phylogenetic analyses, and established a new family, Polycephalomycetaceae. Currently, Hypocreales comprises 27 families: Acremoniopsiaceae, Albomorchellophilaceae, Bionectriaceae, Calcarisporiaceae, Chrysonectriaceae, Clavicipitaceae, Cocoonihabitaceae, Cordycipitaceae, Flammocladiellaceae, Hypocreaceae, Ijuhyaceae, Myrotheciomycetaceae, Nectriaceae, Neoacremoniaceae, Niessliaceae, Nothoacremoniaceae, Ophiocordycipitaceae, Polycephalomycetaceae, Pseudoniessliaceae, Pseudodiploosporeaceae, Sarocladiaceae, Sedecimiellaceae, Stachybotryaceae, Stromatonectriaceae, Tilachlidiaceae, Valsonectriaceae and Xanthonectriaceae. Bionectriaceae was once considered the largest family within Hypocreales, comprising 26 genera with perithecial sexual morphs and five genera with cleistothecial sexual Revision of acremonium-like fungi 273 4 morphs (Rossman et al. 1999). It is characterised by globose to pyriform perithecial ascomata, occasionally cleistothecia, which are either superficial or immersed, with or without a stroma. The colours range from white, yellow, or orange to brown, reddish brown, or purple, and remain unchanged in KOH or lactic acid. The asci are unitunicate, 8-spored, clavate, or globose, with or without an apical ring. Ascospores show variations in shape, septation, and surface ornamentation, with walls that range from hyaline to brown (Rossman et al. 1999). The asexual morphologies, including acremonium-like, penicillium-like, and verticillium-like forms (Rossman et al. 1999, Summerbell et al. 2011), are diverse. According to the phylogenetic analyses conducted by Hou et al. (2023), more than half of the Acremonium species with acremonium-like morphology, as recognised by Gams (1971), were classified within Bionectriaceae. Within Bionectriaceae, the 39 genera accepted in a recent study (Hou et al. 2023) were found to include both sexual and asexual taxa. Most genera are only known from their asexual morphs, which are mainly acremonium-like, including Acremonium s. str., Alloacremonium, Fusariella, Gliomastix, Gossypinidium, Monohydropisphaera, Musananaesporium, Ovicillium, Paragliomastix, Proliferophialis, Proxiovicillium, Pseudoacremonium, Ramosiphorum, Septofusidium, Stanjemonium, Verruciconidia, and Waltergamsia (Hou et al. 2023). Additionally, some genera have perithecial or cleistothecial sexual morphs, along with asexual morphs that are acremonium-like, including Bulbithecium, Emericellopsis, Geonectria, Hapsidospora, Lasionectria, Lasionectriella, Lasionectriopsis, Nectriopsis, Ochronectria, Paracylindrocarpon, Protocreopsis, Roumegueriella, Stilbocrea, and Verrucostoma (Hou et al. 2023). Acremonium sensu lato (s. lat.) species, a diverse group of fungi found in various environmental niches worldwide, are significant in human medicine, the food industry, agriculture, and biotechnology. In the medical field, Acremonium species are primarily recognised as opportunistic pathogens, particularly in immunocompromised individuals, and have been linked to infections such as onychomycosis, keratomycosis, peritonitis, and disseminated infections (Gams 1971, Novicki et al. 2003, Summerbell & Scott 2015, Summerbell et al. 2018). Acremonium falciforme (currently reclassified as Neocosmospora falciformis) has been identified as the etiological agent of upper gastrointestinal tract lesions in a bone marrow transplant recipient with congenital severe combined immunodeficiency, as documented in a case report from Hong Kong (Lau et al. 1995, Summerbell & Scott 2015). Sarocladium kiliense has been documented in a case of esophagitis in an otherwise healthy 11-yr-old boy, suggesting that the infection, like those originating in the lower gut, may have been caused by contamination from food or water or an airborne source (Simon et al. 1991, Summerbell & Scott 2015). In the food industry, species within Acremonium s. lat. are classified as food-spoiling microorganisms, as they have been found in cereal and grain products (such as wheat, barley, and rice), leading to spoilage during storage; are associated with the deterioration of fruits and vegetables (such as bananas with crown rot, Chapter 4 274 fresh vegetables, and soybeans); and have also been reported to contaminate various food categories, including nuts and seeds (such as peanuts, pecans, hazelnuts, and walnuts), dairy products, beverages, and processed foods like frozen meat, salami, and biltong (Gams 1971, Fernández-Trujillo 1997, Fujikawa 1997, Pitt et al. 1993, Pitt & Hocking 1997, 2009, 2022, Samson et al. 2004, Summerbell & Scott 2015, Summerbell et al. 2018). Fujikawa et al. (1997) found that bottled mineral water was contaminated by Acremonium spp. Two species of Acremonium, Acremonium tubakii (now reclassified as Emericellopsis tubakii) and Acremonium rutilum (currently known as Protocreopsis rutila), were identified as postharvest pathogens of peaches (Fernández-Trujillo et al. 1997). Acremonium strictum (currently reclassified as Sarocladium strictum) was found on black bean samples collected during the harvest season in Salta, a northwestern province of Argentina (Castillo et al. 2004). In agriculture, Acremonium s. lat. species exhibit dual roles, ranging from plant pathogens, e.g., Acremonium brown spot of bagged apples in China, a plant disease caused by Acremonium sclerotigenum (currently named Acremonium egyptiacum), causing 1–30% annual yield losses (Li et al. 2014), or beneficial endophytes. In symbiotic relationships Acremonium has been found to enhance plant resilience to environmental stresses such as drought, salt, and pathogen infections (Auer & Ludwig-Müller 2014, Bobeck & Pearce 2017, Sutton & Mason 2017). Furthermore, Acremonium s. lat. species show significant promise in biotechnology, as they are known to produce a variety of bioactive metabolites, with over 600 secondary metabolites identified to date, classified into terpenoids, peptides, polyketides, and others (steroids, amides, and alkaloids), predominantly from marine sources (Qin et al. 2024). This emphasizes the ongoing research into the bioactive compounds produced by Acremonium species, which have primarily been evaluated for their cytotoxic, antibacterial, and anti-inflammatory properties, offering valuable insights for both scientific research and the pharmaceutical industry (Qin et al. 2024). Acremonium-like taxa were studied by Hou et al. (2023), with most species identified as belonging to the genus Acremonium or related genera based on morphological characteristics, including 261 ex-type cultures from Acremonium species and related genera, as documented by Gams (1971) and Summerbell et al. (2011). As a continuation of the work by Hou et al. (2023), our study aims to further investigate acremonium-like fungi, including isolates in the CBS collection preliminarily identified as Acremonium spp. based on morphological characteristics, as well as species listed in Gams’ monograph Cephalosporium-artige Schimmelpilze (Hyphomycetes), particularly those strains of Acremonium and related genera that have not yet been subjected to molecular analyses. Our objectives were as follows: 1) to conduct a comprehensive investigation of acremoniumlike fungi species