A novel, exclusively lichen-inhabiting lineage of hypocrealean fungi revealed in the Sordariomycetes

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ISSN (Online) 1878-9080 https://doi.org/10.3114/persoonia.2025.54.02 Persoonia 54, 2025: 47–91 https://www.persoonia.org RESEARCH ARTICLE A novel, exclusively lichen-inhabiting lineage of hypocrealean fungi revealed in the Sordariomycetes V. Darmostuk1*, J. Etayo2, P. Rodriguez-Flakus1, M. Kukwa3, R. Pino-Bodas4,5, S. Pérez-Ortega6, A. Flakus1 1W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Kraków, Poland 2Navarro Villoslada 16, 3º dcha, 31003 Pamplona, Navarra, Spain 3Department of Plant Taxonomy and Nature Conservation, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, PL-80-308 Gdańsk, Poland 4Instituto de Investigación en Cambio Global (IICG-URJC), Universidad Rey Juan Carlos, Tulipán s/n, 28933 Móstoles, Spain 5Departamento de Biología y Geología, Física y Química Inorgánica, Rey Juan Carlos University, Tulipán s/n, 28933 Móstoles, Madrid, Spain 6Real Jardín Botánico, CSIC, Claudio Moyano 1, E-28014 Madrid, Spain *Corresponding author: [email protected] Abstract: Lichen-inhabiting (lichenicolous) fungi comprise a considerable portion of the Hypocreales (Sordariomycetes), their placement and phylogenetic relationships within the order remain largely unknown due to a lack of available molecular data. This study focuses mainly on tropical lichenicolous hypocrealean fungi which were neglected for a long time. Increasing knowledge of this fungal group is crucial to better understanding the complex evolutionary histories and trophic strategies of the Hypocreales. Through an order-wide phylogeny based on multiple loci, we unveiled a novel lineage within the Hypocreales, composed exclusively of lichenicolous species from genera such as Ovicuculispora and Paranectria, along with Nectriopsis lichenophila and Nectria byssophila-like taxa. Beyond the strong phylogenetic support, the clade is also characterized by its distinct morphology. Here it is introduced as a new family Paranectriaceae characterized by yellow to orange sessile ascomata, featuring a distinct tomentum, and by pigment in ascomata walls that do not change colour in KOH solution. The delimitation of interspecific and generic boundaries within the novel family was based on molecular, morphological and ecological data. As a result, we established nine species and five genera, including two genera new to science (Rossmaniella and Sphaeronectria) and four new species (Rossmaniella coenogonii, R. cryptica, R. filispora, and R. tylophori). Additionally, we reinstated the genus Ciliomyces, with the type species Ciliomyces oropensis, from the synonyms of Paranectria. Our results also show that the genus Neobaryopsis is more closely related to the family Calcarisporiaceae than to the Cordycepitaceae, as originally described. A key to species determination within Paranectriaceae is provided. The present study suggests that neglected lichenicolous fungi are an important component that appears in Hypocreales several times during their evolution, and indicates that their considerable diversity can still be hidden. Citation: Darmostuk V, Etayo J, Rodriguez-Flakus P, Kukwa M, Pino-Bodas R, Pérez-Ortega S, Flakus A (2025). A novel, exclusively licheninhabiting lineage of hypocrealean fungi revealed in the Sordariomycetes. Persoonia 54: 47–91. doi: 10.3114/persoonia.2025.54.02 Received: 14 April 2024; Accepted: 22 March 2025; Effectively published online: 17 April 2025 Corresponding editor: J. Houbraken Key words: Ciliomyces Hypocreales lichenicolous fungi Nectria byssophila new taxa Paranectria Paranectriaceae Rossmaniella Sphaeronectria taxonomy INTRODUCTION Fungi represent one of the three major eukaryotic life forms on our planet, alongside plants and animals (Hawksworth 2001, Hawksworth & Lücking 2017, Burki et al. 2019). With 150000 known species (Phukhamsakda et al. 2022), fungi are essential components of all ecosystems, providing ecological functions ranging from beneficial symbionts through antagonistic pathogens to decomposers of organic matter and ecosystem services (Pringle et al. 2011). One of the largest groups of fungi are Sordariomycetes which are represented by 10500 species (Maharachchikumbura et al. 2016). This fungal class is characterized by perithecioid ascomata and unitunicate asci, and its large part consists of Hypocreales. All major nutritional modes, such as saprotrophs, endophytes, parasites of insects, plants, other fungi, and lichen, are represented in Sordariomycetes (e.g. Hyde et al. 2020). They are ubiquitous, with a cosmopolitan distribution, playing an important role as decomposers and in nutrient cycling (Zhang et al. 2006, Zhang & Wang 2015). However, they constitute one of the least understood groups of fungi (Maharachchikumbura et al. 2016). Hypocreales is comprised of more than 6000 species and 300 genera in several families and has become one of the most diverse orders in Sordariomycetes (Hyde et al. 2020). Members of Hypocreales are characterized by unitunicate asci produced within usually fleshy, lightly to brightly coloured (rarely blackish), uniloculate, typically ostiolate ascomata with various hyphomycetous asexual morphs (Rogerson 1970, Rehner & Samuels 1995). Hypocreales have been extensively studied in recent years, but their taxonomy, phylogeny, diversity, distribution, and ecology still require Persoonia – Volume 54, 202548 thorough revision and are far from being well understood (Giraldo et al. 2015, Maharachchikumbura et al. 2015, 2016, Hongsanan et al. 2017, Gams et al. 2019, Hyde et al. 2020, Hou et al. 2023, Li et al. 2023, Perera et al. 2023, Sun et al. 2023, Yu et al. 2024). Currently, Hypocreales is comprised of 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. The order has a long and complex taxonomical history and, due to its morphological diversity, several attempts have been made to circumscribe it. It was introduced by Lindau (1897) to accommodate the family Hypocreaceae with Hypocrea as the type genus. Subsequently, Seaver (1909) accepted two families, Nectriaceae and Hypocreaceae, in the order. His classification relied on morphological and anatomical features of ascomata, and this taxonomic hypothesis was supported by several researchers (Petch 1938, Kreisel 1969). The family Hypocreaceae includes the species with often disarticulating ascospores and yellow to orange, KOH+ perithecia that are mostly immersed in a stroma or sessile on a subiculum. In contrast, the Nectriaceae include fungi with red to dark purple, KOH+ ascomata, and nonto multiseptate and muriform ascospores. The most recent and comprehensive revision of Nectriaceae was published by Lombard et al. (2015), who, based on a multi-gene phylogeny, re-assessed the family and accepted 47 genera. Currently, Nectriaceae remains one of the best-studied families in Hypocreales and includes numerous species with wide practical applications (Lombard et al. 2015, Crous et al. 2021). Niessliaceae was established to accommodate Niesslia species (Kirschstein 1939) and it was classified as Ascomycota incertae sedis for a long time. Based on hamathecium features, e.g., short paraphyses, periphysate ostiole, dark pigmented peridium and phialidic conidiogenous cells Acremonium or Monocillium-type, as well as molecular relationship, it was emplaced in the order Hypocreales (Samuels & Barr 1997, Jaklitsch & Voglmayr 2012, Maharachchikumbura et al. 2015, 2016, Hongsanan et al. 2017, Sun et al. 2017, Hyde et al. 2020). So far, the family includes 20 odd genera of saprobic, parasitic and lichenicolous fungi (Müller & von Arx 1962, Barr 1990, Samuels & Barr 1997, Hyde et al. 2020, Huang et al. 2021, Crous et al. 2023). However, DNA sequences are lacking for many genera of Niessliaceae, and a comprehensive phylogenetic study is needed to circumscribe it and establish the phylogenetic relationships within the family (Maharachchikumbura et al. 2016, Huang et al. 2021). The family name Clavicipitaceae was initially used by Earle (1901) for a group previously treated as a subfamily within Hypocreaceae. Rogers (1979) revised the clavicipitalen fungi, i.e., species with cylindrical asci, thickened ascus apices and filiform ascospores, which often disarticulate into part-spores, and classified them in to separate order Clavicipitales. However, early phylogenetic analyses showed that the Clavicipitaceae belonged to the order Hypocreales (Spatafora & Blackwell 1993, Rehner & Samuels 1995). Sung et al. (2007) divided the Clavicipitaceae into three families based on the phylogenetic results and morphological features: Clavicipitaceae (with dark to bring-coloured solid ascomata), Cordycipitaceae (with immersed to superficial perithecia on the fleshy stroma or on pallid to bring coloured subiculum) and Ophiocordycipitaceae (with darkly pigmented stromata that are flexible to wiry). Recently, a new family, Polycephalomycetaceae, was described to accommodate genera Polycephalomyces, Perennicordyceps, and Pleurocordyceps, which were previously included in the family Ophiocordycipitaceae (Xiao et al. 2023). This family was separated based on morphological differences, such as stromatic ascomata with a thick peridium and cylindrical secondary spores, as well as the results of multi-locus phylogenetic analyses. The Bionectriaceae was introduced by Rossman et al. (1999) to accommodate genera with immersed to superficial perithecioid ascomata that are white, yellow, orange to tan or brown, without colour reaction in KOH or lactic acid. These authors included 26 genera in Bionectriaceae which primarily consist of herbicolous, corticolous, lichenicolous or fungicolous species. Later, Rossman et al. (2001) conducted the first phylogenetic assessment of this family based on the LSU gene sequences, showing that the family forms a monophyletic lineage within Hypocreales. In the last decade, molecular data has been used to comprehensively revise several groups within the Hypocreales, leading in updated classifications and a significant increase in the number of accepted families. Crous et al. (2014) established the Stachybotryaceae to accommodate the genera Myrothecium, Peethambara and Stachybotrys, which previously were classified as Hypocreales incertae sedis. Species of this family are characterized by a hyphomycetes asexual morphs with mononematous to sporodochial to synnematous conidiomata, with phialidic conidiogenous cells and 0–1-septate conidia in dark green to black slimy masses (Castlebury et al. 2004, Crous et al. 2014, Lombard et al. 2016). Another family, the Tilachlidiaceae, was introduced by Lombard et al. (2015) based on multi-gene phylogenetic analyses and it includes two genera with synnematous conidiomata, Tilachlidium and Septofusidium. Recently, Psychronectria was described (Pawłowska et al. 2017), representing the first sexual genus within Tilachlidiaceae. Flammocladiellaceae (type genus Flammocladiella) was distinguished by Crous et al. (2015) based on the phylogenetic analyses of the LSU gene sequences and morphological characters, such as flame-like conidial masses formed in sporodochia. The genus Flammocladiella is represented by two fungicolous species, Flammocladiella anomiae and F. decora (Crous et al. 2015, Lechat & Fournier 2018). Sun et al. (2017) described Calcarisporiaceae (type genus Calcarisporium) based on multi-locus molecular data and the combination of the following morphological characters: hyaline, erect, verticillate conidiophores and sympodial, polyblastic conidiogenesis. Currently, this family includes the fungicolous genus Calcarisporium and the saprotrophic genus Verticimonosporium (Sun et al. 2017, Perera et al. 2023). In the same year, Cocoonihabitaceae was described by Zhuang & Zeng (2017) to accommodate the genus Cocoonihabitus. The genus formed a distinct clade in a multi-locus phylogeny and it is characterized by the following morphological features: asci with a thickened apical Darmostuk et al.: New lineage of hypocrealean fungi 49 cap penetrated by a narrow pore as well as on specific host preferences (Zhuang & Zeng 2017). The family Sarocladiaceae was introduced by Crous et al. (2018a) to accommodate a distinctive lineage previously included within the Bionectriaceae. This lineage consists of the asexual genera with stained verrucous hyaline to brown phialidic conidiogenous cells and fusiform aseptate conidia, such as Parasarocladium and Sarocladium. Myrotheciomycetaceae was introduced by Crous et al. (2018b) with the type genus Myrotheciomyces. The family, which appeared as a monophyletic distinct lineage in a phylogeny based on LSU sequences, is characterized by the presence of phialidic conidiogenous cells, as well as 0–1-septate conidia aggregated in a slimy mass. These authors also included the asexual genera Emericellopsis, Leucosphaerina, and Trichothecium into this family, which previously classified as Hypocreales incertae sedis. However, recent phylogenetic studies conducted by Hou et al. (2023) placed the genus Emericellopsis in the Bionectriaceae. In addition, several new families have been recently identified through mult-gene phylogenetic analyses and morphological features. These include Ijuhyaceae, Stromatonectriaceae and Xanthonectriaceae, which have been segregated from Bionectriaceae (Perera et al. 2023). Additionally, two families, Albomorchellophilaceae and Pseudodiploosporeaceae, which included fungal parasites were established (Sun et al. 2023, Yu et al. 2024). Furthermore, a comprehensive study of fungal isolates from mangrove sediments allowed to establish the families Acremoniopsiaceae and Sedecimiellaceae (Li et al. 2023). Moreover, a recent revision of Acremonium-like fungi in Hypocreales, based on multi-gene phylogeny, has revealed five new hypocrealean families: Chrysonectriaceae, Neoacremoniaceae, Nothoacremoniaceae, Pseudoniessliaceae and Valsonectriaceae (Hou et al. 2023). The family delimitation within the order Hypocreales remains unclear due to the morphological variability of species, particularly in their asexual morphs (e.g., Acremonium-like, Clonostachyslike species) and the lack of sampling from certain geographic regions. Lichenicolous fungi form a distinct group of fungi that showed parasitic, parasymbiotic, or saprotrophic relationships with lichens, developing sporocarps on their thalli or reproductive structures. These fungi interact with lichens in various ways, ranging from symptomless associations to more aggressive parasitism, influencing the functionality of the lichen (Lawrey & Diederich 2003, Hafellner & Obermayer 2009, Diederich et al. 2018, 2022, Hafellner 2018). Over 2300 species of these fungi have been described, although it is estimated that the total number could be at least twice as high. These fungi primarily belong to Ascomycota, with a smaller portion classified under Basidiomycota, and they showed a high degree of specialization toward their lichen hosts (Diederich et al. 2018, 2022). However, uncertainties in species boundaries can obscure the full extent of host specificity. The exploration of poorly known groups of Hypocreales and specific ecological niches led to the discovery of several novel lineages, which were formally described as the families and listed above. However, further studies in some still neglected groups, including lichen-inhabiting species, may substantially contribute to our knowledge of Hypocreales. Lichenicolous fungi constitute a significant part of Hypocreales with ca 180 currently known species, which is about 7.7 % of all known lichenicolous fungi (Lawrey & Diederich 2003, Diederich et al. 2018). Therefore, it is essential to include a broad sampling of lichenicolous fungi in further phylogenetic studies to clarify their systematics and gain a better understanding of the evolutionary patterns associated with lifestyles in Hypocreales. Based on morphological and anatomical features many lichenicolous fungi in the order Hypocreales were assigned to Bionectriaceae, Nectriaceae and Niessliaceae (Rossman et al. 1999, Diederich et al. 2018). Several of these fungi were classified into previously known genera, which include fungicolous or saprotrophic species. However, recent molecular studies have revealed that some of these taxa belong to other genera and families within Hypocreales (Lawrey et al. 2015, Flakus et al. 2019a, Haldeman & Darmostuk 2024). In this study, we focused on determining the phylogenetic placement of lichenicolous fungi within Hypocreales using a five-loci phylogeny (nuSSU, ITS, LSU, tef1 and rpb1). Most of them have previously been assigned to the family Bionectriaceae. To achieve this aim, we reconstructed the phylogeny of the Hypocreales using a representative dataset containing 228 taxa. Our specific goals were as follows: i) to reconstruct a more comprehensive phylogeny of Hypocreales; ii) to resolve the phylogenetic placement and clarify the taxonomy of lichenicolous species from the genera Ovicuculispora, Paranectria, Nectriopsis lichenophila, and Nectria byssophila-like species; and iii) to clarify the phylogenetic relationship between the new lichenicolous lineage and other families of the order Hypocreales. MATERIALS AND METHODS Taxon sampling and morphological studies This study is based on freshly collected material of lichenicolous species belonging to Hypocreales, complemented by specimens deposited at K-M, KHER, KRAM, LPB, UGDA and UPS herbaria and personal herbarium of J. Etayo (hb. Etayo). Morphological and anatomical characters were examined using standard dissectingand compound-microscopes (Nikon SMZ 800, Nikon Eclipse 80i DIC; and Leica S9i and S9D). Sections were prepared manually using a razor blade, or freezing sliding microtome Microm HM 430 (Thermo Fisher Scientific, USA) combined with a BFS-MP freezing stage and a BFS3MP controller. Sections and squash mounts were examined in distilled water, 10 % KOH (K) or lactophenol cotton blue (LPCB; Fluka, no. 61335-100ML). All photomicrographs showing anatomical characters were made using transmitted differential interference contrast (DIC) microscopy. Amyloid reactions of anatomical structures were tested using Lugol’s solution (I) (Fluka, no. 62650-1L-F), or with Lugol’s solution preceded by a 10 % KOH treatment (K/I). All measurements were made in distilled water or LPCB. Measurements are given as (min.–)x–SD–x+SD(–max.), where x is the average and SD is the standard deviation. Persoonia – Volume 54, 202550 DNA extraction, PCR amplification, and DNA sequencing Lichen thalli with ascomata of lichenicolous fungi were stored at -20 °C until processing. The ascomata were removed from the host thallus and carefully cleaned in double distilled water on a microscope slide under sterile conditions to remove host tissues and other visible impurities using ultra-thin tweezers and a razor blade. Genomic DNA was extracted from 4 to 10 clean ascomata or hymenia, depending on each specimen, using the QIAamp DNA Investigator Kit (Qiagen, Germany), the E.Z.N.A Forensic DNA Isolation kit (Omega Bio-Tek) or the DNeasy Plant Mini Kit following the manufacturer’s instructions. We amplified and sequenced the small ribosomal subunit nuc rDNA (nuSSU) using primer pair N24 and NS1 (White et al. 1990, Gargas & Taylor 1992), nuc rDNA internal transcriber spacers (ITS = ITS1+5.8S+ITS2) and nuc rDNA large subunit (LSU) using the primer pairs ITS1F and LR5 or ITS1F/ITS4 and LROR/LR5 (White et al. 1990, Gardes & Bruns 1993), a fragment of the region coding for protein synthesis elongation factor 1 alpha (tef1) with the primer pair EF1-1983F and EF1-2228R (Rehner & Samuels 1995) and a fragment of the region coding for the RNA polymerase largest subunit (rpb1) using the primers pair RPB1cf and RPB1Af (Stiller & Hall 1997). The amplification parameters and additional detailed information on PCR, visualization of amplicons, and preparation of samples can be found in Rodriguez-Flakus & Printzen (2014) and Flakus et al. (2019a). The PCR amplicons were sequenced in both directions by Macrogen (Amsterdam, the Netherlands or Madrid, Spain). The newly generated sequences were carefully checked, assembled, and edited manually using Geneious Pro v. 8.0. (Biomatters Ltd) and deposited in GenBank. Detailed information on the sequences used in this study is provided in Table 1. Phylogenetic analyses and taxon selection All sequences generated were firstly checked by BLAST nucleotides searches (Altschul et al. 1990) to discard potential contaminations by any unrelated fungi. Alignments for each region were generated using MAFFT (Katoh & Standley 2013) implemented on the GUIDANCE2 Web server (Penn et al. 2010). We used the default cut-off score of 0.93 in all single gene alignments. The single-locus phylogenies for all loci were generated (results not shown) to detect topology incongruences. The intron regions of nuSSU were delimited and removed from the alignment manually. The coding domain sequence (CDS) of the protein-coding regions were detected by Augustus web-tool (Stanke et al. 2008). Two multi-locus datasets were assembled: 1) the Hypocreales order-wide matrix (LSU+tef1+rpb1 +rpb2+tub2), to reveal the phylogenetic placement and relationship of target group of lichenicolous species with other Hypocreales fungi. The dataset includes representatives of all families of the order, e.g. 321 specimens of 228 species, with four species from the order Sordariales selected as outgroup taxa, e.g. Achaetomium macrosporum, Chaetomium elatum, Neurospora crassa and N. tetrasperma (available at https:// doi.org/10.6084/m9.figshare.25324963); 2) the Paranectriaceae multi-gene matrix (5.8S+LSU +nuSSU+tef1+rpb1), to delimit species and elucidate their relationships within the Paranectriaceae. The dataset included target lichenicolous specimens (28 specimens of 9 species), as well as representatives of the CylindromoniumTrichonectria clade (10 specimens of 6 species). Three specimens of the Niessliaceae selected as outgroup taxa (available at https://doi.org/10.6084/m9.figshare.25324936). PartitionFinder 2 (Lanfear et al. 2017) was used to select the best partition scheme for our dataset and substitution models for each partition. The single substitution model was selected for each region for two datasets under a greedy search algorithm and the Akaike information criterion (AIC) (Lanfear et al. 2012). For the four protein-coding gene regions (tef1, rpb1, rpb2 and tub2), each codon position was analysed as a distinct partition, the first, second and third codon position. The final partition scheme used in the analyses is presented in Table 2. Maximum Likelihood (ML) analyses were carried out using a heuristic search as implemented in IQ-TREE v. 2.1.2 on the CIPRES Science Gateway (Ronquist et al. 2012) and 1000 ultrafast bootstrap replicates were selected to estimate branch support (Nguyen et al. 2015, Minh et al. 2020). The Bayesian inference (BI) phylogenetic tree was generated in MrBayes v. 3.2.6 on the CIPRES Science Gateway (Ronquist et al. 2012) using the partitions and substitution models obtained by PartitionFinder v. 2. The posterior probabilities were calculated by sampling trees using the Markov chain Monte Carlo (MCMC) approach. Two independent parallel runs were started each with four incrementally heated chains (temperature parameter for MCMC chains was 0.15). This MCMC was allowed to run for 100 million generations, sampling every 1000th tree and discarding the first 50 % of the