using both morphological characteristics and molecular data, including unidentified strains from the CBS collection and those not previously examined in Gams’ monograph; 2) to reassess and refine the classification of Acremonium spp. and related taxa within the context of the current phylogenetic framework, clarifying their placement Revision of acremonium-like fungi 275 4 within Hypocreales and related orders; 3) to provide detailed analyses and descriptions for those undescribed taxa, and to assign correct names to acremonium-like fungi that have been poorly understood due to their reduced morphological features. MATERIALS AND METHODS Isolates All cultures of acremonium-like fungi obtained from the CBS culture collection of the Westerdijk Fungal Biodiversity Institute (WI; Utrecht, the Netherlands) were included in this study. Most of these cultures were identified as species in the genus Acremonium or associated genera based on morphological characteristics or initial DNA sequence data from the CBS collection, as well as the strains being listed in Gams’ monograph Cephalosporiumartige Schimmelpilze (Hyphomycetes) (Supplementary Table S1). DNA extraction, PCR amplification and sequencing To extract genomic DNA, fungal colonies were cultured on oatmeal agar (Crous et al. 2019a) at room temperature for 2 wk. Genomic DNA was then isolated using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA), following the manufacturer’s protocol. Four gene regions were amplified using the methods outlined in Zhao et al. (2025). The ITS region was amplified with the primer pair ITS5/ITS4 (White et al. 1990), the LSU region with LR0R/LR5 primers (Vilgalys & Hester 1990, Rehner & Samuels 1994), and the RPB2 and TEF1 genes using the primer sets RPB2-5F2/RPB2-7CR (Liu et al. 1999, Sung et al. 2007b) and EF-983F/EF-2218R (Rehner & Buckley 2005), respectively. The consensus sequences for each isolate were generated by assembling the forward and reverse reads using Geneious Prime v. 2022 (Biomatters Inc., New Zealand). The corresponding GenBank accession numbers for the newly generated sequences are listed in Table S1. Phylogenetic analyses Alignments for the four loci (ITS, LSU, RPB2, TEF1) were generated using MAFFT version 7 with default settings on the online server (https://mafft.cbrc.jp/alignment/server/ index.html) (Kuraku et al. 2013, Katoh et al. 2019). When necessary, manual editing of the alignments was performed using MEGA v. 7.0.21 (Kumar et al. 2016). Phylogenetic inferences of the concatenated alignments (ITS, LSU, RPB2, and TEF1) were performed using two Maximum Likelihood (ML) methods: RAxML and IQ-TREE. Phylogenetic analyses were performed using RAxML (Maximum Likelihood) on the CIPRES Science Gateway portal v. 3.3 (https://www.phylo.org/; Miller et al. 2012) and RAxML-HPC2 on ACCESS v. 8.2.12 (Stamatakis 2014), employing the default GTR substitution model and 1000 rapid Chapter 4 276 bootstrap replicates. Additional maximum likelihood analyses were carried out with IQTREE v. 2.1.3 (Nguyen et al. 2015, Minh et al. 2020), utilising UFBoot2 bootstrapping (ultrafast bootstrapping, with ≥ 95 % considered significant) for branch support estimation (Hoang et al. 2018). The optimal evolutionary model for each partition was determined using ModelFinder (Kalyaanamoorthy et al. 2017, Minh et al. 2020), as implemented in IQ-TREE. The resulting trees were visualized using FigTree v. 1.4.2, and the concatenated data of the four loci (ITS, LSU, RPB2, and TEF1) and the phylogenetic trees were uploaded to figshare (doi: 10.6084/m9.figshare.28705967). Morphology Macroscopic characteristics of colonies were assessed on oatmeal agar (OA), malt extract agar (MEA), potato dextrose agar (PDA), and synthetic nutrient-poor agar (SNA) following incubation in darkness at 25 °C for 14 d. The upper and reverse surface colours of the colonies were determined using Rayner’s (1970) colour charts for standardized evaluation. Microscopic structures were examined from 14-d-old colonies grown on OA or SNA under near-UV light at room temperature. Clear lactic acid was used as the mounting medium for observing the microstructures. Micro-morphological characteristics were examined using a Nikon Eclipse 80i compound microscope equipped with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope. Photomicrographs and measurements were captured with a Nikon DS-Ri2 high-definition colour digital camera, utilising NIS-Elements D software v. 4.50 (Nikon, Tokyo, Japan). Descriptive data, illustrations, and nomenclatural information were submitted to MycoBank (www. MycoBank.org; Crous et al. 2004), and corresponding specimens were preserved in the CBS Fungarium. Abbreviations used for genera in the text are as follows: A. = Acremonium, Allo = Allomusicillium, Aur. = Aurantidochium, B. = Brunneomyces, Ca. = Cannomyces, Chl. = Chlamydocillium, Chlam. = Chlamydosporiella, Cl. = Clavatomyces, Lag. = Lagenariomyces, L. = Lasionectria, N. = Neochrysonectria, Para. = Parasarocladium, Pil. = Pilgeriellomyces, Poly. = Polyphialocladium, Pt. = Protocreopsis, R. = Roumegueriella, S. = Sarocladium, Spor. = Sporodochius, T. = Titanomyces, V. = Verruciconidia. RESULTS Phylogenetic analyses In this study, we investigated acremonium-like fungi taxa, analysing a total of 402 isolates. To understand the phylogenetic relationships within Hypocreales and related orders, an overview phylogenetic tree was constructed using ITS, LSU, RPB2, and TEF1 sequences, which positioned the different families within the orders (Dataset 1). This was followed by more detailed phylogenetic analyses focusing on specific families, including Bionectriaceae, Revision of acremonium-like fungi 277 4 Sarocladiaceae, and Trichosphaeriaceae, incorporating all available cultures from these families (Datasets 2–4). The same phylogenetic methods used for Dataset 1 were also used for Datasets 2–4, ensuring consistent and comparable results across all analyses. Dataset 1: The concatenated and aligned ITS, LSU, RPB2, and TEF1 sequences from four genes and 638 taxa, belonging to the order Hypocreales and Trichosphaeriales, and its related orders, were utilised to define the boundaries at the family and order levels and to optimize the clarity and structure of the phylogenetic tree, with Saccharata proteae (CBS 115206) serving as the outgroup (Dothideomycetes, Botryosphaeriales, Saccharataceae). The alignment contained a total of 3869 characters (including gaps), with the following partitions: ITS: 1–1142, 1142 bp; LSU: 1143–1968, 826 bp; RPB2: 1969–2918, 950 bp; TEF1: 2919–3869, 951 bp. Of these, 1256 character sites were conserved (ITS: 228, LSU: 400, RPB2: 165, TEF1: 463), 2402 were variable (ITS: 816, LSU: 415, RPB2: 702, TEF1: 469), and 2031 were parsimony-informative characters (ITS: 699, LSU: 331, RPB2: 592, TEF1: 409). The phylogeny presented in Fig. 1 was the RAxML tree based on the combined dataset, with bootstrap support values from both RAxML (RAxML-BS > 50 %) and IQ-TREE (IQ-TREEBS > 90 %) analyses plotted on the branches. The phylogenetic tree (Fig. 1) mainly contains Clades I–XXXI, which show the wellsupported family Trichosphaeriaceae within Trichosphaeriales, along with 