sampled tree as a burn-in factor. The analysis was stopped when the standard deviation of split frequencies had dropped below 0.01. The resulting ML and BI phylogenetic trees were visualized in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/ software/figtree/) and Inkscape v. 1.4 (https://inkscape.org/). RESULTS Phylogenetic relationship of Hypocreales at the family level In this study, 115 new sequences from 35 specimens of lichenicolous fungi were generated (22 of nuSSU, 28 of ITS, 27 of LSU, 22 of tef1 and 16 of rpb1). The combined Hypocreales order-wide matrix consisted of 4 584 characters (843 of LSU, 1065 of tef1, 669 of rpb1, 1101 of rpb2, 906 of tub2), of which 2053 are parsimony-informative sites, 392 singleton sites and 2139 constant sites. The orderlevel backbone tree of Hypocreales (Fig. 1) showed similar topology from ML and BI analyses and therefore the ML tree was selected to represent and discuss the phylogenetic relationships among taxa. Our Hypocreales order-wide phylogenetic matrix based on five genomic regions showed high support (bootstrap, bp/posterior probability, pp – 100/1) for the ingroup of 228 Hypocreales species. This clade further consisted of 29 wellsupported subclades, which corresponded to the 27 families previously recognized in the order (Hyde et al. 2020, Hou et al. 2023, Li et al. 2023, Perera et al. 2023, Xiao et al. 2023) and two of these subclades represent the Cylindromonium- Darmostuk et al.: New lineage of hypocrealean fungi 51 Table 1. GenBank accession numbers for Hypocrealaes sequences used in the phylogenetic analyses. Sequences newly generated in this study are shown in bold. The asterisk indicates type materials. Voucher specimen includes collector, collector number, herbarium or culture collection and isolation number. Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 Achroiostachys humicola CBS 868.73* Turkey soil — — KU845819 KU845854 —KU845837 KU845760 DAOM 226830 Canada soil — — KU845822 KU845857 —KU845838 KU845763 Acremoniopsis suttonii CBS 138708* Spain soil — — MH877666 OQ470733 — OQ453831 — Acremonium rutilum CBS 396.66* Germany moist greenhouse wall — — HQ232124 OQ471142 — — — CBS 229.70 Germany moist greenhouse wall ——MH871348 OQ471141 — — — Akanthomyces lecanii CBS 101247 West Indies Coccus viridis ——KM283794 — DQ522407 KM283859 — Akanthomyces tuberculatus OSC 111002 n/a Lepidoptera ——DQ518767 DQ522338 DQ522384 DQ522435 DQ522499 Albomorchellophila morchellae KUNCC21-10005* China Morchella sp. — — OP580862 OP585424 ——— KUNCC21-10100 China Morchella sp. — — OR420021 OR420838 ——— Albonectria rigidiuscula CBS 315.73 Malaysia Theobroma cacao — — KM231677 KM231938 KM232229 KM232378 KM232071 CBS 122570 Cameroon bark — — KM231676 KM231937 KM232228 KM232377 KM232070 Allantonectria miltina CBS 121121 Italy Agave americana ——HM484572 HM484524 HM484587 KM232409 HM484609 CBS 474.69 Spain Agave americana — — KM231716 KM231973 KM232269 KM232408 — Aquanectria penicillioides CBS 257.54 USA Acer sp., leaf — — MH868856 KM231865 KM232135 KM232299 — Aquanectria submersus CBS 394.62* UK unknown — — KM231612 — KM232134 HQ897728 KM231999 Aschersonia calendulina SM 00186.01 Thailand leaves — — JN940909 —JN987887 — — Balansia claviceps CBS 501.70 India Cyrtococcum oxyphyllum ——MH871588 — — — — Balansia pilulaeformis AEG 94-2 n/a n/a — — AF543788 DQ522319 DQ522365 DQ522414 — Beauveria brongniartii ARSEF 617* France Melolontha melolontha ———HQ880991 HQ880854 HQ880926 — BCC 16585 n/a Anomala cuprea ——JF415967 JF416009 JN049885 JF415991 — Beauveria scarabaeidicola ARSEF 5689 n/a Scarabaeidae ——AF339524 DQ522335 DQ522380 DQ522431 DQ522496 Bisifusarium dimerum CBS 108944* Netherlands human — — JQ434514 KR673912 KM232212 KM231919 EU926400 Bisifusarium nectrioides CBS 176.31 Honduras humus — — EU926245 EU926312 JX171477 JX171591 EU926378 Brevistachys subsimplex ATCC 32888 USA water hyacinth — — AY489711 AY489606 AY489639 EF692519 — Bullanockia australis CPC 28976 Australia Kingia australis — — KY173506 OQ470804 —OQ451835 — Caespitomonium euphorbiae CBS 147075* Namibia Euphorbia sp. — — OK663737 OK651197 — OK651157 OK651201 Caespitomonium hyalinulum CBS 271.36 USA air in hospital — — HQ232045 — — — — Calcarisporium arbuscula CBS 144.52 UK Russula sp. — — MH868488 — — — — CBS 518.66 Netherlands Boletus sp. — — MH870517 — — — — Persoonia – Volume 54, 202552 Table 1. (Continued). Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 CBS 900.68 Germany decayed agaric fungi — — KX442598 KX442596 —KX442597 — Calcarisporium cordycipiticola CGMCC 3.17904 China Cordyceps militaris — — KX442604 KX442605 — KX442607 — CGMCC 3.17905* China Cordyceps militaris — — KX442599 KX442593 —KX442594 — Calcarisporium xylariicola HMAS 276836* Italy Xylaria sp. — — KX442601 KX442595 — KX442600 — Calonectria brassicae CBS 111869 Indonesia Argyreia splendens — — GQ280698 FJ918567 KM232181 KM232308 AF232857 Calonectria daldiniana CBS 749.70* Netherlands Ilex aquifolium, leaf litter — — GQ280706 GQ267312 — — — Calostilbe striispora CBS 133491 French Guiana bark — — KM231653 KM231918 KM232204 KM232361 KM232048 Campylocarpon fasciculare CBS 112613* South Africa Vitis vinifera — — HM364313 JF735691 HM364331 KM232322 AY677221 Campylocarpon pseudofasciculare CBS 112679* South Africa Vitis vinifera — — HM364314 JF735692 HM364332 KM232323 AY677214 Capitofimbria compacta CBS 111739* Brazil rotten leaf — — MH874460 KU846378 -KU846349 KU846404 Chaetomium elatum CBS 374.66 USA decomposing leaf — — MH870466 KF001730 KF001775 KF001820 KC109776 Chaetopsina fulva CBS 142.56* Italy Cedrus deodara — — KM231637 KM231902 KM232188 — — CBS 137301 Spain dead leaves — — KY853492 — — — — Chrysonectria crystallifera CBS 102567* Japan Monstera sp. — — OQ055444 OQ470825 —OQ453920 — Chrysonectria finisterrensis JPP17021* France Quercus sp. — — MF611689 — — — — Ciliomyces oropensis Darmostuk 957 Ukraine Lecania croatica —PP513162 PP513187 PP583632 ——— Etayo 29106 Spain Parmelina tiliacea PP513209 PP513163 PP513188 —PP594991 — — Flakus 26387 Bolivia Normandina pulchella PP513210 PP513164 PP513189 PP583633 PP594992 — — Etayo 31139 Spain Physconia perisediosa —PP513224 PP513228 PQ031220 ——— Etayo 31593 Spain Anaptychia ciliaris —PP513225 PP513229 — — — — Etayo 31861 Spain Hypotrachyna revoluta —PQ012595 PQ012597 — — — — Cladobotryum asterophorum CBS 676.77* Japan agaric — — AJ583469 FN868712 FN868776 FN868649 — Cladobotryum penicillatum CBS 407.80* Netherlands Sebacina effusa — — MH873046 FN868725 FN868788 FN868661 — Claviceps fusiformis ATCC 26019 n/a Poaceae — — U17402 DQ522320 DQ522366 — DQ522477 Claviceps paspali ATCC 13892 n/a Poaceae — — U47826 DQ522321 DQ522367 DQ522416 — Clonostachys miodochialis CBS 997.69* Netherlands agricultural soil — — MH871287 OQ944658 —OQ927715 OQ982677 Clonostachys rosea CBS 114056 Venezuela bark — — AY489176 AY489611 — DQ522415 DQ522476 Coccinonectria pachysandricola CBS 501.63* Germany Pachysandra terminalis — — KM231640 KM231905 KM232190 KM232350 KM232033 Darmostuk et al.: New lineage of hypocrealean fungi 53 Table 1. (Continued). Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 CBS 476.92 Netherlands Pachysandra terminalis — — KM231641 KM231906 KM232191 MH936693 KM232034 Cocoonihabitus sinensis voucher 8039* China cocoon of Saturniidae — — KY924869 — — — — voucher 9880 China cocoon of Saturniidae — — MF687396 — — — — Collarina aurantiaca CBS 138274* Spain sediments — — MH877656 — — OQ560707 — CBS 110646 Netherlands Reed sandy soil — — OQ055447 OQ470828 ——— Corallocytostroma ornithocopreoides WAC 8705 Australia Astrebla pectinata — — — LT216546 — LT216598 FJ711476 Corallonectria jatrophae CBS 913.96* Puerto Rico tree — — KM231611 KM231863 KM232132 KM232298 KC479787 Cordyceps militaris YFCC 6587 China n/a — — MN576818 MN576988 MN576878 MN576932 — Cordyceps subtenuipes YFCC 6051* China Lepidoptera — — MN576775 MN576945 MN576835 MN576891 — Cosmospora arxii CBS 748.69 Germany Hypoxylon sp. — — KM231694 KM231950 KM232245 — — Cosmospora coccinea CBS 343.70 Germany Inonotus radiatus — — MH871456 — — — — CBS 341.70 Germany Inonotus nodulosus — — KM231692 KM231947 KM232242 HQ897777 KM232086 Cosmospora khandalensis AR 4770 Argentina Annulohypoxylon sp. — — KJ676180 — KJ676217 — KJ676256 Cosmosporella cavisperma CBS 172.31* Norway Pinus sylvestris — — MW827645 — JX171465 MW834000 — Curvicladiella cignea CBS 109167* French Guiana leaf litter — — — KM231867 KM232142 KM232311 KM232002 Cyanonectria buxi CBS 130.97 France Buxus sempervirens — — KM231679 HQ728150 KM232233 HM626690 KM232075 Cyanonectria cyanostoma CBS 115512* France Buxus sempervirens — — MH874353 HM626647 GQ506017 HQ897759 HM484611 Cylindrocladiella camelliae CPC 234 South Africa Eucalyptus grandis — — JN099249 JN099087 KM232139 KM232304 AY793471 Cylindrocladiella lageniformis CBS 340.92* Brazil Eucalyptus sp — — JN099165 JN099003 JN989491 KM232303 — Cylindromonium eugeniicola CBS 146075* South Africa Eugenia capensis — MN562142 MN567649 OQ470832 — OQ560710 — Cylindromonium everniae CPC 40760* Netherlands Evernia prunastri — OK664736 OK663775 OK651192 — OK651171 — Cylindromonium lichenicola CBS 303.70 Germany Alnus glutinosa, on bark —MH859675 MH871429 OQ470833 —OQ453927 — CBS 415.70A Netherlands Desmococcus sp., on bark —MH859774 MH871536 — — — — Cylindromonium rhabdosporum CBS 438.66* Austria old Cladonia furcata —MH858850 HQ232120 OQ470835 —OQ453929 — Dactylonectria macrodidyma CBS 112615* South Africa Vitis vinifera — — KM515900 JF268750 HM364333 JF268710 AY677233 Didymostilbe aurantiospora CBS 616.85* Japan Arenga tremula, petioles — — MH873595 — — — — Elaphocordyceps fracta OSC 110990 n/a n/a — — DQ518759 DQ522328 DQ522373 DQ522425 DQ522487 Persoonia – Volume 54, 202554 Table 1. (Continued). Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 Elaphocordyceps japonica OSC 110991 n/a n/a — — DQ518761 DQ522330 DQ522375 DQ522428 DQ522490 Eucasphaeria capensis CBS 120028* South Africa Eucalyptus sp., living leaves — — MH874626 OQ470896 —OQ453986 — CBS 120027 South Africa Eucalyptus sp., living leaves — — MH874625 OQ470897 —OQ453987 — Eucasphaeria protea CBS 146815* South Africa Protea neriifolia — — MW175397 MW173129 — MW173116 — Eucasphaeria rustici CBS 142085* Australia Eucalyptus creta, leaves — — KY173501 OQ470898 —OQ453988 — Flammocladiella anomiae CBS 142775 Bulgaria Massaria anomia — — MN597426 MW890089 —MW890062 MW890128 CBS 144256* France Massaria anomia — — MN597425 MW890090 —MW890090 MW890129 SOMF 30203 Bulgaria Massaria anomia — — MN597424 — — — — Flammocladiella decora CLL16020 France Massaria inquinans — — MF614949 OQ470901 ——— CBS 138906 Germany Acer platanoides, twigs — — KR611901 MW890088 —MW890061 MW890127 Fusariella atrovirens CBS 311.73 Algeria desert soil — — MH872395 OQ470904 —OQ453993 — Fusariella hughesii CBS 435.70 Netherlands agricultural soil — — MH871547 OQ470906 —OQ453995 — Fusicolla melogrammae CBS 141092* UK Melogramma campylosporum — — KY092489 ———MW834305 Fusicolla violacea CBS 634.76* Iran Diaspidiotus perniciosus — — KM231700 KM231956 KM232251 HQ897696 KM232095 Geejayessia atrofusca CBS 125482 Canada Staphylea trifolia, twigs — — MH875066 MW834282 MW834196 HQ897775 — Geejayessia celtidicola CBS 125502* Canada Celtis occidentalis, twigs — — HM626669 HM626638 KM232232 HM626685 KM232074 Gelasinospora tetrasperma CBS 178.33 Canada dung of Lagopus sp., grouse — — DQ470980 —DQ471178 DQ470932 — Gliomastix murorum CBS 154.25* n/a Malus sylvestris — — HQ232063 OQ470923 — OQ454012 — CBS 127397 USA n/a — — MH876020 — — — — Gliomastix tumulicola CBS 127532* Japan white salt-like masses on painting — — OQ055547 OQ470950 —OQ454039 — K5916-10-3 Japan viscous substances on the stone wall — — AB540476 — — — — Grandibotrys hyalinus MFLUCC 17-1076* Thailand decaying wood — — MF346064 — — — — Grandibotrys pseudotheobromae CBS 136170* Nepal decaying wood — — KU846161 KU846216 —KU846188 KU846241 Grandibotrys xylophila CBS 136179* Nepal decaying wood — — KU846163 KU846217 —KU846190 — Gregatothecium humicola CBS 205.96* Papua New Guinea soil — — KU846347 KU846402 —KU846376 KU846432 Darmostuk et al.: New lineage of hypocrealean fungi 55 Table 1. (Continued). Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 Heleococcum aurantiacum CBS 201.35 n/a mushroom compost — — JX158441 JX158397 —JX158463 — Heteroacremonium album CGMCC 3.22405* China sediment — — OQ758137 OQ809041 —OQ809001 — CGMCC 3.22409 China sediment — — OQ758138 OQ809042 —OQ809002 — Heteroacremonium rugosum CGMCC 3.22520* China sediment — — OQ758139 OQ809043 —OQ809003 — Hevansia novoguineensis NHJ 11923 n/a Spider (Arachnida) — — EU369032 EU369013 EU369052 EU369072 — Hirsutella thompsonii ARSEF 2800 USA Acari — — KM652142 KM652023 KM652058 — — ARSEF 3323 Costa Rica Dolichotetranychus floridanus — — KM652143 KM652024 KM652059 — — Hydropisphaera peziza BPI 802846 USA bark — — AY489730 AY489625 AY489661 DQ522444 — CBS 123792 Netherlands n/a — — MH874857 — — — — Hypocrella discoidea BCC 8237 n/a n/a — — DQ384937 DQ384977 DQ385000 DQ452461 — Hypomyces semitranslucens CBS 458.71 Russia Lentinus sp. — — MH871985 — — — — Ijuhya chilensis CBS 102803 USA Nolina micrantha — — KY607553 — KY607579 —KY684187 Ijuhya paraparilis MAFF241404 Japan n/a — — GQ506012 — GQ506041 — — Ilyonectria capensis CBS 132815* South Africa Protea sp. — — KM515908 JX231119 KM232171 KM232336 JX231103 Ilyonectria destructans CBS 264.65 Sweden Cyclamen persicum — — KM515927 JF735695 KM232169 KM232334 — Lasionectria lecanodes CBS 139482 France Peltigera sp. — — KP899119 — — — — Lasionectria mantuana A.R. 4029 Finland n/a — — GQ505994 HM484844 GQ506024 — — CBS 114291* Finland decortivated wood — — OQ055596 OQ471001 — OQ454092 — Lichenobarya usneae Buck 61451 Canada dead Usnea — — KP899625 — — — — Macroconia leptosphaeriae CBS 717.74 France Sordariomycetes sp. — — KM231707 — KM232257 KM232390 KM232099 Macroconia papilionacearum CBS 125495 USA Ascomycota sp. — — KM231704 KM231958 KM232254 HQ897776 KM232096 Mariannaea humicola CBS 740.95* Brazil soil — — KM231619 KM231880 KM232153 KM232328 KM232012 Mariannaea elegans HU0029* Netherlands Pinus sylvestris, decayed bark — — KX986139 ———KX986145 Melanopsamma pomiformis BPI843533 UK Ulmus sp. — — AY489709 AY489604 AY489637 EF692511 — UAMH 10484 Canada dead twig — — — DQ676611 — DQ676598 — CBS 101322* Czech Republic Fagus sylvatica — — KU846068 — — KU846078 — Memnoniella echinata CBS 216.32* UK cotton yarn — — KU846169 KU846222 —KU846196 KU846245 Metapochonia bulbillosa CBS 145.70* Denmark Picea abies, root 7.5 mm thick — — AF339542 EF468796 EF468902 EF468943 — Metapochonia goniodes CBS 891.72* Germany Pulcherricium caeruleum — — AF339550 DQ522354 DQ522458 DQ522401 DQ522521 Persoonia – Volume 54, 202562 Table 1. (Continued). Taxa Herbarium/Strain number Country Substrate/host nuSSU ITS LSU tef-1 rpb1 rpb2 tub2 ATCC 36477 n/a n/a — — U69891 — — — - Valsonectria inflata CBS 212.69 UK intertidal salt marsh mud — — HQ232050 OQ471228 — OQ560706 - CBS 604.68 Germany soil — — OQ430149 OQ471229 —OQ454295 - CBS 497.82 Netherlands agricultural soil — — OQ430150 OQ471230 — OQ454296 - Valsonectria roseola CBS 416.81 UK Dianthus caryophyllus — — OQ430151 OQ471231 — OQ454297 - CBS 289.62* UK Dactylis glomerata — — OQ430152 OQ471232 — OQ454298 - Valsonectria soli CBS 770.69* France agricultural soil — — OQ430158 OQ471306 — OQ454304 - CBS 106.70 Netherlands agricultural soil — — MH878244 OQ471237 — OQ454303 - Verrucostoma freycinetiae MAFF 240100* Japan Freucinetia boninensis, dead leaves — — GQ506013 HM484853 GQ506018 —HM484885 Verrucostoma martinicensis CBS 138731* Martinique Danaea elliptica, dead stem ——MH877668 — — — — Virgatospora echinofibrosa AR2824 Mexico litter — — AY489706 AY489601 AY489634 — — Xanthonectria pseudopeziza CBS 140160 France Suaeda vera ——KU593583 OQ471291 —OQ454358 — CBS 141245 France Smyrnium olusatrum ——KU946964 OQ471290 — OQ454357 — CBS 126104 France Buxus sempervirens ——MH875476 — — — — MFLU 16-0513 Italy Robinia pseudoacacia — — ON230061 ON238005 ——— Xenoacremonium falcatus CBS 400.85* New Zealand Pinus radiata — — KM231712 KM231967 KM232263 OQ454359 KM232104 Xenoacremonium recifei CBS 137.35* Brazil human — — HQ232106 KP012646 KM232264 KM232397 KM232105 Xenocylindrocladium guianense CBS 112179* French Guiana plant litter — — JQ666073 KM231895 KM232166 KM232314 AF320197 Xenocylindrocladium serpens CBS 128439 Ecuador bark — — KM231688 KM231894 KM232165 — — Xenogliocladiopsis cypellocarpa CBS 133814* Australia Eucalyptus cypellocarpa — — KM231623 KM231885 KM232158 KM232332 KM232017 CPC 17153 Australia Eucalyptus sp. — — KM231624 KM231884 KM232159 KM232333 KM232018 Xepicula leucotricha BPI 843408 USA decaying grass leaf — — AY489707 AY489602 AY489635 — — Darmostuk et al.: New lineage of hypocrealean fungi 63 Nectriaceae Neoacremoniaceae Sedecimiellaceae Chrysonectriaceae Nothoacremoniaceae Nothoacremonium exiguum CBS 587.73* Nothoacremonium vesiculophorum CBS 397.70B* Nothoacremonium subcylindricum CBS 190.70 Nothoacremonium subcylindricum CBS 781.69 Nothoacremonium subcylindricum CBS 416.68* Chrysonectria finisterrensi JPP 17021* Chrysonectria crystallifera CBS 102567* Heteroacremonium rugosum CGMCC 3.22520 Heteroacremonium album CGMCC 3.22409 Heteroacremonium album CGMCC 3.22405* Sedecimiella taiwanensis CY5100* Sedecimiella funiculosa CGMCC 3.22348* Sedecimiella funiculosa CGMCC 3.22356 Neoacremonium vitellinum CBS 793.69 Neoacremonium vitellinum CBS 792.69* Neoacremonium flavum CBS 452.70* Neoacremonium flavum CBS 398.70 Neoacremonium distortum CBS 665.75 Neoacremonium distortum CBS 314.72* Allantonectria miltina CBS 474.69 Allantonectria miltina CBS 121121 Thyronectria quercicola CBS 128976* Thyronectria lamyi CBS 417.89 Thyronectria caudata CBS 136003* Nectria mariae CBS 125294* Nectria nigrescens CBS 125148 Nectria cinnabarina CBS 125165* Cosmospora arxii CBS 748.69 Rectifusarium ventricosum CBS 748.79* Rectifusarium robinianum CBS 430.91* Bisifusarium nectrioides CBS 176.31 Bisifusarium dimerum CBS 108944* Calostilbe striispora CBS 133491 Chaetopsina fulva FMR 13129 Chaetopsina fulva CBS 142.56* Sarcopodium circinatum CBS 587.92* Sarcopodium circinatum CBS 100998 Coccinonectria pachysandricola CBS 476.92 Coccinonectria pachysandricola CBS 501.63* Xenoacremonium recifei CBS 137.35* Xenoacremonium falcatus CBS 400.85* Paracremonium inflatum CBS 485.77* Paracremonium apiculatum CGMCC 3-19309* Pseudocosmospora eutypellae GJS 10-294 Pseudocosmospora eutypellae CBS 133966* Cosmosporella cavisperma CBS 172.31* Cosmospora khandalensis AR4770 Cosmospora coccinea CBS 343.70 Cosmospora coccinea CBS 341.70 Fusicolla violacea CBS 634.76* Fusicolla melogrammae CBS 141092* Macroconia papilionacearum CBS 125495 Macroconia leptosphaeriae CBS 717.74 Microcera coccophila CBS 310.34 Microcera rubra CBS 638.76* Microcera larvarum CBS 738.79* Geejayessia celtidicola CBS 125502* Geejayessia atrofusca CBS 125482 Cyanonectria cyanostoma CBS 101734* Cyanonectria buxi CBS 130.97 Albonectria rigidiuscula CBS 315.73 Albonectria rigidiuscula CBS 122570 Neocosmospora illudens CBS 147303 Neocosmospora vasinfecta CBS 325.54 Neocosmospora ambrosia CBS 571.94* Neonectria ramulariae CBS 151.29 Neonectria neomacrospora CBS 118984 Neonectria faginata CBS 217.67* Dactylonectria macrodidyma CBS 112615* Ilyonectria capensis CBS 132815* Ilyonectria radicicola CBS 264.65 Ilyonectria destructans CBS 264.65 Corallonectria jatrophae CBS 913.96* Aquanectria submersus CBS 394.62* Aquanectria penicillioides CBS 257.54 Cylindrocladiella lageniformis CBS 340.92* Cylindrocladiella camelliae CPC 234 Xenocylindrocladium serpens CBS 128439 Xenocylindrocladium guianense CBS 112179* Curvicladiella cignea CBS 109167* Calonectria daldiniana CBS 749.70* Calonectria brassicae CBS 111869 Xenogliocladiopsis cypellocarpa CPC 17153 Xenogliocladiopsis cypellocarpa CBS 133814* Thelonectria discophora CBS 125153 Thelonectria veuillotiana MAFF 241544 Thelonectria olida CBS 215.67* Mariannaea humicola CBS 740.95* Mariannaea elegans HU0029* Rugonectria rugulosa CBS 129158 Rugonectria rugulosa CBS 126565 Campylocarpon pseudofasciculare CBS 112679* Campylocarpon fasciculare CBS 112613* 100/1 100/1 100/1 100/1 100/1 94/1 97/1 100/1 100/1 100/1 100/1 77/- 95/1 98/1 77/0.97 94/1 78/- 97/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 93/0.99 90/0.97 96/0.97 89/0.99 100/1 100/1 100/1 100/1 100/1 100/1 77/0.97 98/1 74/- 87/- 99/0.97 95/1 99/1 98/1 100/1 100/1 100/1 96/0.98 86/- 81/- 100/1 71/- 77/- 82/0.97 100/1 98/1 100/1 100/1 83/0.97 96/0.99 95/1 100/1 100/1 100/1 83/0.97 83/0.97 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 87/0.99 80/1 98/1 98/1 97/0.99 95/1 0.1 Fig. 1. Phylogenetic relationships within the order Hypocreales inferred from a Maximum likelihood analysis (ML) of a combined LSU, tef-1, rpb1, rpb2 and tub2 data set. Achaetomium macrosporum, Chaetomium elatum, Neurospora crassa and N. tetrasperma were used as the outgroup. Lichenicolous fungi are highlighted in blue. Bootstrap support value from ML/posterior probability from Bayesian analyses at branches. Thickened branches represent either bootstrap support values ≥ 70 % and/or Bayesian posterior probabilities ≥ 0.97, lower values inducated by ‘-‘. The asterisk indicates type materials. Persoonia – Volume 54, 202564 Bionectriaceae Flammocladiellaceae Ijuhyaceae Xanthonectriaceae Tilachlidiaceae Stromatonectriaceae Myrotheciomycetaceae Paranectriaceae CylindromoniumTrichonectria clade Cylindromonium eugeniicola CBS 146075* Trichothecium hongkongens CBS 102186 Trichothecium hongkongens CBS 101444 Trichothecium crotocinigenum CBS 129.64* Trichothecium sympodiale CBS 227.76* Trichothecium sympodiale ATCC 36477 Myrotheciomyces corymbiae CPC 33206* Trichothecium roseumUAMH 7839 Trichothecium roseum RiT1 Trichothecium roseumPH6 Trichothecium roseum LCP 50 627 Trichothecium roseum KUNCC 21-10015 Trichothecium roseum DAOM 57205 Trichothecium roseum PG Trichothecium roseum DAOM 208997 Stromatonectria caraganae CBS 127387* Stromatonectria caraganae CBS 125579 Tilachlidium brachiatum CBS 505.67 Tilachlidium brachiatum CBS 506.67 Tilachlidium brachiatum CBS 363.97 Bullanockia australis CPC 28976* Xanthonectria pseudopeziza CBS 126456 Xanthonectria pseudopeziza CBS 126104 Xanthonectria pseudopeziza CBS 141245 Xanthonectria pseudopeziza CBS 140160 Ijuhya paraparilis Tua h52 Ijuhya chilensis CBS 102803 Flammocladiella decora CLL16020 Flammocladiella decora CBS 138906 Flammocladiella anomiae CBS 142775 Flammocladiella anomiae SOMF 30203 Flammocladiella anomiae CBS 144256* Stephanonectria keithii GJS 92-133 Clonostachys rosea CCFC 226708 Clonostachys miodochialis CBS 997.69* Nectriopsis lindauiana CBS 897.70* Nectriopsis lindauiana CBS 839.70 Nectriopsis exigua BPI 748377 Nectriopsis violacea CBS 914.70* Nectriopsis violacea CBS 849.70 Ochronectria thailandica MFLUCC 15-0140* Ochronectria calami CBS 445.96 Ochronectria calami ATCC 46692 Protocreopsis freycinetiae CBS 573.76* Acremonium rutillum CBS 396.66* Acremonium rutillum CBS 229.70 Septofusidium herbarum CBS 265.58 Septofusidium berolinense CBS 731.70 Lasionectria mantuana CBS 114291* Lasionectria mantuana AR 4029 Caespitomonium hyalinulum CBS 271.36 Caespitomonium euphorbiae CBS 147075* Synnemellisia urenae BRIP 71652* Synnemellisia aurantia COAD 2070* Fusariella hughesii CBS 435.70 Fusariella atrovirens CBS 311.73 Verrucostoma martinicensis CBS 138731* Verrucostoma freycinetiae MAFF 240100* Roumegueriella rufula GJS 91-164 Roumegueriella rufula CBS 346.85 Heleococcum aurantiacum CBS 201.35 Hydropisphaera peziza CBS 123792 Hydropisphaera peziza BPI 802846 