27 known families, the positionally ambiguous clade Trichonectria-Cylindromonium, and two new families with wellor moderately supported clades within the order Hypocreales. The topologies observed in both the RAxML and IQ-TREE analyses were largely consistent within each genus clade of the families, except for a few family clades that shifted to different positions between two analyses, which were instances of low support between different family clades within the order Hypocreales. The following clades include taxonomic novelties and strains of known species that were examined in this study. Clade I (RAxMLBS = 100 %, IQ-TREE-BS = 100 %) encompasses species belonging to Trichosphaeriaceae within Trichosphaeriales. An individual phylogenetic tree that provides further insights into the phylogeny of Trichosphaeriaceae is presented in Fig. 2. Clades II–XXXI represent families within Hypocreales. The new family Aurantidochiaceae (Clade II; RAxML-BS = 94 %, IQ-TREE-BS = 100 %) comprises the two novel genera Aurantidochium, with its type species Aur. nolinae, and Lagenariomyces, which contains two new species, Lag. collarulis and Lag. varioconidialis. Clade III (RAxML-BS = 100 %, IQ-TREE-BS = 100 %) corresponds to the family Sarocladiaceae, and additional details on the phylogeny of Sarocladiaceae are illustrated in a separate phylogenetic tree provided in Fig. 3. Clade V (RAxML-BS = 100 %, IQ-TREE-BS = 100 %) includes species from the genera Trichonectria and Cylindromonium, which belong, respectively, to the Hypocreales genera incertae sedis and Nectriaceae. The status of Clade V remains to be determined. Niessliaceae (Clade VI; RAxML-BS = 89 %, IQ-TREE-BS = 99 %) includes species of Niesslia, which are polyphyletic and intermixed with other species from the genera Eucasphaeria, Myrtacremonium, Neoeucasphaeria, Chapter 4 278 Lagenariomyces collarulis CBS 122558 Sarocladium nubiaquae CBS 117135T Titanomyces triconidiogenes CBS 630.94T Chlamydosporiella restricta CBS 178.40T Chordomyces albus CBS 987.87T Parasarocladium funiculosum CBS 141.62T Allomusicillium domschii CBS 764.69T Sodiomyces alcalophilus CBS 114.92T Plectosphaerella cucumerina CBS 131739T Sodiomyces alkalinus CBS 110278T Chlamydosporiella restricta CBS 177.40 Chlamydosporiella aerina CBS 598.70T Phialemonium thermophilum CBS 733.71 Sarocladium hirsutum CBS 376.70KT Chlamydocillium guttulatum CBS 523.72 Acrostalagmus annulatus CBS 121.84 Titanomyces triconidiogenes CBS 681.94 Chlamydocillium viridicolor CBS 113439T Furcasterigmium furcatum CBS 116548 Chlamydocillium theobromae CBS 112098T Lagenariomyces collarulis CBS 122985T Chlamydocillium curvulum CBS 430.66T Parasarocladium gamsii CBS 726.71T Acrostalagmus luteoalbus CBS 112.16 Chlamydocillium simulans CBS 128171 Saccharata proteae CBS 115206 Chlamydocillium soli CBS 347.76T Parasarocladium breve CBS 150.62T Sarocladium alniphilum CBS 113444T Polyphialocladium margaretcollinsiae BRIP62736aT Sarocladium summerbellii CBS 430.70T Lagenariomyces varioconidialis CBS 122798T Sarocladium oryzae CBS 180.74 Brunneomyces romanianus CBS 490.81T Gibellulopsis serrae CBS 892.70T Chlamydocillium cyanophilum CBS 246.74AT Parasarocladium kislosladkoense CBS 143528T Sarocladium kiliense CBS 400.52 Brunneomyces polyphialidens CBS 166.80T Sarocladium strictum CBS 334.77 Parasarocladium kislosladkoense CBS 143529 Sarocladium zeae CBS 800.69T Furcasterigmium furcatum CBS 122.42T Summerbellia oligotrophica CBS 657.94T Gibellulopsis nigrescens CBS 120949T Brunneochlamydosporium terrestre CBS 112777T Sarocladium bactrocephalum CBS 749.69T Sarocladium catenulatum CBS 125892T Polyphialocladium fusisporum CBS 406.66T Parasarocladium wereldwijsianum CBS147224 Sarocladium bacillisporum CBS 425.67T Brunneomyces brunnescens CBS 559.73T Plectosphaerella plurivora CBS 131742T Coniochaeta lignicola CBS 267.33 Brunneomyces pseudozeylanicum CBS 560.73T Chlamydocillium curvulum CBS 229.75 Allomusicillium malicola CBS 359.80T Aurantidochium nolinae CBS 110134T Brunneochlamydosporium nepalense CBS 971.72T Sarocladium limosialveum CBS 143532T Sarocladium humicola CBS 446.54T Coniochaeta psammospora CBS 590.63 Chlamydocillium lolii CBS 214.70T Phialemonium obovatum CBS 109043 Parasarocladium kislosladkoense CBS 143522 Sarocladium gamsii CBS 707.73T Phialemonium obovatum CBS 279.76 Chordomyces antarcticus CBS 120045T Chlamydocillium guttulatum CBS 104.78T Sarocladium kiliense CBS 122.29T Parasarocladium wereldwijsianum CBS147226T Gibellulopsis fusca CBS 560.65T Parasarocladium radiatum CBS 142.62T Chlamydocillium viridicolor CBS 113575 Sarocladium strictum CBS 346.70T Phialemonium thermophilum CBS 734.71 Sarocladium glaucum CBS 796.69T Brunneochlamydosporium macroclavatum CBS 101249T 4× 4× 2× 62/- 54/- 50/- 93/99 99/100 94/100 99/100 80/100 -/95 99/100 II. Aurantidochiaceae I.Trichosphaeriaceae 84/98 94/100 81/100 III. Sarocladiaceae 98/100 98/100 55/98 59/98 70/100 92/99 92/100 71/- 74/- 99/100 87/100 91/100 71/100 74/100 95/100 Cephalothecaceae (Cephalothecales) Cephalothecaceae (Coniochaetales) Trichosphaeriales Hypocreales 2× 2× 2× Fig. 1. See legend on following page. Revision of acremonium-like fungi 279 4 and Rosasphaeria. The Neochrysonectriaceae clade (Clade VIII) was recognised as a novel monophyletic family, containing the novel genus Neochrysonectria, with type species Neochrysonectria humicola. The family Stachybotryaceae (Clade XXIII; RAxML-BS = 60 %, IQ-TREE-BS = 95 %) contains 37 known genera and one novel genus, Sporodochius, along with the new combination Spor. pironii. All cultures of this species were previously recognised as Nectriella pironii and form a lineage within the family Stachybotryaceae. The family Bionectriaceae (Clade XXXI; RAxML-BS = 53 %, IQ-TREE-BS = 96 %) with a low support value in the RAxML analysis, but with two well-support subclade (RAxML-BS = 89 %, IQ-TREE-BS = 100 %) and (RAxML-BS = 98 %, IQ-TREE-BS = 100 %) is also presented in a separate tree (Fig. 4). Clade IX (Chrysonectriaceae; RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XI (Sedecimiellaceae; RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XII (Nectriaceae; RAxML-BS = 98 %, IQ-TREE-BS = 100 %), Clade XIV (Ophiocordycipitaceae: RAxML-BS = 79 %, IQ-TREE-BS = 99 %), Clade XVII (Clavicipitaceae: RAxML-BS = 90 %, IQ-TREE-BS = 100 %), Clade XXI (Cordycipitaceae: RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XXII (Pseudoniessliace: RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XXIV (Valsonectriaceae: RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XXV (Myrotheciomycetace: RAxML-BS = 100 %, IQ-TREE-BS = 100 %), Clade XXVII (Xanthonectriaceae: RAxML-BS = 91 %, IQ-TREEBS = 100 %), Clade XXVIII (Ijuhyaceae: RAxML-BS = 100 %, IQ-TREE-BS = 100 %) all include cultures analysed in our study. Dataset 2: The concatenated and aligned ITS, LSU, RPB2, and TEF1 sequences from four genes and 122 taxa of the Trichosphaeriaceae were included, with Monilochaetes infuscans (CBS 379.77 and CBS 869.96), serving as outgroups (Chaetosphaeriales, Australiascaceae; Fig. 2). The alignment contained a total of 2987 characters (including gaps), with the following partitions: ITS: 1–606, 606 bp; LSU: 607–1396, 790 