Paracylindrocarpon aloica CPC 27362* Paracylindrocarpon pandanicola KUMCC 17-0272* Paracylindrocarpon nabanheensis KUMCC 16-0147* Gliomastix tumulicola K5916-10-3 Gliomastix tumulicola CBS 127532* Gliomastix murorum CBS 154.25 Gliomastix murorum CBS 127397* 100/1 100/1 100/1 99/1 96/1 100/1 97/1 100/1 100/1 85/0.98 99/1 100/1 71/- 100/1 99/1 99/1 98/1 89/0.97 93/0.98 100/1 100/1 90/0.98 100/1 100/1 100/1 100/1 97/1 94/0.98 88/0.97 100/1 100/1 100/1 95/1 100/1 100/1 100/1 100/1 100/1 96/1 99/1 98/1 100/1 100/1 100/1 97/0.98 70/- 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 90/0.98 75/0.97 100/1 100/1 100/1 100/1 97/1 79/0.98 78/0.98 Fig. 1. (continued) Darmostuk et al.: New lineage of hypocrealean fungi 65 Niessliaceae Acremoniopsiaceae Clavicipitaceae Pseudoniessliaceae Ophiocordycipitaceae Polycephalomycetaceae Cocoonihabitaceae Calcarisporiaceae Albomorchellophilaceae Cordycipitaceae Hypocreaceae Pseudodiploosporeaceae Pseudodiploospora longispora KUNCC 21-10020 Pseudodiploospora longispora KUNCC 21-10026 Pseudodiploospora longispora KUNCC 21-10027 Pseudodiploospora longispora CGMCC 3.23771 Mycogone rosea YBM050 Trichoderma viride CBS 127113 Trichoderma viride CBS 757.71 Hypomyces semitranslucens CBS 458.71 Protocrea pallida TFC 99-209 Protocrea farinosa TFC 97-168 Cladobotryum penicillatum CBS 407.80* Cladobotryum asterophorum CBS 676.77* Simplicillium lamellicola CBS 116.25* Simplicillium lanosoniveum IMI 317442 Simplicillium lanosoniveum CBS 704.86 Hevansia novoguineensis NHJ 11923 Syspastospora parasitica IMI 255607 Samsoniella inthanonensis TBRC 7915* Akanthomyces lecanii CBS 101247 Akanthomyces tuberculatus OSC 111002 Cordyceps subtenuipes YFCC 6051* Cordyceps militaris YFCC 6587 Beauveria scarabaeidicola ARSEF 5689 Beauveria brongniartii BCC 16585 Beauveria brongniartii ARSEF 617* Albomorchellophila morchellae KUNCC 21-10100 Albomorchellophila morchellae KUNCC 21-10005* Calcarisporium xylariicola HMAS 276836* Calcarisporium cordycipiticola CGMCC 3-17905* Calcarisporium cordycipiticola CGMCC 3-17904 Calcarisporium arbuscula CBS 518.66 Calcarisporium arbuscula CBS 900.68 Calcarisporium arbuscula CBS 144.52 Cocoonihabitus sinensis 9880 Cocoonihabitus sinensis 8039* Polycephalomyces formosus NBRC 100686 Polycephalomyces formosus CGMCC 5-2205 Polycephalomyces formosus CGMCC 5-2206 Ophiocordyceps sinensis EFCC 7287 Ophiocordyceps lanpingensis YHOS 0705 Hirsutella thompsonii ARSEF 3323 Hirsutella thompsonii ARSEF 2800 Ophiocordyceps heteropoda OSC 106404 Ophiocordyceps heteropoda EFCC 10125 Elaphocordyceps japonica OSC 110991 Elaphocordyceps fracta OSC 110990 Pseudoniesslia minutispora CBS 148 70 Pseudoniesslia minutispora CBS 735 69 Pseudoniesslia minutispora CBS 267 89 Pseudoniesslia minutispora CBS 246.82* Metarhizium ellipsoideum BCC 49285* Metarhizium blattodeae MY 00896 Metapochonia goniodes CBS 891.72* Metapochonia bulbillosa CBS 145.70* Neobarya sp. Buck 26786 Moelleriella oxystoma BCC 8406 Hypocrella discoidea BCC 8237 Aschersonia calendulina SM 00186.01 Balansia pilulaeformis AEG 94-2 Balansia claviceps CBS 501.70 Neobarya parasitica Corallocytostroma ornithocopreoides WAC 8705 Claviceps paspali ATCC 13892 Claviceps fusiformis ATCC 26019 Collarina aurantiaca CBS 110646 Collarina aurantiaca CBS 138274* Acremoniopsis suttonii CBS 138708* Nothoacremoniopsis sedimenticola CGMCC 3.22383* Nothoacremoniopsis sedimenticola CGMCC 3.22385 Niesslia exilis CBS 357.70 Niesslia constricta CBS 760.69* Niesslia nordinii CBS 116.70 Niesslia nordinii CBS 101.63* Eucasphaeria proteae CBS 146815* Rosasphaeria moravica CBS 124270* Neoeucasphaeria eucalypti CBS 145075* Eucasphaeria rustici CBS 142085* Eucasphaeria capensis CBS 120028* Eucasphaeria capensis CBS 120027 100/1 100/1 100/1 100/1 70/- 99/1 99/1 78/0.98 80/- 100/1 100/1 100/1 88/0.98 98/- 100/1 100/1 100/1 91/0.98 87/0.97 87/0.98 98/0.98 84/0.99 100/1 100/1 100/1 100/1 100/1 100/1 95/- 70/- 100/1 100/1 86/0.99 98/1 100/1 98/1 100/1 92/1 81/0.98 98/1 98/1 90/0.98 100/1 97/1 97/1 100/1 100/1 95/1 99/1 100/1 100/1 98/0.98 77/0.97 84/0.97 100/1 100/1 100/1 90/0.97 86/- 90/0.97 80/- Fig. 1. (continued) Trichonectria clade as well as a newly described family (Fig. 1).The families Albomorchellophilaceae, Bionectriaceae, Chrysonectriaceae, Clavicipitaceae, Flammocladiellaceae, Ijuhyaceae, Nectriaceae, Neoacremoniaceae, Ophiocordycipitaceae, Polychephalomycetaceae, Pseudodiploosporeaceae, Sedecimiellaceae, Stachybotriaceae, Stromatonectriaceae, Tilachlidiaceae and Xanthonectriaceae formed well-supported clades and phylogenetic relationships between them are consistent with those found in previous studies (Hou et al. 2023, Li et al. 2023, Perera et al. 2023, Persoonia – Volume 54, 202566 Stachybotriaceae Valsonectriaceae Sarocladiaceae outgroup Neurospora crassa CMP 6360 Gelasinospora tetrasperma AFTOL 1287* Chaetomium elatum CBS 374.66 Achaetomium macrosporum CBS 532.94 Sarocladium ochraceum CBS 428.67* Sarocladium strictum CBS 346.70* Sarocladium oryzae CBS 180.74* Parasarocladium radiatum CBS 142.62* Parasarocladium tasmanniae CPC 38162* Parasarocladium debruynii CBS 144942* Valsonectria inflata CBS 212.69 Valsonectria inflata CBS 604.68 Valsonectria inflata CBS 497.82 Valsonectria soli CBS 770.69* Valsonectria soli CBS 106.70 Valsonectria roseola CBS 416.81 Valsonectria roseola CBS 289.62* Brevistachys subsimplex ATCC 32888 Memnoniella echinata CBS 216.32* Striatibotrys eucylindrospora CBS 203.61 Stachybotrys chlorohalonata UAMH 6417 Stachybotrys chartarum CBS 182.80* Grandibotrys hyalicus MFLUCC 17-1076* Didymostilbe aurantiospora CBS 616.85* Achroiostachys humicola DAOM 226830 Achroiostachys humicola CBS 868.73* Grandibotrys xylophila CBS 136179* Grandibotrys pseudotheobromae CBS 136170* Sirastachys phaeospora CBS 100155* Melanopsamma pomiformis CBS 101322* Melanopsamma pomiformis UAMH 10484 Melanopsamma pomiformis ATCC 18873 Peethambara sundara CBS 646.77* Septomyrothecium uniseptatum MUCL 52942 Parvothecium terrestre CBS 198.89* Peethambara spirostriata CBS 110115 Didymostilbe echinofibrosa AR 2824 Myxospora masonii CBS 174.73* Myrothecium inundatum IMI 158855 Myrothecium inundatum CBS 616.70 Gregatothecium humicola CBS 205.96* Xepicula leucotrichum BPI 843408 Capitofimbria compacta CBS 111739* 78/- 100/1 100/1 100/1 100/1 99/1 94/0.98 91/0.97 99/1 99/1 99/1 98/1 80/1 100/1 96/0.98 99/1 75/- 96/0.99 100/1 70/- 100/1 100/1 100/1 100/1 100/1 100/1 100/1 76/0.98 100/1 100/1 99/1 Fig. 1. (continued) Xiao et al. 2023). Details of the phylogenetic relationships of targeted lichenicolous species are given in the next section. In our phylogeny, the 66 species of the family Nectriaceae formed a well-supported clade (95/1). Additionally, two specimens of the genus Roselliniella, formed a well-supported clade (100/1) within Nectriaceae. This genus was previously classified as Hypocreales insertae sedis; however, as the generic type of Roselliniella (R. nephromatis) has not been sequenced yet, the inclusion within Nectriaceae is tentative. The species of the family Myrotheciomycetaceae formed a well-supported clade (100/1). Myrotheciomycetaceae showed a close relationship to the clade comprising families Bionectriaceae, Flammocladiellaceae, Stromatonectriaceae, Tilachlidiaceae and Xanthonectriaceae, with strong statistical support (99/1). The genera Calcarisporium (with three species, including the generic type C. arbuscula) and the monotypic Neobaryopsis, which previously has been placed within the Cordycipitaceae by Flakus et al. (2019a), formed a well-supported clade (100/1) corresponding to the family Calcarisporiaceae. This clade showed a highly supported (92/1) sister relationship to the family Albomorchellophilaceae. Similar to previous studies, the phylogenetic relationship between the families Cocoonihabitaceae, Cordycipitaceae, Hypocreaceae, Polychephalomycetaceae and Pseudoniessliaceae received moderate or low statistical support (Hou et al. 2023, Perera et al. 2023, Xiao et al. 2023). The species previously assigned to the family Cordycipitaceae and Hypocreaceae were placed in a well-supported clade (98/0.98 and 84/0.97 respectively). Members of the recently established Pseudodiploosporeaceae were resolved in the well-supported clade (100/1) and showed a sister relationship to the family Hypocreaceae. Pseudoniessliaceae is represented in our analyses by four specimens of Pseudoniesslia minutispora, which showed a low-supported sister relationship to the family Clavicipitaceae. Two specimens of Cocoonihabitus species (Cocoonihabitaceae) are resolved in a well-supported clade and showed a sister relationship (86/0.99) to the clade comprised by families Ophiocordycipitaceae and Polychephalomycetaceae. Specimens of the family Nothoacremoniaceae clustered in a highly supported clade (100/1) and it was closely related to the clade (80/0.97) comprising families Chrysonectriaceae, Neoacremoniaceae and Sedecimiellaceae. The Valsonectriaceae is represented by three species of Valsonectria in our data set, forming a distinct and well-supported clade (100/1). The family Sarocladiaceae (100/1) showed a sister relationship to the remaining families of the order Hypocreales. This is the first phylogenetic study that includes a wide sampling of lichenicolous species of the genera Ovicuculispora and Paranectria, as well as Nectriopsis lichenophila, and Nectria byssophila-like species, which in our phylogenetic analyses are nested together in a monophyletic highly supported clade (100/1). This new lichenicolous clade demonstrates a well-supported sister relationship (79/0.98) to the clade consisting of species from the asexual morph genus Cylindromonium (generic type C. eugeniicola) and a few species from the sexual genus Trichonectria (generic type T. hirta, but not sequenced so far). This clade represents an independent lineage with strong statistical support (100/1), and perhaps needs to be uploaded to the family level. However, Trichonectria appears to be a polyphyletic assemblage and no sequences of T. hirta, which is the type species of the genus, are available. Because of this, we decided not to introduce any nomenclature changes until further studies resolve the phylogenetic placement of T. hirta. Our analyses included sequences of 9 specimens of the family Niessliaceae which were resolved as a monophyletic clade with a high support (99/1). The family Niessliaceae Darmostuk et al.: New lineage of hypocrealean fungi 67 Rossmaniella Paranectria Ciliomyces Sphaeronectria Ovicuculispora CylindromoniumTrichonectria clade Outgroup Paranectriaceae Niesslia exilis CBS 560.74 Niesslia cladoniicola CBS 960.73* Niesslia exilis CBS 357.70 Cylindromonium eugeniicola CBS 146075* Trichonectria rectipila CBS 132.87* Cylindromonium lichenicola CBS 303.70 Cylindromonium rhabdosporum CBS 438.66* Cylindromonium everniae CBS 148255* Trichonectria setadpressa Flakus 29612.2 Trichonectria setadpressa Etayo 20-13 Trichonectria setadpressa Flakus 29612.1 Trichonectria setadpressa Flakus 29617 Trichonectria setadpressa Flakus 28886 Ovicuculispora cf. macrospora Flakus 29165 Ovicuculispora cf. macrospora Flakus 29171 Ovicuculispora parmeliae LE 261143 Ovicuculispora parmeliae Flakus 26252 Ovicuculispora parmeliae Flakus 26009 Ovicuculispora parmeliae Flakus 27025 Ovicuculispora parmeliae Flakus 27067 Sphaeronectria lichenophila Flakus 27360 Sphaeronectria lichenophila Flakus 28086 Sphaeronectria lichenophila Flakus 26977 Sphaeronectria lichenophila Flakus 27650 Ciliomyces oropensis Flakus 26387 Ciliomyces oropensis Etayo 29106 Ciliomyces oropensis Etayo 31861 Ciliomyces oropensis Etayo 31593 Ciliomyces oropensis Etayo 31139 Ciliomyces oropensis Darmostuk 957 Paranectria affinis UPS F-561663 Paranectria affinis K(M)-253675 Rossmaniella cryptica Flakus 26967* Rossmaniella tylophori Flakus 26805* Rossmaniella coenogonii Kukwa 15078a* Rossmaniella filispora Flakus 27389* Rossmaniella filispora Etayo 30600a Rossmaniella filispora Etayo 30600b Rossmaniella filispora Etayo 31862.2 Rossmaniella filispora Etayo 31862.1 Rossmaniella filispora Flakus 27467 100/1 87/0.97 100/1 x2 x2 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 100/1 87/1 92/0.97 99/1 98/0.98 99/0.98 70/- 88/0.97 91/0.97 70/- Neurospora crassaI CMP6360 Gelasinospora tetrasperma AFTOLID 1287 Chaetomium elatum CBS 374 66 Achaetomium macrosporum CBS 532 94 Sarocladium ochraceum CBS42867 Sarocladium strictum CBS 346 70 Sarocladium oryzae CBS180 74 Parasarocladium radiatum CBS 142 62 Parasarocladium tasmanniae CPC 38162 Parasarocladium debruynii CBS 144942 Stachybotrys subsimplex ATCC 32888 Memnoniella echinata CBS 216 32 Striatibotrys eucylindrospora CBS203 61 Stachybotrys chlorohalonata UAMH6417 Stachybotrys chartarum CBS182 80 Grandibotrys hyalicus MFLUCC 17 1076 Didymostilbe aurantiospora CBS 616 85 Achroiostachys humicola DAOM 226830 Achroiostachys humicola CBS 868 73 Grandibotrys xylophila strain CBS 136179 Grandibotrys pseudotheobromae CBS 136170 Sirastachys phaeospora CBS 100155 Melanopsamma pomiformis CBS 101322 Melanopsamma pomiformis UAMH 10484 Melanopsamma pomiformis ATCC 18873 Peethambara sundara CBS 646 77 Septomyrothecium uniseptatum MUCL52942 Parvothecium terrestre CBS 198 89 Peethambara spirostriata CBS 110115 Didymostilbe echinofibrosa AR2824 Myxospora masonii CBS 174 73 Myrothecium inundatum IMI158855 Myrothecium inundatum CBS 616 70 Gregatothecium humicola CBS 205 96 Myrothecium leucotrichum AR3506 Capitofimbria compacta CBS111739 Valsonectria inflata CBS 212 69 Valsonectria inflata CBS 604 68 Valsonectria inflata CBS 497 82 Valsonectria soli CBS 770 69 Valsonectria soli CBS 106 70 Valsonectria roseola CBS 416 81 Valsonectria roseola CBS 289 62 Pseudoniesslia minutispora CBS 148 70 Pseudoniesslia minutispora CBS 735 69 Pseudoniesslia minutispora CBS 267 89 Pseudoniesslia minutispora CBS 246 82 Pseudodiploospora longispora KUNCC21 10026 Pseudodiploospora longispora KUNCC21 10020 Pseudodiploospora longispora KUNCC21 10027 Pseudodiploospora longispora CGMCC 3 23771 Mycogone rosea YBM050 Hypomyces semitranslucens CBS 458 71 Protocrea pallida TFC 99 209 Protocrea farinosa TFC 97 168 Lichenobarya usneae Buck 61451 Cladobotryum penicillatum CBS 407 80 Cladobotryum asterophorum CBS 676 77 Simplicillium lamellicola CBS 116 25 Simplicillium lanosoniveum IMI 317442 Simplicillium lanosoniveum CBS 704 86 Akanthomyces novoguineensis NHJ 11923 Syspastospora parasitica IMI255607 Samsoniella inthanonensis TBRC 7915 Lecanicillium lecanii CBS 101247 Akanthomyces tuberculatus OSC 111002 Cordyceps subtenuipes YFCC 6051 Cordyceps militaris YFCC 6587 Beauveria scarabaeidicola ARSEF 5689 Cordyceps brongniartii BCC16585 Beauveria brongniartii ARSEF 617 Cocoonihabitus sinensis 9880 Cocoonihabitus sinensis 8039 Polycephalomyces formosus NBRC100686 Polycephalomyces formosus CGMCC 5 2205 Polycephalomyces formosus CGMCC52206 Ophiocordyceps sinensis EFCC 7287 Ophiocordyceps lanpingensis YHOS 0705 Hirsutella thompsonii ARSEF 3323 Hirsutella thompsonii ARSEF 2800 Ophiocordyceps heteropoda OSC 106404 Ophiocordyceps heteropoda EFCC 10125 Elaphocordyceps japonica OSC 110991 Elaphocordyceps fracta OSC 110990 Albomorchellophila morchellae KUNCC21 10100 Albomorchellophila morchellae KUNCC21 10005 Neobaryopsis andensis Etayo 20 11 Neobaryopsis andensis Flakus 25967 2 Neobaryopsis andensis Flakus 25967 1 Calcarisporium cordycipiticola CGMCC 3 17905 Calcarisporium xylariicola HMAS 276836 Calcarisporium cordycipiticola CGMCC 3 17904 Calcarisporium arbuscula CBS 518 66 Calcarisporium arbuscula CBS 900 68 Calcarisporium arbuscula CBS 144 52 Metarhizium ellipsoideum BCC 49285 Metarhizium blattodeae MY00896 Metapochonia goniodes CBS 891 72 Metapochonia bulbillosa CBS 145 70 Neobarya sp Buck 26786 Moelleriella oxystoma BCC 8406 Hypocrella discoidea BCC 8237 Aschersonia calendulina SM00186 01 Balansia pilulaeformis AEG 94 2 Balansia claviceps CBS 501 70 Neobarya parasitica Corallocytostroma ornithocopreoides WAC 8705 Claviceps paspali ATCC 13892 Claviceps fusiformis ATCC 26019 Collarina aurantiaca CBS 110646 Collarina aurantiaca CBS 138274 Acremoniopsis suttonii CBS 138708 Nothoacremoniopsis sedimenticola CGMCC 3 22383 Nothoacremoniopsis sedimenticola CGMCC 3 22385 Niesslia cladoniicola CBS 960 73 Niesslia exilis CBS 357 70 Niesslia constricta CBS 760 69 Niesslia nordinii CBS 116 70 Niesslia nordinii CBS 101 63 Eucasphaeria proteae CPC 38661 Rosasphaeria moravica LMM Neoeucasphaeria eucalypti CBS145075 Eucasphaeria rustici CPC 28946 Eucasphaeria capensis CBS 120028 Eucasphaeria capensis CBS 120027 Cylindromonium lichenicola CBS 425 66 Cylindromonium lichenicola CBS 303 70 Cylindromonium rhabdosporum CBS 438 66 Cylindromonium everniae CPC 40760 Cylindromonium eugeniicola CBS 146075 Trichonectria setadpressa A F 29617 Trichonectria setadpressa A F 29612 2 Trichonectria setadpressa J E 20 13 Trichonectria setadpressa A F 29612 1 Trichonectria setadpressa A F 28886 Ovicuculispora cf macrospora Flakus 29171 Ovicuculispora cf macrospora Flakus 29165 Ovicuculispora parmeliae Flakus 27067 Ovicuculispora parmeliae Flakus 27025 Ovicuculispora parmeliae Flakus 26252 Sphaeronectria lichenophila Flakus 27360 Sphaeronectria lichenophila Flakus 27977 Sphaeronectria lichenophila Flakus 27086 Ciliomyces oropensis Flakus 26387 Ciliomyces oropensis Etayo 29106 Ciliomyces oropensis Darmostuk 657 Paranectria affinis UPS F 561663 Paranectria affinis RP670 Rossmaniella tylophoroniae Flakus 26805 Rossmaniella filispora Flakus 26556 Rossmaniella filispora Flakus 27468 Rossmaniella filispora Etayo 30600b Rossmaniella filispora Etayo 30600a Stromatonectria caraganae CBS 127387 Stromatonectria caraganae CBS 125579 Tilachlidium brachiatum CBS 505 67 Tilachlidium brachiatum CBS 506 67 Tilachlidium brachiatum CBS 363 97 Trichothecium hongkongens CBS 102186 Trichothecium hongkongens CBS 101444 Trichothecium crotocinigenu CBS 129 64 Trichothecium sympodiale CBS 227 76 Trichothecium sympodiale ATCC36477 Myrotheciomyces corymbiae CPC 33206 Trichothecium roseum UAMH 7839 Trichothecium roseum KUNCC21 10015 Trichothecium roseum RiT1 Trichothecium roseum PH6 Trichothecium roseum LCP 50 627 Trichothecium roseum DAOM 57205 Trichothecium roseum PG Trichothecium roseum DAOM 208997 Bullanockia australis CPC28976 Xanthonectria pseudopeziza CBS 126456 Xanthonectria pseudopeziza CBS 126104 Xanthonectria pseudopeziza CBS 141245 Xanthonectria pseudopeziza CBS 140160 Ijuhya paraparilis Tua h52 Ijuhya chilensis strain CBS 102803 Flammocladiella decora CLL16020 Flammocladiella aceris CPC 24422 Flammocladiella anomiae SOMF 30203 Flammocladiella anomiae JF17087 Flammocladiella anomiae CLL16017 Stephanonectria keithii GJS92 133 Clonostachys rosea CCFC226708 Clonostachys miodochialisCBS 997 69 Nectriopsis lindauiana CBS 897 70 Nectriopsis lindauiana CBS 839 70 Nectriopsis exigua 98 32 Nectriopsis violacea CBS 914 70 Nectriopsis violacea CBS 849 70 Ochronectria thailandica MFLUCC 15 0140 Ochronectria calami CBS 445 96 Ochronectria calami ATCC46692 Protocreopsis freycinetiae CBS 573 76 Pronectria robergei CBS 128021 Acremonium rutillum CBS 396 66 Acremonium rutillum CBS 229 70 Septofusidium herbarum CBS 265 58 Septofusidium berolinense CBS 731 70 Lasionectria lecanodes CLL14062 Lasionectria mantuana CBS 114291 Lasionectria mantuana A R 4029 Synnemellisia urenae BRIP 71652 Synnemellisia aurantia COAD2070 Caespitomonium hyalinulum CBS 271 36 Caespitomonium euphorbiae CPC 39083 Paracylindrocarpon aloica CPC 27362 Paracylindrocarpon pandanicola KUMCC 17 0272 Paracylindrocarpon nabanheensis KUMCC 16 0147 Fusariella hughesii strain CBS 435 70 Fusariella atrovirens CBS 311 73 Heleococcum aurantiacum CBS201 35 Hydropisphaera peziza CBS123792 Hydropisphaera peziza BPI802846 Gliomastix murorum CBS 154 25 Gliomastix murorum CBS 127397 Gliomastix tumulicola CBS 127532 Gliomastix tumulicola Verrucostoma martinicensis CBS 138731 Verrucostoma freycinetiae MAFF240100 Roumegueriella rufula GJS 91 164 Roumegueriella rufula CBS 346 85 Nothoacremonium exiguum CBS 587 73 Nothoacremonium vesiculophorum CBS 397 70B Nothoacremonium subcylindricum CBS 190 70 Nothoacremonium subcylindricum CBS 781 69 Nothoacremonium subcylindricum CBS 416 68 Chrysonectria finisterrensi JPP17021 Chrysonectria crystallifera CBS 102567 Heteroacremonium rugosum CGMCC 3 22520 Heteroacremonium album CGMCC 3 22409 Heteroacremonium album CGMCC 3 22405 Sedecimiella taiwanensis CY5100 Sedecimiella funiculosa CGMCC 3 22348 Sedecimiella funiculosa CGMCC 3 22356 Neoacremonium vitellinum CBS 793 69 Neoacremonium vitellinum CBS 792 69 Neoacremonium flavum CBS 452 70 Neoacremonium flavum CBS 398 70 Neoacremonium distortum CBS 665 75 Neoacremonium distortum CBS 314 72 Allantonectria miltina CBS 474 69 Allantonectria miltina A R 4391 Thyronectria quercicola CBS 128976 Thyronectria lamyi CBS 417 89 Thyronectria caudata CBS 136003 Roselliniella euparmeliicola DLH 2009a Roselliniella atlantica Coppins15370 Nectria mariae CBS 125294 Nectria nigrescens CBS 125148 Nectria cinnabarina CBS 125165 Cosmospora arxii CBS 748 69 Rectifusarium ventricosum NRRL 20846 Rectifusarium robinianum CBS 430 91 Xenoacremonium recifei CBS 137 35 Xenoacremonium falcatus CBS 400 85 Paracremonium inflatum CBS 485 77 Paracremonium apiculatum CGMCC3 19309 Pseudocosmospora eutypellae GJS 10 294 Pseudocosmospora eutypellae CBS 133966 Cosmosporella cavisperma CBS 172 31 Cosmospora khandalensis AR4770 Cosmospora coccinea CBS 343 70 Cosmospora coccinea CBS 341 70 Fusicolla violacea CBS 634 76 Fusicolla melogrammae CBS 141092 Macroconia papilionacearum CBS 125495 Macroconia leptosphaeriae CBS 717 74 Microcera coccophila CBS 310 34 Microcera physciae CPC 41038 Microcera rubra CBS 638 76 Microcera larvarum CBS 738 79 Bisifusarium nectrioides CBS 176 31 Bisifusarium dimerum CBS 108944 Calostilbe striispora CBS 133491 Chaetopsina fulva FMR 13129 Chaetopsina fulva CBS142 56 Sarcopodium circinatum CBS 587 92 Sarcopodium circinatum CBS 100998 Coccinonectria pachysandricola CBS 476 92 Coccinonectria pachysandricola CBS501 63 Geejayessia celtidicola CBS 125502 Geejayessia atrofusca CBS 125482 Cyanonectria cyanostoma CBS 101734 Cyanonectria buxi CBS 130 97 Albonectria rigidiuscula CBS 315 73 Albonectria rigidiuscula CBS 122570 Neocosmospora illudens CBS 147303 Neocosmospora vasinfecta CBS 325 54 Neocosmospora ambrosia CBS 571 94 Neonectria ramulariae CBS 151 29 Neonectria neomacrospora CBS 118984 Neonectria faginata CBS 217 67 Dactylonectria macrodidyma CBS 112615 Ilyonectria capensis CBS 132815 Ilyonectria radicicola CBS 264 65 Ilyonectria destructans CBS 264 65 Corallonectria jatrophae CBS 913 96 Aquanectria submersus CBS 394 62 Aquanectria penicillioides CBS 257 54 Cylindrocladiella lageniformis CBS 340 92 Cylindrocladiella camelliae CPC 234 Xenocylindrocladium serpens CBS 128439 Xenocylindrocladium guianense CBS112179 Curvicladiella cignea CBS 109167 Calonectria daldiniana CBS 749 70 Calonectria brassicae CBS 111869 Xenogliocladiopsis cypellocarpa CPC 17153 Xenogliocladiopsis cypellocarpa CBS133814 Thelonectria discophora CBS 125153 Thelonectria veuillotiana MAFF241544 Thelonectria olida CBS 215 67 Mariannaea humicola CBS 740 95 Mariannaea elegans HU0029 Rugonectria rugulosa CBS 129158 Rugonectria rugulosa CBS 126565 Campylocarpon pseudofasciculare CBS 112679 Campylocarpon fasciculare CBS 112613 0.02 Fig. 2. Phylogenetic relationships within the family Paranectriaceae and sister Cylindromonium-Trichonectria clade inferred from a Bayesian Inference analysis (BI) of a combined 5.8S, LSU, nuSSU, tef-1 and rpb1 data set. Niesslia spp. were used as the outgroup. Bootstrap