bp; RPB2: 1397–2173, 777 bp; TEF1: 2174–2987, 814 bp. Of these, 1803 character sites were conserved (ITS: 299, LSU: 584, RPB2: 376, TEF1: 544), 1168 were variable (ITS: 293, LSU: 206, RPB2: 399, TEF1: 270), and 1052 were parsimony informative characters (ITS: 263, LSU: 192, RPB2: 353, TEF1: 244). The phylogeny presented in Fig. 2 was the RAxML tree based on the combined dataset, with bootstrap support values from both RAxML (RAxML-BS > 50 %) and IQ-TREE (IQ-TREE-BS > 90 %) analyses plotted on the branches. The topologies observed in both the RAxML and IQ-TREE analyses were largely consistent. Fig. 1. The phylogenetic tree was constructed using Maximum Likelihood (RAxML) analysis, based on aligned and concatenated ITS, LSU, RPB2, and TEF1 sequences from 638 strains representing the orders Hypocreales and Trichosphaeriales, along with related orders and the outgroup. Numbers on the nodes represent Maximum Likelihood bootstrap values: RAxML-BS (≥ 50 %) listed first, followed by IQ-TREE support values (IQ-TREE-BS ≥ 90 %). Newly described species are labeled in red, and new combinations are shown in blue. The strains analysed in this study are highlighted in bold. Colored boxes indicate families, while Roman numerals correspond to families as indicated in the legend. “T” denotes ex-type strains. The tree is rooted with Saccharata proteae (CBS 115206) (Dothideomycetes, Botryosphaeriales, Saccharataceae). The scale bar represents the expected number of substitutions per site. Chapter 4 280 Trichonectria setadpressa AF28886 Niesslia tenuis CBS 201.70 Cylindromonium lichenicola CBS 777.69 Nothoacremonium subcylindricum CBS 416.68T Rosasphaeria moravica CBS 124270T Niesslia mucida CBS 127344 Niesslia ilicifolia CBS 460.74 Niesslia nordinii CBS 116.70 Niesslia tenuis CBS 203.70 Nothoacremoniopsis sedimenticola CGMCC 3.22383T Niesslia aemula CBS 261.70 Niesslia ligustica CBS 359.70 Niesslia mucida CBS 306.70C Niesslia indica CBS 313.61T Niesslia rhizomorpharum CBS 642.85T Niesslia tenuis CBS 197.70 Niesslia heterophora CBS 150.70 Niesslia mucida CBS 193.70 Niesslia tenuis CBS 476.67 Niesslia tenuis CBS 200.70 Niesslia artocarpi CBS 582.73T Eucasphaeria capensis CBS 120028 Myrtacremonium eucalypti CBS 142161T Niesslia aeruginosa CBS 264.89T Niesslia xanthorrhoeae CBS 287.93T Niesslia constricta CBS 760.69T Trichonectria setadpressa AF29617 Niesslia tenuis CBS 772.69 Nothoacremonium exiguum CBS 587.73T Niesslia mucida CBS 404.66T Cylindromonium eugeniicola CBS 108953 Neoeucasphaeria eucalypti CBS 145075T Niesslia indica CBS 182.65 Phaeocollarina guttulata CGMCC 3.22396T Acremoniopsis suttonii CBS 138708T Niesslia exilis CBS 357.70 Niesslia tenuis CBS 432.66T Eucasphaeria rustici CBS 142085T Eucasphaeria capensis CBS 120027T Phaeocollarina guttulata CGMCC 3.22401 Niesslia mucida CBS 306.70B Niesslia tenuis CBS 773.69 Niesslia grisescens CBS 599.88T Niesslia clarkii CBS 170.74T Niesslia tenuis CBS 199.70 Cylindromonium lichenicola CBS 303.70 Niesslia ilicifolia CBS 390.70 Cylindromonium eugeniicola CBS 146075T Nothoacremoniopsis sedimenticola CGMCC 3.22385 Nothoacremonium subcylindricum CBS 190.70 Cylindromonium lichenicola CBS 188.70 Niesslia aeruginosa CBS 994.69 Niesslia exigua CBS 152.68 Niesslia cladoniicola CBS 960.73T Niesslia ilicifolia CBS 459.74 Niesslia exilis CBS 358.70 Niesslia nordinii CBS 101.63T Nothoacremoniopsis irregularis CGMCC 3.22386T Cylindromonium rhabdosporum CBS 438.66T Niesslia loricata CBS 778.69T Niesslia tenuis CBS 202.70 Trichonectria rectipila CBS 132.87T Collarina aurantiaca CBS 138274T Niesslia heterophora CBS 149.70 Niesslia mucida CBS 192.70 Niesslia ligustica CBS 684.95T Cylindromonium lichenicola CBS 776.69 Collarina aurantiaca CBS 110646 Niesslia tenuis CBS 198.70 Niesslia indica CBS 605.69 Nothoacremoniopsis irregularis CGMCC 3.22388 Nothoacremonium vesiculophorum CBS 397.70BT 100/100 -/96 IV. Nothoacremoniaceae V. Trichonectria -Cylindromonium VI. Acremoniopsidaceae VII. Niessliaceae 90/100 67/100 95/100 78/98 96/100 82/100 99/100 89/99 -/98 63/98 -/94 -/94 68/- 71/96 97/100 54/97 87/100 -/94 Hypocreales (continued) Fig. 1. (Continued). Revision of acremonium-like fungi 287 4 Hapsidospora irregularis CBS 510.70T Emericellopsis salmosynnemata CBS 182.56T Acremonium proliferatum CBS 486.86T Acremonium soli CBS 144381T Acremonium multiramosum CBS 147436T Acremonium gamsianum CBS 881.73T Hapsidospora flava CBS 596.70T Acremonium longiphialidicum CBS 451.70T Geosmithia morbida CBS 124664T Circumfusicillium cavernae MUM 20.31T Proliferophialis apiculata CBS 365.64 Acremonium alternatum CBS 407.66T Acremonium ecuadorense CBS 113632T Acremonium brachypenium CBS 866.73T Waltergamsia fusidioides CBS 840.68T Ovicillium subglobosum CBS 101963T Proxiovicillium blochii CBS 324.33 Geosmithia lavendula CBS 344.48T Acremonium tapetis CBS 220.70T Acremonium brunneisporum CBS 413.76T Stilbocrea colubrensis CBS 141857T Physaromyces sterilis CBS 139048T Acremonium egyptiacum CBS 124.42T Stanjemonium grisellum CBS 655.79T Proxiovicillium blochii CBS 427.93 Stilbocrea walteri CBS 144627T Alloacremonium humicola CBS 613.82T Pilgeriellomyces brasiliensis CBS 100345 Acremonium synnematoferum CBS 147431T Emericellopsis terricola CBS 120.40T Amphichorda felina CBS 250.34 Waltergamsia fusidioides CBS 113.69 Amphichorda felina CBS 648.66 Bulbithecium arxii CBS 737.84T Acremonium egyptiacum CBS 740.69 Stanjemonium ochroroseum CBS 656.79T Bulbithecium hyalosporum CBS 318.91T Circumfusicillium cavernae MUM 20.32 Emericellopsis glabra CBS 119.40T Alloacremonium ferrugineum CBS 102877T Proliferophialis apiculata CBS 303.64T Ovicillium attenuatum CBS 399.86T Pilgeriellomyces brasiliensis CBS 100346T Acremonium charticola CBS 117.25T 2x XXXI. Bionectriaceae 67/100 53/96 89/100 99/100 -/98 58/- 50/- 50/- 53/- 99/100 87/100 98/100 -/97 -/98 -/98 78/100 97/100 -/96 87/100 82/100 Hypocreales (continued) Fig. 1. (Continued). novelties and strains of known species that were examined in this study. Clade III-1 (RAxMLBS = 100 %, IQ-TREE-BS = 100 %) accommodated the genus Polyphialocladium containing one know species Poly. fusisporum, and one new combination Poly. margaretcollinsiae (basionym: Chlamydocillium margaretcollinsiae). Clade III-2 (RAxML-BS = 100 %, IQ-TREEBS = 100 %) comprised the type species, Chlamydocillium cyanophilum, and additional eight known species, Chl. acacia, Chl. antarcticum, Chl. curvulum, Chl. guttulatum, Chl. lolii, Chl. simulans, Chl. soli, Chl. terrestris, and two novel species, Chl. theobromae and Chl. viridicolor. Clade III-3 (RAxML-BS = 100 %, IQ-TREE-BS = 100 %) was represented by 15 accepted species of Parasarocladium (included the type species Para. radiatum), and one novel species, Para. kislosladkoense. The genus Sarocladium (Clade III-4; RAxML-BS = 100 %, IQ-TREE-BS = 100 %) comprises 32 previously known species, along with six newly proposed species: S. alniphilum, S. catenulatum, S. hirsutum, S. humicola, S. limosialveum, and S. nubiaquae. Dataset 4: The concatenated and aligned ITS, LSU, RPB2, and TEF1 sequences from four Chapter 4 288 Verruciconidia maritima CBS 385.96T Sesquicillium buxi CBS 202.69 Ramosiphorum sporodochiale CBS 554.80T Collarimyces guttiformis CBS 133486T Protocreopsis polyphialidica CBS 116130T Hydropisphaera peziza CBS 135908 Verrucostoma martinicensis CBS 138731T Lasionectriella herbicola CBS 140156T Clavatomyces prestoeae CBS 101691T Ramosiphorum polyporicola CBS 109.87 Clavatomyces korfii CBS 138710 Protocreopsis rutila CBS 396.66T Protocreopsis freycinetiae CBS 573.76T Lasionectria olida CBS 799.69T Paragliomastix luzulae CBS 935.69 Protocreopsis physciae CBS 149678T Fusariella atrovirens CBS 310.73 Paracylindrocarpon pandanicola KUMCC 17-0272T Paracylindrocarpon aloicola CBS 141300T Clavatomyces korfii CBS138733T Verruciconidia terricola CBS 431.66T Paragliomastix luzulae CBS 495.67 Sesquicillium buxi CBS 696.93T Protocreopsis spinulosa CBS 591.97T Protocreopsis caricicola CBS 140572T Hydropisphaera peziza CBS 139487 Lasionectriella rubioi CBS 140157T Clonostachys rosea CBS 710.86T Lasionectriopsis dentifera CBS 574.76T Verruciconidia indonesiana CBS 737.94T Septofusidium berolinense CBS 731.70 Ramosiphorum polyporicola CBS 123779T Protocreopsis finnmarkica CBS 147428T Clonostachys solani CBS 697.88T Clavatomyces pycnidialis CBS 102156T Nectriopsis violacea CBS 914.70T Verruciconidia thailandica CBS139715T Lasionectriopsis germanica CBS 143538T Paracylindrocarpon foliicola CBS 140758T Stephanonectria keithii CBS 943.72 Mycocitrus phyllostachydis CBS 330.69 Protocreopsis ellipsoidea CBS 112.70T Lasionectria mantuana CBS 114291 Pseudoacremonium sacchari CBS 137990T Ochronectria calami CBS 445.96 Verruciconidia verruculosa CBS 989.69T Lasionectria eichhorniae CBS 211.74T Protocreopsis vulpina CBS 565.76 Gliomastix masseei CBS 794.69T Geonectria subalpina CBS 143540T Hydropisphaera fungicola CBS 122304T Fusariella atrovirens CBS 311.73 Protocreopsis phormiicola CBS 567.76T Verruciconidia unguis CBS 424.93T Cannomyces spinulosus CBS 726.87T Lasionectria antillana CBS 122797T Nectriopsis violacea CBS 849.70 Lasionectria sp. LZ01 Lasionectria mantuana CBS 142926 Protocreopsis helvetica CBS 127989T Ochronectria thailandica MFLUCC 15-0140T Gliomastix roseogrisea CBS 134.56T Verruciconidia guizhouensis SQT04T Verruciconidia persicina CBS 310.59T Stephanonectria keithii CBS 100007 Lasionectria sansevieriae CBS 146973T Roumegueriella rufula CBS 346.85 Mycocitrus odorus CBS 100104T Verruciconidia siccicapita CBS 378.70AT Gliomastix murorum CBS 154.25T 98/100 52/- 96/100 91/100 0.2 74/- 99/100 76/98 96/100 56/98 -/98 98/100 87/100 72/100 97/100 74/100 95/100 70/100 56/100 -/93 66/99 90/100 -/100 92/100 76/100 56/100 54/96 59/99 76/100 51/- 99/100 98/99 99/100 59/99 -/98 78/97 60/99 96/100 99/100 92/100 XXXI. Bionectriaceae Hypocreales (continued) Fig. 1. (Continued). Revision of acremonium-like fungi 289 4 Lectera colletotrichoides IMI 303685 Stachylidium pallidum BCC 79031 Brunneomyces brunnescens CBS 559.73T Chlamydosporiella restricta CBS 178.40T Nigrocephalum collariferum CBS 124586T Brunneomyces hominis CBS 769.69 Lectera phaseoli IMI 366179T Lectera longa IMI 181698T Sodiomyces tronii CBS 137620 Brunneomyces europaeus CBS 560.86 Paramusicillium asperulatum CBS 120158 Verticillium alfalfae CBS 130.51 Chlamydosporiella restricta CBS 177.40 Verticillium isaacii CBS 100839 Acrostalagmus annulatus CBS 185.70 Monilochaetes infuscans CBS 869.96 Allomusicillium domschii CBS 764.69T Brunneomyces pseudozeylanicus CBS 560.73T Chlamydosporiella aerina CBS 598.70T Lectera colletotrichoides IMI 332702 Sodiomyces magadii CBS 137619T Brunneomyces hominis CBS 140682 Verticillium albo-atrum CBS 130340T Stachylidium pallidum DAOMC 226658 Acrostalagmus luteoalbus CBS 112.16 Verticillium albo-atrum CBS 120947 Nigrocephalum collariferum CBS 124585 Chlamydosporiella restricta CBS 443.66 Chlamydosporiella restricta CBS 988.69 Allomusicillium malicola CBS 359.80T Sodiomyces alcalophilus CBS 114.92T Verticillium alfalfae CBS 130341T Verticillium zaregamsianum CBS 130342T Musicillium tropicale CBS 458.51 Brunneomyces hominis FMR 10429T Brunneomyces polyphialidens CBS 166.80T Musicillium elettariae CBS 252.80T Lectera capsici CBS 142534T Verticillium zaregamsianum CBS 100842 Musicillium theobromae CBS 968.72T Brunneomyces europaeus CBS 100351 Sodiomyces alkalinus CBS 133680 Acrostalagmus luteoalbus CBS 121214 Monilochaetes infuscans CBS 379.77 Acremoniisimulans thailandensis MFLUCC 16-0372T Brunneomyces romanianus CBS 490.81T Verticillium isaacii CBS 130343T Titanomyces triconidiogenes CBS 681.94 Brunneomyces europaeus CBS 652.96T Sodiomyces tronii CBS 137618T Titanomyces triconidiogenes CBS 630.94T Sodiomyces alkalinus CBS 110278T Acrostalagmus annulatus CBS 121.84 Lectera humicola IMI 265740T Stachylidium bicolor CBS 121802T 94/99 98/100 92/98 73/95 82/97 86/99 88/99 84/97 68/97 90/100 90/100 90/98 74/95 56/91 55/92 94/99 99/100 4× 4× 2× 2× 2× I-1. Acrostalagmus I-2. Sodiomyces I-3. Titanomyces 2× I-4. Verticillium I-5. Brunneomyces 2× I-6. Allomusicillium I-7. Paramusicillium I-8. Musicillium I-9. Stachylidium I-10. Acremoniisimulans I-11. Nigrocephalum I-12. Chlamydosporiella I-13. Lectera 2× 2× 2× 2× 100/99 Fig. 2. The phylogenetic tree was constructed using Maximum Likelihood (RAxML) analysis, based on aligned and concatenated ITS, LSU, RPB2, and TEF1 sequences from 122 strains representing the Trichosphaeriaceae within the Trichosphaeriales, along with the outgroup. Numbers on the nodes represent Maximum Likelihood bootstrap values: RAxML-BS (≥ 50 %) listed first, followed by IQ-TREE support values (IQ-TREE-BS ≥ 90 %). Newly described species are labeled in red, and new combinations are shown in blue. The strains analysed in this study are highlighted in bold. Colored boxes indicate genera, while Roman numerals combined with Arabic numerals indicate genera nested within families, as shown in the legend. “T” denotes ex-type strains. The tree is rooted with Monilochaetes infuscans (CBS 379.77 and CBS 869.96) (Chaetosphaeriales, Australiascaceae). The scale bar represents the expected number of substitutions per site. Chapter 4 290 0.08 Chordomyces antarcticus CBS 138120 Plectosphaerella melonis CBS 683.88 Plectosphaerella cucumerina CBS 101014 Plectosphaerella melonis CBS 407.95 Plectosphaerella melonis CBS 409.95 Gibellulopsis fusca CBS 560.65T Plectosphaerella melonis CBS 410.95 Plectosphaerella melonis CBS 459.95 Summerbellia oligotrophica CBS 299.70G Paragibellulopsis chrysanthemi MAFF 243430 Musidium stromaticum CBS 135.74B Plectosphaerella melonis CBS 525.93 Summerbellia oligotrophica CBS 657.94T Musidium stromaticum CBS 863.73T Fuscohypha expansa CBS 103.95 Brunneochlamydosporium nepalense CBS 255.75A Sayamraella subulata BCC 78964T Gibellulopsis serrae CBS 127328 Brunneochlamydosporium nepalense CBS 971.72T Plectosphaerella cucumerina CBS 126914 Gibellulopsis serrae CBS 416.76 Brunneochlamydosporium macroclavatum CBS 413.80 Paragibellulopsis chrysanthemi MAFF 243429 Theobromium fuscum CBS 112271T Furcasterigmium furcatum CBS 299.70A Brunneochlamydosporium macroclavatum CBS 609.76 Chordomyces albus CBS 205.70 Fuscohypha expansa CBS 418.89T Summerbellia oligotrophica CBS 299.70H Trichosphaeria pilosa CBS 149698 Gibellulopsis serrae CBS 892.70T Plectosphaerella plurivora CBS 131742T Brunneochlamydosporium nepalense CBS 255.75C Gibellulopsis serrae