value from ML/posterior probability from Bayesian analyses at branches. Thickened branches represent either bootstrap support values ≥ 70 % and/ or Bayesian posterior probabilities ≥ 0.97, lower values inducated by ‘-‘. The asterisk indicates type materials. Persoonia – Volume 54, 202568 showed a sister relationship to the clade comprising the new family Paranectriaceae and Cylindromonium-Trichonectria clade. The members of the family Acremoniopsiaceae clustered in a well-supported clade (100/1) and showed moderately supported sister relationships to the aforementioned families. Phylogenetic relationships within the new family The Paranectriaceae multi-gene dataset was analysed to determinate the interspecific relationships among genera representing the target lichenicolous lineage of the order Hypocreales. This dataset consisted of 42 specimens with sequences of the 5.8S, LSU, nuSSU, tef-1, rpb1 regions. This five-locus alignment contained 4209 characters (158 5.8S, 886 LSU, 1 631 nuSSU, 960 tef-1, 594 rpb1), of which 640 were parsimony-informative, 257 singleton sites, and 3312 constant sites. The tree topology of ML and Bayesian analysis was the same and only the 50 % majority rule consensus tree is shown in Fig. 2. The new lineage of lichenicolous fungi, proposed here as a new family Paranectriaceae, formed a strongly supported clade (100/1), showing a sister relationship to the Cylindromonium-Trichonectria clade. The new family consists of five genera represented by lichenicolous species. The nine lichenicolous specimens characterized by having filiform ascospores formed a well-supported clade of Rossmaniella genus (88/0.97). Morphologically, due to byssoid ascomata grouped on a distinct subiculum and long filiform ascospores, the specimens resemble such lichenicolous fungi as Lichenobarya, Nectria byssophila and Neobaryopsis, but they are phylogenetically not related. Therefore, the new genus Rossmaniella, with four newly recognized species is introduced for this group. This clade shows a sister relationship to the morphologically different genus Paranectria, although without significant statistical support. The phylogeny revealed the species of the genus Paranectria clustered in two separate clades. Two specimens of P. affinis, the generic type of Paranectria, growing on Ephebe lanata formed a separate, highly supported (100/1) clade (Fig. 2) and showed a relationship to the genus Rossmaniella, but with low support. In contrast, six specimens of Paranectria oropensis (under the name Ciliomyces oropensis in the Fig. 2) formed a separate and well-supported clade (100/1), which seems to be not related to the sequences of P. affinis; however, the relationships are not well resolved. Due to differences in morphology of those two species and together with phylogenetic placement, the genus Ciliomyces is reinstated for this species. The four specimens of the tropical species Nectriopsis lichenophila clustered in a well-supported (100/1) clade sister to the larger clade consisting of Ciliomyces, Paranectria and Rossmaniella. This fungus appears to be not related to the genus Nectriopsis s. str. (generic type N. violaceae growing on Fuligo septica) which belongs to the family Bionectriaceae (see Fig. 1). Thereby, based on the phylogenetic and morphological differences between Nectriopsis lichenophila and other species in Nectriopsis s. str., the new monotypic genus Sphaeronectria is described here. The genus Ovicuculispora is represented by two species in our phylogeny, O. parmeliae (generic type, represented by five specimens) and O. cf. macrospora (two specimens). They both cluster in a well-supported monophyletic clade (100/1) showing a sister relationship to the remaining taxa of the new family. The sequenced specimens of O. parmeliae were collected on different host species (see Table 1) Table 2. Partition scheme used in the phylogenetic analyses. Dataset Region Codon position Model Hypocreales dataset LSU — GTR+I+G tef-1 The first codon position TVM+I+G tef-1 The second codon position TVM+I+G tef-1 The third codon position GTR+I+G rpb1 The first codon position GTR+I+G rpb1 The second codon position TVM+I+G rpb1 The third codon position TVM+I+G rpb2 The first codon position GTR+I+G rpb2 The second codon position TVMEF+I+G rpb2 The third codon position GTR+I+G tub2 The first codon position GTR+I+G tub2 The second codon position TRNEF+G tub2 The third codon position SYM+I+G Paranectriaceae dataset 5.8S — TRNEF+I+G LSU — TRNEF+I+G nuSSU — K80-I tef-1 The first codon position TIM+I+G tef-1 The second codon position TIM+I+G tef-1 The third codon position GTR+G rpb1 The first codon position TIM+I+G rpb1 The second codon position TRNEF+I+G rpb1 The third codon position TVM+G Darmostuk et al.: New lineage of hypocrealean fungi 69 belonging to the families Parmeliaceae (Lecanorales) and Lobariaceae (Peltigerales). However, all the specimens grouped in a single well-supported clade (99/1), indicating a non-specific phylogenetic correlation with the host type. Despite growing on several unrelated host genera, the genetic variation among loci seems to be low and indicates that all examined specimens belong to the same lichenicolous species. Additional specimens of Ovicuculispora parmeliae (Flakus 26010, 26011) collected on Sticta sp. from Bolivia were not included in the tree, due to the fact that only ITS sequences were obtained. However, we have tried to use morphological data and the ITS region (not included here) to place this species within the new family circumscription and it showed 100 % similarity with a sequence obtained from the specimen on Sticta sp. (KRAM L-74677), as well as 99 % similarity to sequences for specimens on other hosts (Crocodia, Heterodermia and Hypotrachyna s.lat). TAXONOMY Paranectriaceae Darmostuk, Etayo & Flakus, fam. nov. MycoBank MB 858383. Etymology: Referring to the name of the type genus. Type genus: Paranectria Sacc. Fig. 3. Ascomata morphology of the family Paranectriaceae. A. Ciliomyces oropensis (Darmostuk 957). B. Ovicuculispora parmeliae (Etayo 32636). C. Ovicuculispora cf. macrospora (Flakus 29165). D. Paranectria affinis (UPS F-561663). E. Sphaeronectria lichenophila (Flakus 26997). F. Rossmaniella filispora (Flakus 27467). G. Rossmaniella filispora (Etayo 30600); H. Rossmaniella tylophori (Flakus 26805). I. Rossmaniella cryptica (Flakus 26967). Scale bars = 250 μm. Persoonia – Volume 54, 202570 Ascomata perithecioid, superficial, globose to pyriform, yellow to bright orange, scattered or forming groups, with more or less developed whitish subiculum in the lower part of ascomata. Ascomatal wall composed of several layers of irregular cells, hyaline to yellowish, not changing colour in KOH. Asci unitunicate, cylindrical to subclavate, 4–8-spored. Ascospores hyaline, ellipsoid to filiform, 1–3-septate to muriform, also two types (macroand microspores) can occur in the same ascus. All the genera included in this family are lichenicolous species. Included genera: Ciliomyces, Ovicuculispora, Paranectria, Rossmaniella, Sphaeronectria. Ciliomyces Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 115: 673. 1906. MycoBank MB 1057. Type species: Ciliomyces oropensis (Ces.) Höhn. Ascomata perithecioid, globose to ovoid, superficial, scattered, totally covered by whitish tomentum, with visible ostiole. Ascomatal wall up to 35 µm thick, composed of pseudoparenchymatous thick-walled cells. Asci clavate, short-stalked, 8-spored (rarely 4-spored). Ascospores hyaline, ovoid to narrowly ellipsoid, with elongate appendages at each end, submuriform to muriform. Asexual morph acremoniumlike with 1–3-septate, hyaline, ellipsoid to subcylindrical conidia. Notes: Our phylogeny shows that the sequences of Paranectria oropensis clustered in a well-supported monophyletic clade are only distinctly related to the type of the genus Paranectria, P. affinis. Furthermore, we observed morphological differences in ascomata and ascospores of both species. Therefore Ciliomyces, with the type species Ciliomyces oropensis, is reinstated here from the synonyms of Paranectria. The genus Ciliomyces was established by Höhnel (Höhnel & Litschauer 1906) to accommodate the lichenicolous species Ciliomyces oropensis and was later accepted by Keissler (1930) and Samuels (1976) as a separate genus. However, referring to the presence of elongate appendages at each end of ascospores in both genera, Hawksworth & Pirozynski (1977) considered Ciliomyces to be a heterotypic synonym of Paranectria. Nevertheless, the generic type of Paranectria, P. affinis, is characterized by white to pale luteous ascomata with arachnoid hyphae appearing only at their lower part and ascospores with transverse septa only. In contrast, the reinstated Ciliomyces is characterized by yellow orange ascomata fully covered by whitish tomentum and submuriform to muriform ascospores (Hawksworth 1982, Rossman 1983, Rossman et al. 1999, Zhurbenko 2009). Although these differences have been briefly discussed in several works, not considered to have enough taxonomic value to separate both genera (Rossman 1983, Rossman et al. 1999). No molecular data are available for Paranectria alstrupii and P. superba. However, based on their similar muriform ascospores to C. oropensis and lichenicolous lifestyle, we consider that these species can be affiliated with Ciliomyces (Hawksworth 1982, Zhurbenko 2009, Zhurbenko & Dillman 2010). We are awaiting further data before proposing formal changes. Ciliomyces oropensis (Ces.) Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 115: 673. 1906. MycoBank MB 232256. Figs 3A, 4C. Basionym: Sphaeria (Nectria) oropensis Ces., in Rabenhorst, Klotzschii Herb. Viv. Mycol., Edn Nov, Ser. Sec., Cent. 6: no. 524. 1857. Synonyms: Cucurbitaria oropensis (Ces.) Kuntze, Revis. gen. pl. (Leipzig) 3(3): 461. 1898. Dialonectria oropensis (Ces.) Cooke, Grevillea 12(64): 111. 1884. Nectria oropensis (Ces.) Sacc., Syll. Fung. (Abellini) 2: 511. 1883. Paranectria oropensis subsp. parviseptata M.S. Cole & D. Hawksw., Mycotaxon 77: 324. 2001. Paranectria oropensis (Ces.) D. Hawksw. & Piroz., Canad. J. Bot. 55(19): 2555. 1977. Typus: Italy, Province of Biella, Piedmont, near the great Sanctuary of the Blessed Virgin Maria on Mount Oropa, Sep. 1856, V. Cesati (M, not examined). Nectria lichenicola P. Crouan & H. Crouan, Florule de Finistère (Paris): 256. 1867. Dialonectria lichenicola (P. Crouan & H. Crouan) Cooke, Grevillea 12(64): 111. 1884. Pleonectria lichenicola (P. Crouan & H. Crouan) Sacc., Michelia 1 (3): 325. 1878. Typus: France, Finistère, on the granular thallus of a lichen, on the trunk of a beech tree (type not located). Pleonectria appendiculata Vouaux, Bulletin de la Société Mycologique de France 28: 193. 