CBS 127338 Chordomyces antarcticus CBS 120045T Brunneochlamydosporium nepalense CBS 682.74 Brunneochlamydosporium macroclavatum CBS 373.93 Furcasterigmium furcatum CBS 116548 Plectosphaerella melonis CBS 411.95 Chordomyces albus CBS 741.69 Gibellulopsis nigrescens CBS 120949T Paragibellulopsis chrysanthemi MAFF 242621 Phialoparvum bifurcatum CBS 299.70B Brunneochlamydosporium terrestre CBS 112777T Brunneochlamydosporium cibotii CBS 109240T Chordomyces albus CBS 124675 Chordomyces albus CBS 206.70 Plectosphaerella cucumerina CBS 131739T Chordomyces albus CBS 742.69 Furcasterigmium furcatum CBS 128188 Musidium stromaticum CBS 133.74 Chordomyces albus CBS 299.70E Chordomyces antarcticus CBS 120042 Furcasterigmium furcatum CBS 299.70C Brunneochlamydosporium nepalense CBS 255.75B Parafuscohypha proliferata CBS 308.74T Chordomyces antarcticus CBS 610.69 Plectosphaerella ramiseptata CBS 131861T Plectosphaerella ramiseptata CBS 131743 Plectosphaerella melonis CBS 489.96T Furcasterigmium furcatum CBS 122.42T Brunneochlamydosporium macroclavatum CBS 101249T Brunneochlamydosporium nepalense CBS 277.89 Musidium stromaticum CBS 132.74 Chordomyces albus CBS 204.70 Chordomyces albus CBS 987.87T Brunneochlamydosporium nepalense CBS 107.75 71/- 98/99 81/90 57/- 73/- 79/- 82/98 91/97 65/- 95/99 I-14. Gibellulopsis I-15. Chordomyces I-16. Furcasterigmium I-17. Theobromium I-18. Musidium I-19. Sayamraella I-20. Summerbellia I-21. Phialoparvum I-22. Paragibellulopsis I-23. Trichosphaeria I-26. Brunneochlamydosporium I-27. Plectosphaerella I-24. Parafuscohypha I-25. Fuscohypha 2× 2× 94/100 -/97 63/- 73/- 96/100 99/100 93/100 73/95 Fig. 2. (Continued). Revision of acremonium-like fungi 291 4 Parasarocladium kislosladkoense CBS 143525 Chlamydocillium acaciae CBS 523.72T Parasarocladium mabikii SFC20240607-M027T Chlamydocillium cyanophilum CBS 102685 Polyphialocladium fusisporum CBS 114602 Chlamydocillium simulans CBS 152313T Chlamydocillium cyanophilum CBS 246.74AT Parasarocladium aestuarinum CMG30T Chlamydocillium curvulum CBS 761.69 Parasarocladium mabikii SFC20240607-M026 Chlamydocillium viridicolor CBS 113575 Parasarocladium multimorphologicum SFC20240607-M024T Chlamydocillium terrestris CBS 110514T Parasarocladium kislosladkoense CBS 143522 Parasarocladium wereldwijsianum CBS 147226T Parasarocladium wereldwijsianum CBS 147224 Chlamydocillium curvulum CBS 117528 Parasarocladium kislosladkoense CBS 144937 Parasarocladium gamsii CBS 423.81 Chlamydocillium cyanophilum CBS 888.73 Parasarocladium dipikae BRIP68235aT Chlamydocillium curvulum CBS 430.66T Chlamydocillium simulans CBS 128171 Chlamydocillium curvulum CBS 333.92 Parasarocladium sp. CBS 147.49 Chlamydocillium curvulum CBS 825.70 Parasarocladium gamsii CBS 726.71T Chlamydocillium curvulum CBS 229.75 Gibellulopsis nigrescens CBS 120949T Parasarocladium kislosladkoense CBS 143519 Polyphialocladium fusisporum CBS 406.66T Chlamydocillium viridicolor CBS 113439T Parasarocladium tasmanniae CBS 146807T Plectosphaerella cucumerina CBS 131739T Parasarocladium funiculosum CBS 141.62T Parasarocladium sinense CGMCC 3.25521 Polyphialocladium margaretcollinsiae BRIP62736aT Parasarocladium debruynii CBS 144942T Parasarocladium kislosladkoense CBS 143523 Parasarocladium kislosladkoense CBS 143524 Chlamydocillium theobromae CBS 112098T Parasarocladium chondroidum CBS 652.93T Parasarocladium alavariense CMG32T Parasarocladium multimorphologicum SFC20240607-M023 Chlamydocillium curvulum CBS 101442 Chlamydocillium guttulatum CBS 384.70C Chlamydocillium antarcticum CBS 120502T Parasarocladium breve CBS 150.62T Parasarocladium fusiforme CMG36T Parasarocladium aestuarinum CMG31 Parasarocladium kislosladkoense CBS 143528T Chlamydocillium guttulatum CBS 104.78T Chlamydocillium lolii CBS 214.70T Chlamydocillium curvulum CBS 898.85 Chlamydocillium soli CBS 347.76T Parasarocladium kislosladkoense CBS 143529 Parasarocladium radiatum CBS 142.62T Plectosphaerella cucumerina CBS 101014 Parasarocladium breve CBS 102443 Parasarocladium sinense ZY22.072T Chlamydocillium curvulum CBS 127311 82/98 63/98 89/100 52/- 78/95 63/- 63/- 62/- 98/100 96/100 93/96 85/98 99/100 97/100 89/100 80/99 83/96 4× 4× 2× III-1. Polyphialocladium 2× 2× 2× III-2.Chlamydocillium III-3. Parasarocladium Fig. 3. The phylogenetic tree was constructed using Maximum Likelihood (RAxML) analysis, based on aligned and concatenated ITS, LSU, RPB2, and TEF1 sequences from 162 strains representing Sarocladiaceae within Hypocreales, along with the outgroups. Numbers on the nodes represent Maximum Likelihood bootstrap values: RAxML-BS (≥ 50 %) listed first, followed by IQ-TREE support values (IQ-TREE-BS ≥ 90 %). Newly described species are labeled in red, and new combinations are shown in blue. The strains analysed in this study are highlighted in bold. Colored boxes indicate genera, while Roman numerals combined with Arabic numerals indicate genera nested within families, as shown in the legend. “T” denotes ex-type strains. The tree is rooted with Gibellulopsis nigrescens (CBS 120949), Plectosphaerella cucumerina (CBS 101014 and CBS 131739) (Trichosphaeriales, Trichosphaeriaceae). The scale bar represents the expected number of substitutions per site. Chapter 4 292 Sarocladium strictum CBS 319.70A Sarocladium hirsutum CBS 376.70KT Sarocladium bifurcatum CBS 137658T Sarocladium bacillisporum CBS 388.67 Sarocladium summerbellii CBS 430.70T Sarocladium strictum CBS 334.77 Sarocladium strictum CBS 286.70G Sarocladium dejongiae CBS 144929T Sarocladium strictum CBS 128169 Sarocladium strictum CBS 376.70G Sarocladium nubiaquae CBS 117135T Sarocladium ochraceum CBS 428.67T Sarocladium citri CBS 145044T Sarocladium fuscum CBS 334.80T Sarocladium subulatum MUCL 9939T Sarocladium strictum CBS 287.70H Sarocladium strictum CBS 436.66 Sarocladium caricicola CBS 150792T Sarocladium liquanense ACCC39306T Sarocladium bactrocephalum CBS 749.69T Sarocladium strictum CBS 287.70J Sarocladium terricola CBS 131566 Sarocladium mali ACCC39308T Sarocladium limosialveum CBS 143532T Sarocladium glaucum CBS 796.69T Sarocladium strictum CBS 376.70E Sarocladium theobromae CBS 683.94 Sarocladium alniphilum CBS 113444T Sarocladium summerbellii CBS 797.69 Sarocladium strictum CBS 376.70H Sarocladium strictum CBS 287.70K Sarocladium dejongiae CBS 287.70G Sarocladium catenulatum CBS 125892T Sarocladium bacillisporum CBS 425.67T Sarocladium terricola CBS 131567 Sarocladium terricola CBS 243.59T Sarocladium mali ACCC39307 Sarocladium sasijaorum CBS 147213T Sarocladium strictum CBS 288.70A Sarocladium strictum CBS 376.70F Sarocladium strictum CBS 288.70B Sarocladium implicatum CBS 128323 Sarocladium implicatum CBS 959.72T Sarocladium theobromae CBS 113440T Sarocladium dejongiae CBS 110721 Sarocladium strictum CBS 346.70T Sarocladium strictum CBS 376.70I Sarocladium pseudostrictum CBS 137660T Sarocladium gamsii CBS 707.73T Sarocladium implicatum CBS 109930 87/96 70/95 90/97 88/100 96/100 94/100 80/92 99/100 95/100 99/100 86/100 98/100 89/99 91/99 99/100 99/100 91/97 93/99 68/- III-4. Sarocladium Fig. 3. (Continued). genes and 540 taxa belong to the Bionectriaceae, with Tilachlidium brachiatum (CBS 363.97, CBS 505.67), Flammocladiella anomiae (CBS 142775), F. aceris (CBS 138906) and F. decora (CBS 142776) serving as outgroups (Hypocreales, Tilachlidiaceae and Flammocladiellaceae; Fig. 4). The final alignment consisted of 3277 characters (including gaps), with the following partitions: ITS: 1–871, 871 bp; LSU: 872–1663, 792 bp; RPB2: 1664–2459, 796 bp; TEF1: 2460–3277, 818 bp. Of these, 1528 character sites were conserved (ITS: 270, LSU: 556, RPB2: 263, TEF1: 439), 1670 were variable (ITS: 534, LSU: 228, RPB2: 530, TEF1: 378), and Revision of acremonium-like fungi 293 4 0.2 Sarocladium humicola CBS 446.54T Sarocladium kiliense CBS 116434 Sarocladium zeae CBS 293.70 Sarocladium zeae CBS 292.70B Sarocladium zeae CBS 119968 Sarocladium agaricicola CBS 126941 Sarocladium kiliense CBS 125062 Sarocladium kiliense CBS 400.52 Sarocladium attenuatum CBS 399.73T Sarocladium kiliense CBS 155.61 Sarocladium kiliense CBS 377.70G Sarocladium kiliense CBS 139.37 Sarocladium junci CBS 148277T Sarocladium kiliense CBS 123.29 Sarocladium kiliense CBS 377.70B Sarocladium kiliense CBS 377.70I Sarocladium kiliense CBS 152.61 Sarocladium kiliense CBS 185.63 Sarocladium kiliense CBS 377.70H Sarocladium kiliense CBS 156.61 Sarocladium sparsum BCRC FU31121T Sarocladium zeae CBS 119967 Sarocladium agaricicola CBS 113717T Sarocladium kiliense CBS 145.62 Sarocladium kiliense CBS 158.61 Sarocladium kiliense CBS 125069 Sarocladium kiliense CBS 104.64 Sarocladium kiliense CBS 377.70J Sarocladium spinificis BCRC 34941T Sarocladium kiliense CBS 377.70C Sarocladium spinificis CBS 102676 Sarocladium kiliense CBS 142819 Sarocladium kiliense CBS 377.70A Sarocladium kiliense CBS 122.29T Sarocladium spirale BCRC FU31117T Sarocladium oryzae CBS 180.74T Sarocladium kiliense CBS 127.32 Sarocladium zeae CBS 802.69 Sarocladium hominis CBS 137659T Sarocladium kiliense CBS 131115 Sarocladium zeae CBS 800.69T Sarocladium zeae CBS 119969 Sarocladium kiliense CBS 377.70E Sarocladium kiliense CBS 377.70F Sarocladium kiliense CBS 128.21 Sarocladium kiliense CBS 124.26 Sarocladium kiliense CBS 135.35 Sarocladium brachiariae CGMCC 2192T Sarocladium zeae CBS 801.69 Sarocladium kiliense CBS 157.61 Sarocladium ferrugineum CBS 102673T 99/100 99/100 58/- 83/94 91/100 99/100 94/100 75/90 III-4. Sarocladium 1475 were parsimony informative characters (ITS: 458, LSU: 193, RPB2: 488, TEF1: 336). The phylogeny presented in Fig. 4 was the RAxML tree based on the combined dataset, with bootstrap support values from both RAxML (RAxML-BS > 50 %) and IQ-TREE (IQ-TREEBS > 90 %) analyses plotted on the branches. The topologies observed in both the RAxML and IQ-TREE analyses were largely consistent. Fig. 3. (Continued). Chapter 4 294 Nectriopsis cribrariae CBS 828.70T Flammocladiella decora CBS 142776 Nectriopsis sporangiicola CBS 385.78 Mycocitrus coccicola BUcCo Sesquicillium buxi CBS 202.69 Nectriopsis microspora CBS 954.72 Sesquicillium candelabrum CBS 119045T Nectriopsis rexiana CBS 305.70A Nectriopsis fuliginicola CBS 400.82T Mycocitrus coxeniae BRIP 49599aT Nectriopsis didymii CBS 852.70AT Mycocitrus odorus CBS 100104T Clonostachys epichloe CBS 101037T Nectriopsis fuliginicola CBS 491.67 Nectriopsis rexiana CBS 248.70 Nectriopsis violacea CBS 744.83 Nectriopsis ellipsoidea CBS 358.78T Nectriopsis rexiana CBS 401.66 Tilachlidium brachiatum CBS 363.97 Nectriopsis violacea CBS 914.70T Nectriopsis candicans CBS 701.79T Sesquicillium rossmaniae CBS 211.93T Tilachlidium brachiatum CBS 505.67 Mycocitrus phyllostachydis CBS 330.69 Sesquicillium candelabrum CBS 513.67 Mycocitrus odorus CBS 120610 Clonostachys rogersoniana CBS 139289 Nectriopsis sporangiicola CBS 166.74T Nectriopsis didymii CBS 579.73 Nectriopsis violacea CBS 849.70 Clonostachys solani f. nigrovirens CBS 183.30T Sesquicillium thailandense CBS 139546T Clonostachys buxicola CBS 102419T Nectriopsis lindauiana CBS 897.70T Mycocitrus synnematus CBS 126677T Stephanonectria keithii CBS 943.72 Mycocitrus odorus CBS 109504 Nectriopsis floccosa CBS 126110T Clonostachys rosea f. catenulata CBS 154.27T Nectriopsis microspora CBS 933.69T Sesquicillium rossmaniae CBS 221.93 Nectriopsis fuliginicola CBS 399.82 Clonostachys miodochialis CBS 997.69T Collarimyces guttiformis CBS 133486T Mycocitrus coccicola BUcS Stephanonectria chromolaenae MFLUCC 18-0589T Sesquicillium sesquicillii CBS 180.88T Nectriopsis rexiana CBS 305.70C Nectriopsis candicans CBS 440.65 Clonostachys pityrodes CBS 102033T Stephanonectria keithii CBS 100007 Flammocladiella aceris CBS 138906T Sesquicillium buxi CBS 696.93 Nectriopsis cribrariae CBS 645.82 Flammocladiella anomiae CBS 142775 Clonostachys grammicospora CBS 209.93T Clonostachys rogersoniana CBS 920.97T Clonostachys solani CBS 697 88T Mycocitrus zonatus CBS 400.70T Nectriopsis didymii CBS 326.79 Sesquicillium symmetricum CBS 485.78 Clonostachys rosea CBS 710.86T Sesquicillium symmetricum CBS 124.79T 99/100 9 8/100 87/98 86/100 79/100 50/97 71/- XXXI-6.Clonostachys XXXI-5.Sesquicillium XXXI-1.Nectriopsis XXXI-3.Stephanonectria XXXI-4.Mycocitrus XXXI-2.Collarimyces 81/98 95/99 58/- 56/92 90/100 61/98 95/100 81/95 87/98 91/99 92/98 88/100 76/100 98/100 99/100 Fig. 4. The phylogenetic tree was constructed using Maximum Likelihood (RAxML) analysis, based on aligned and concatenated ITS, LSU, RPB2, and TEF1 sequences from 540 strains representing Bionectriaceae within Hypocreales, along with the outgroups. Numbers on the nodes represent Maximum Likelihood bootstrap values: RAxML-BS (≥ 50 %) listed first, followed by IQ-TREE support values (IQ-TREE-BS ≥ 90 %). Newly described species are labeled in red, and new combinations are shown in blue. The strains analysed in this study are highlighted in bold. Colored boxes indicate families,while Roman numerals combined with Arabic numerals indicate genera nested within families, Revision of acremonium-like fungi 295 4 Protocreopsis phormiicola CBS 567.76T Ramosiphorum thailandicum CBS 101914T Lasionectriopsis dentifera CBS 574.76T Protocreopsis gallica CBS 135079T Lasionectriella marigotensis CBS 131606T Protocreopsis polyphialidica CBS 116130T Lasionectriella rubioi CBS 140157T Ochronectria thailandica MFLUCC 15-0140T Lasionectriopsis stereicola CBS 101910 Protocreopsis physciae CBS149679 Lasionectriopsis germanica CBS 113762 Protocreopsis loweniae Rodriguez-Flakus 4000T Ochronectria calami CBS 445.96 Protocreopsis finnmarkica CBS 147427 Ochronectria calami CBS 454.96 Clavatomyces prestoeae CBS 101691T Protocreopsis rutila CBS 225.70 Lasionectriopsis dentifera CBS 650.75 Protocreopsis chlamydospora CBS 141859 Lasionectriopsis germanica CBS 143538T Protocreopsis freycinetiae CBS 573.76T Protocreopsis caricicola CBS 110505 Clavatomyces palmarum HKAS115734 Clavatomyces palmarum HKAS115709T Ramosiphorum echinoporiae CBS 115288T Ochronectria calami CBS 123492 Protocreopsis rutila CBS 229.70 Ramosiphorum polyporicola CBS 109.87 Protocreopsis euphorbiae CPC 38896T Protocreopsis globulosa KRAM L-75079T Protocreopsis finnmarkica CBS 147428T Lasionectriopsis pteridii CBS 579.90 Protocreopsis gallica CBS 141241 Protocreopsis physciae CBS149678T Lasionectriella herbicola CBS 140156T Ramosiphorum polyporicola CBS 123779T Clavatomyces pycnidialis CBS 102156T Protocreopsis spinulosa CBS 591.97T Protocreopsis caricicola CBS 140572T Protocreopsis euphorbiae CBS 146972 Lasionectriopsis pteridii CBS 782.69T Lasionectriella rubioi CBS 132543 Protocreopsis vulpina CBS 565.76 Ramosiphorum sporodochiale CBS 554.80T Protocreopsis ellipsoidea CBS 112.70T Clavatomyces korfii CBS 138710 Lasionectriopsis stereicola CBS 123299T Protocreopsis helvetica CBS 127989T Protocreopsis pertusa CBS 568.76 Lasionectriella arenuloides CBS 576.76T Protocreopsis chlamydospora CBS 144254T Protocreopsis rutila CBS 399.66 Clavatomyces korfii CBS 138733T Lasionectriopsis pteridii CBS 138752 Protocreopsis rutila CBS 396.66T Ramosiphorum echinoporiae CBS 120607 100 2× XXXI-7. Protocreopsis XXXI-8.Clavatomyces XXXI-9.Ochronectria XXXI-10.Lasionectriella XXXI-11.Ramosiphorum XXXI-12.Lasionectriopsis 78/100 95/100 90/99 99/100 58/99 61/- 78/98 52/95 -/95 -/97 53/92 69/99 75/100 67/100 90/100 88/99 54/- 70/- 54/- 98/100 95/100 99/100 99/100 Fig. 4. (Continued). The Bionectriaceae phylogenetic tree (Fig. 4) contains Clades XXXI-1–XXXI-53, which represent the 51 well-supported, previously accepted genera of Bionectriaceae, along with two genera proposed in this study. The following clades include taxonomic novelties and strains of known species that were examined in this study. Clade XXXI-7 (RAxML-BS = 90 as shown in the legend. “T” denotes ex-type strains. The tree is rooted with Tilachlidium brachiatum (CBS 363.97, CBS 505.67), Flammocladiella anomiae (CBS 142775), F. aceris (CBS 138906) and F. decora (CBS 142776) (Hypocreales, Tilachlidiaceae & Flammocladiellaceae). The scale bar represents the expected number of substitutions per site. Chapter 4 296 Geonectria alni CBS 140756T Verruciconidia guizhouensis SQT04T Lasionectria cerealis CBS 208.70 Lasionectria olida CBS 798.69 Lasionectria boothii CBS 129747 Hydropisphaera aurantiaca CBS 201.35 Lasionectria antillana CBS 122797T Paragliomastix venezuelana CBS 102074T Verruciconidia unguis CBS 424.93T Geonectria quercus CBS 137308T Lasionectria chondroid CBS 565.73T Lasionectria phormii CBS 102040T Pseudoacremonium sacchari CBS 137990T Hydropisphaera heliconiae CBS 138704T Lasionectria sylvana CBS 566.76 Verruciconidia persicina CBS 310.59T Hydropisphaera solani CBS 147425T Lasionectria cerealis CBS 393.66 Hydropisphaera suffulta CBS 122.87 Verruciconidia verruculosa CBS 989.69T Hydropisphaera peziza CBS 139487 Paragliomastix znieffensis CBS 140584T Lasionectria sansevieriae CBS 146973T Hydropisphaera armeniaca CBS 135905T Verruciconidia guizhouensis SQT05 Lasionectria castaneicola CBS 122792T Lasionectria mantuana CBS 114291 Verruciconidia thailandica CBS 139715T Verruciconidia verruculosa CBS 398.73 Paragliomastix luzulae CBS 495.67 Verruciconidia maritima CBS 385.96T Lasionectria olida CBS 799.69T Verruciconidia saulensis CLLG21159T Lasionectria mantuana CBS 142926 Lasionectria atrorubra CBS 123502T Paragliomastix chiangraiensis MFLUCC 14-0397T Verruciconidia quercina CBS 114227 Hydropisphaera cyatheae CBS 575.76 Paragliomastix chiangraiensis CBS 277.80B Paragliomastix rosea CBS 277.80AT Cannomyces spinulosus CBS 726.87T Verruciconidia erythroxyli CBS 378.70D Geonectria subalpina CBS 143540T Lasionectria sp. LZ01 Verruciconidia unguis CBS 378.70E Verruciconidia persicina CBS 113716 Lasionectria krabiense MFLUCC 15-0673T Lasionectria bisepta CBS 227.70 Lasionectria lecanodes CBS 139482 Verruciconidia quercina CBS 469.67T Parageonectria arachispora CBS 118.87T Hydropisphaera aurantiaca CBS 397.67 Verruciconidia erythroxyli CBS 728.87T Lasionectria eichhorniae CBS 211.74T Septofusidium berolinense CBS 731.70 Verruciconidia quercina CBS 183.78 Hydropisphaera martinicensis CBS 140578 Verruciconidia infuscata CBS 100888T Verruciconidia terricola CBS 431.66T Hydropisphaera martinicensis CBS 136679T Lasionectria bisepta CBS 750.69T Verruciconidia terricola CBS 568.74 Hydropisphaera peziza CBS 135908 Hydropisphaera fungicola CBS 123912 Hydropisphaera fungicola CBS 122304T Verruciconidia indonesiana CBS 737.94T Paragliomastix luzulae CBS 935.69 Verruciconidia persicina CBS 295.70N Lasionectria antillana CBS 685.94 Hydropisphaera gossypina CBS 140581T Lasionectria bisepta CBS 753.69 Verruciconidia persicina CBS 295.70E Verruciconidia siccicapita CBS 378.70AT 97/100 53/94 98/100 87/100 -/99 55/94 86/100 94/100 -/99 4× 2× 2× 2× XXXI-13.Cannomyces XXXI-14.Lasionectria XXXI-15.Verruciconidia XXXI-16.Pseudoacremonium XXXI-17.Septofusidium XXXI-18. Paragliomastix XXXI-19.Parageonectria XXXI-20.Geonectria XXXI-21. Hydropisphaera 2× 99/100 98/100 69/100 52/- 89/100 94/97 78/97 99/100 98/100 97/100 62/99 98/100 98/100 97/100 88/99 97/100 97/100 81/100 64/100 60/96 72/97 77/97 60/97 60/98 79/100 99/100 99/100 89/100 97/100 89/100 2× 2× Fig. 4. (Continued). Revision of acremonium-like fungi 303 4 TREE-BS = 100 %), Clade XXXI-49 (Waltergamsia; RAxML-BS = 100 %, IQ-TREE-BS = 100 %), and Clade XXXI-52 (Emericellopsis; RAxML-BS = 75 %, IQ-TREE-BS = 99 %). Taxonomy Phylogenetic analyses based on multi-locus sequence alignments were conducted on 402 strains of acremonium-like fungi. These strains represent 148 species, belonging to 17 families within Hypocreales, and one family, Trichosphaeriaceae, within Trichosphaeriales. This study proposes two new families and seven new genera, along with the description of 34 new species and three new combinations. One new sterile species is described based on DNA sequence data, following the methodology of Hou et al. (2023). Taxonomic arrangement follows phylogenetic positions, with families and genera arranged according to the clade numbering system (Figs 1–4). In Fig. 1, families are numbered with Roman numerals. In Figs 2–4, genera within Trichosphaeriaceae (Fig. 2), Sarocladiaceae (Fig. 3), and Bionectriaceae (Fig. 4) are arranged using a combination of Roman and Arabic numerals to indicate genera nested within families. Species within each genus are listed alphabetically. Clade I. Trichosphaeriaceae G. Winter [as ‘Trichosphaerieae’], Rabenh. Krypt.-Fl., Edn 2 (Leipzig): 191. 1885. Synonym: Plectosphaerellaceae W. Gams et al., Nova Hedwigia 85: 476. 2007. Classification: Trichosphaeriales, Sordariomycetes. Type genus: Trichosphaeria Fuckel Clade I-3. Titanomyces Lin Zhao & Crous, gen. nov. MycoBank MB 858416. Etymology: Referring to the ancient Greek word τίτανος (títanos), meaning “lime” or “alkaline earth” materials such as gypsum and white clay, it references the alkaline nature of the substrate from which the strains of this genus were isolated. Mycelium consisting of branched, hyaline, septate, smooth or rough, thin-walled hyphae. Sporulation abundant, phalacrogenous, nematogenous. Conidiophores arising from the agar surface and aerial hyphae, solitary or aggregated, (sub-)erect, unbranched or basitonously branched, commonly with repeated percurrent or sympodial proliferation, showing conidiogenous cells as short lateral and cylindrical asymmetrical projections, hyaline, smooth-walled. Conidiogenous cells monophialidic or polyphialides, terminal or lateral, cylindrical, or subulate, straight or curved, hyaline, thickand smooth-walled, with conspicuous collarette and periclinal thickening at conidiogenous locus; with percurrent or [Document text truncated for crawler view.]