1912. Typus: France,France, on a thin unidentified thallus, on an old oak near Docelles in the Vosges, J. Harmand J. Harmand (type not located). For detailed description see Hafellner & Obermayer (2009) and Navarro-Rosinés & Llimona (2018). Distribution, habitat and host range: This is a common generalist species in the northern hemisphere, with scattered records from the southern hemisphere. It has been reported on many unrelated corticolous lichens (Diederich 2003, Hafellner & Obermayer 2009, Brackel 2014) and rarely on saxicolous ones (Navarro-Rosinés & Llimona 2018). Specimens examined: Bolivia, Chuquisaca Department Chuquisaca, Zudañez Province, El Palmar Integrated Management Natural Area, Salviatójo near Lomán,, 18º45’51”S, 64º50’09”W, 2836 m a.s.l., disturbed BolivianoTucumano forest with Podocarpus and shrubs, on Normandina pulchella, 14 Jul. 2015, A. Flakus 26387 (KRAM L-74672; LPB). Spain, Cáceres, Monfragüe National Park, Saltos del Torrejón, near Tres Caños, Quercus ilex ssp. ballota wood pasture with quartzite stones.39º50’39”N, 06º00’05”W, 230 m a.s.l., on Parmelina tiliacea on Quercus ilex, 9 May 2015, J. Etayo 29106 (hb. Etayo); Guipúzcoa, Peñas de Aia, Lesakako Bidea road, just before the tunnel 43º16’25”N, 1º47’35.5”W, 500 m a.s.l., on Hypotrachyna revoluta on Fagus sylvatica, 31 Aug. 2019, J. Etayo 31862 (hb. Etayo, sub Rossmaniella filispora); Navarra, Basaburúa Mayor valley, between Aizároz and Arrarás, track to Bergañe, 550 m a.s.l., on unidentified lichen and bryophytes on Fagus, 20 Nov. 1994, J. Etayo 35899 (hb. Etayo); Lizarrusti pass, between Etxarri Aranaz and Beasain, 42º57’20”N, 2º05’00”W, 565 m a.s.l., on Physconia Darmostuk et al.: New lineage of hypocrealean fungi 71 perisidiosa growing on Quercus robur, 5 Jan. 2017, J. Etayo 31139 (hb. Etayo); Ciaúrriz, track a few meters before the junction of the N-411 with the entrance to the town, oak grove with wet boxwood, 42º55’49”N, 1º37’18”W, 625 m a.s.l., on Anaptychia ciliaris on Quercus faginea, 25 Nov. 2018, J. Etayo 31593 (hb. Etayo); Teruel, between Albarracín and Bezas, 40º22’52”N, 1º24’02”W, 1350 m a.s.l., on Physconia grisea on sandstones under Pinus pinaster, 16 Aug. 2020, J. Etayo 32605 (hb. Etayo). Ukraine, Ternopil region, Ternopil district, vicinity of the Posukhiv village, 49.41122 N, 24.93305 E, on thallus of Lecania croatica, on Fagus bark, 5 Sept. 2020, V. Darmostuk 957 & O. Sira (KRAM L-74673). Fig. 4. Ascospores morphology of the family Paranectriaceae. A. Rossmaniella cryptica (Flakus 26967). B. Paranectria affinis (UPS F-561663). C. Ciliomyces oropensis (Darmostuk 957). D. Sphaeronectria lichenophila (Kukwa 16271). E. Macrospores of Ovicuculispora cf. macrospora (Flakus 29165). F. Macrospores of Ovicuculispora parmeliae (Flakus 26011). Scale bars = 25 μm. Persoonia – Volume 54, 202578 Fig. 8. Rossmaniella cryptica (holotype Flakus 26967, except B from K-M000454728). A, B. Ascomata on the host thallus. C. Squashed ascomata (in water). D. Section of the ascomata (in LPCB). E. Asci (in Congo Red). F. Ascomatal wall (in LPCB). G. Asci apex (in Congo Red and LPCB). H. Ascospores (first and second in water, other in LPCB). Scale bars: A, B = 250 μm; C, D = 50 μm; E, H = 25 μm; F, G = 10 μm. Darmostuk et al.: New lineage of hypocrealean fungi 79 Fig. 9. Rossmaniella filispora (holotype Flakus 26556, except B from Flakus 29557). A–D. Ascomata on the host thallus. E. Squashed ascomata (in water). F. Section of the ascomata (in LPCB). G. Ascomata wall (in LPCB). H. Hairs (in Congo Red). I. Ascus apex (in Congo Red). J. Asci with ascospores (in Congo Red). Scale bars: A–D = 250 μm; E, F =100 μm; G, H, I = 10 μm; J = 25 μm. Notes: This species is morphologically very similar to Rossmaniella filispora, but can be distinguished by the combination of morphological features and phylogenetic position. Rossmaniella cryptica has somewhat bigger, vivid orange ascomata, mostly in a group up to 4(–7) [in R. filispora (5–)7–12 ascomata in a group], bright orange ostiolate part without distinct hairs (distinct hairs present in R. filispora), somewhat shorter ascospores (160–)165–175(–180) μm [in R. filispora (165–)180–200(–210) μm] with longer, (13.2– )15.2–21.0(–23.8) μm, individual cells [in R. filispora (11.0– )11.5–13.7(–14.5) μm]. Persoonia – Volume 54, 202580 Rossmaniella filispora Darmostuk, Etayo & Flakus, sp. nov. MycoBank MB 858387. Figs 3F, 3G, 9. Etymology: Named after the filiform ascospores. Typus: Bolivia, Chuquisaca Department, Belisario Boeto Province, close to Padilla between Nuevo Mundo and Santa Rosa, 18º57’12”S, 64º16’37”W, 1790 m a.s.l., transition between Boliviano-Tucumano forests and dry interandean vegetation, on corticolous Punctelia sp., 16 Jul. 2015, A. Flakus 26556 (holotype KRAM L-74684, isotype LPB). Ascomata perithecioid, ovoid to pyriform, not collapsed when dry, superficial, clustered in dense groups of (5–)7– 12 ascomata, pale to bring orange, ostiolate part the same colour as ascomatal surface, the lover part covered by hyaline tomentum, becoming more compact towards the top of ascomata, (220–)270–340(–365) × (190–)200–235(–270) µm (n = 20), sometimes individual ascomata has irregular form in the group due to compression by other ascomata. Ascomatal surface near the ostiole with short, cylindric, hyaline, 1–2-septate hairs, sometimes with inflated tips, 20– 55 × 5–7 μm. Ascomatal wall 20–35 μm thick, thicker at the upper part, hyaline to pale yellow, composed of two layers of cells: external layer composed of 3–5 layers of isodiametric to irregular, thin-walled cells, 5–7 μm in diam; an inner region with 3–5 layers of elongated thin-walled cells, 10–15 × 3–4.5 μm, KOH–. Asci narrowly cylindrical, without apical thickness, 8-spored, (164–)180–210(–230) × (8.5–)9.0–13.0(–14.4) μm (n = 15). Ascospores hyaline, 8–12-septate, septa often hardly visible, filiform, straight or curved, parallel to twisted in the ascus proximal ends rounded, distal end pointed, (165– )180–200(–210) × (2.8–)3.0–3.4(–3.8) μm (n = 30), individual cells (11.0–)11.5–13.7(–14.5) μm (n = 30) long. Conidiomata not observed. Distribution, habitat and host range: This species was reported from scattered localities in Europe (Spain) and South America (Bolivia). Rossmaniella filispora is probably not a host-specific taxon as it was found on several lichen hosts belonging to the Parmeliaceae (Flavopunctelia, Parmotrema, Hypotrachyna), Peltigeraceae (Peltigera) and Ramalinaceae (Phyllopsora). This species did not show any pathogenic effect on the host. Specimens examined: Bolivia, Chuquisaca Department, Belisario Boeto Province, close to Padilla between Nuevo Mundo and Santa Rosa, 18º57’06”S, 64º16’14”W, 1936 m a.s.l., transition between Boliviano-Tucumano forests and dry interandean vegetation, on Flavopunctelia sp., 16 Jul. 2015, A. Flakus 26594 (LPB); ibid., 18º57’12”S, 64º16’37”W, 1790 m a.s.l., transition between Boliviano-Tucumano forests and dry interandean vegetation, on corticolous Flavopunctelia sp., 16 Jul. 2015, A. Flakus 26557 (LPB); Cochabamba Department, Carrasco Province, Carrasco National Park, Korikaza close to Monte Punku, 17º33’30”S, 65º16’32”W, 2880 m a.s.l., lower montane Yungas cloud forest, on corticolous Parmotrema sp., 27 Nov. 2014, J. Etayo 29951 (LPB); Tarija Department, Burnet O’Connor Province, close to los Pinos, old road between Entre Ríos and Tarija, 21º24’50”S, 64º18’33”W, 2149 m a.s.l., Boliviano-Tucumano forest dominated by shrubs, with Alnus acuminata, Podocarpus and Ericaceae, on Phyllopsora sp., 29 Jul. 2015, A. Flakus 27468 (KRAM L-74686, LPB); ibid., on Parmotrema sp., M. Kukwa 16861a (UGDA L, LPB); ibid., 21º27’48”S, 64º13’24”W, 1943 m a.s.l., Boliviano-Tucumano forest with Podocarpus, on corticolous Punctelia sp., 28 Jul. 2015, A. Flakus 27389a (LPB); ibid., 21º25’07”S, 64º18’50”W, 2190 m a.s.l., Boliviano-Tucumano forest dominated by shrubs, Andino Montano belt, on Peltigera didactyla, 29 Jul. 2015, J. Etayo 30600 (LPB); ibid., 112 km from Tarija on the way to Entre Ríos, near San Diego, 21°26’28”S, 64°14’37”W, 1620 m a.s.l., TucumanoBoliviano montano forest, on Parmotrema sp. on twig, 9 Aug. 2012, J. Etayo 28594 (hb. Etayo); near Soledad, 21°39’52”S, 64°07’22”W, 1700 m, Tucumano-Boliviano montano forest, on Parmotrema reticulatum on twig, 11 Aug. 2012, J. Etayo 28777 (LPB). Spain, Guipuzcoa, Peñass de Aia, Lesakako Bidea, antes del tunel, 43 16’25”N, 1 47’35.5”W, 500 m a.s.l., on Hypotrachyna revoluta on Fagus sylvatica, 31 Aug. 2019, J. Etayo 31862 (hb. Etayo). Notes: The species is morphologically very similar to Rossmaniella cryptica, but it can be distinguished by specific morphological features and its phylogenetic position. However, R. filispora does not seem to be a rare species in the tropical forests of Bolivia and is known from several localities. Rossmaniella tylophori Darmostuk & Flakus, sp. nov. MycoBank MB 858388. Figs 3H, 10. Etymology: Named after the host lichen genus, Tylophoron. Typus: Bolivia, Chuquisaca Department, Luis Calvo Province, Iñao National Park and Integrated Management Natural Area, between Ticucha and Entre Ríos, 19º31’09”S, 63º53’31”W, 1373 m a.s.l., disturbed area with shrubs, on corticolous Tylophoron protrudens, 19 Jul. 2015, A. Flakus 26805 (holotype KRAM L-74687, isotype LPB) Ascomata perithecioid, ovoid to pyriform, superficial, solitary, lemon yellow, (200–)230–270(–300) × (150–)160–175(– 185) µm (n = 15), covered by not dense whitish tomentum, composed of simple to branched, hyaline, septate, thinwalled, verruculose hyphae, 4–5 μm thick. Ascomata surface evenly coloured, without hairs near ostiole part. Ascomatal wall 18–25 μm thick, slightly thicker at the upper part, hyaline to pale yellow, composed of two layers: an external layer of globose, thin-walled cells, 2–4 × 2–3 μm; an inner region of flattened, thin-walled cells, 3–12 × 1–2 μm, KOH–. Asci cylindrical, without apical thickness, 8-spored, (170– )175–180(–200) × (12.6–)13.0–13.8(–14.2) µm (n = 15). Ascospores hyaline, 8–12-septate, filiform, straight or rarely slightly curved, parallel to rarely twisted in the ascus, both ends rounded, (125–)140–155(–180) × (2.8–)3.4–4.4(–4.6) μm (n = 30), individual cells (10.0–)12.0–15.6(–16.8) μm (n = 30) long. Conidiomata not observed. Distribution, habitat and host range: This species is known only from the type locality in Bolivia. This fungus grows on corticolous Tylophoron protrudens and the infection does not cause visible damage to the host thallus. Darmostuk et al.: New lineage of hypocrealean fungi 81 Notes: Rossmaniella tylophori can be distinguished from other Rossmaniella species by solitary lemon-yellow ascomata, shorter and wider ascospores and host specificity. However, since the species is known only from a single collection, the host range is unknown. Previously only two lichenicolous species were reported on Tylophoron species, i.e. Taeniolella serusiauxii reported from Europe and tropical regions (Heuchert et al. 2018) and Chaenothecopsis pilosa described from Tanzania (Tibell & Ryman 1995). Fig. 10. Rossmaniella tylophori (holotype Flakus 26805). A–C. Ascomata on the host thallus. D. Squashed ascomata (in water). E. Ascomatal wall (in LPCB). F. Asci apex (in Congo Red). G. Asci (in Congo Red). H. Ascospores (in Congo Red and LPCB). Scale bars: A–C = 250 μm; D = 100 μm; E, F = 10 μm; G, H = 25 μm. Persoonia – Volume 54, 202582 Sphaeronectria Darmostuk, Etayo & Flakus, gen. nov. MycoBank MB 858389. Etymology: Referring to the globose ascomata. Type species: Sphaeronectria lichenophila (Speg.) Darmostuk, Etayo & Flakus Ascomata perithecioid, superficial, globose to subglobose, constricted laterally or apically, yellowish to orange, with tiny tomentum on the lower part of ascomata, 150–250 μm diam., covered by abundant septate hyaline hyphae, 50–100 µm long. Ascomatal wall formed by several layers of cells, hyaline to pale yellowish in the outer part, up to 20 μm thick, superficially paraplectenchymatous, KOH–. Asci unitunicate, subcylindrical, 4-spored, without apical thickness. Ascospores hyaline, 1-septate, ellipsoid, with pointed ends, slightly constricted at the septa, smooth-walled. Conidiomata not observed. Notes: Previously the type species of Sphaeronectria (S. lichenophila) was included in the genus Nectriopsis, typified by N. violacea, but based on our phylogenetic results, S. lichenophila showed a different phylogenetic position in Paranectriaceae, whereas Nectriopsis is a member of Bionectriaceae. Currently, the genus is monotypic. Sphaeronectria lichenophila (Speg.) Darmostuk, Etayo & Flakus, comb. nov. MycoBank MB 858390. Figs 3E, 4D. Basionym: Nectria lichenophila Speg., Boln Acad. Nac. Cienc. Córdoba 11(4): 525. 1889. Synonyms: Nectria spegazzinii Vouaux, Bull. Soc. Mycol. Fr. 28: 189. 1912. Nectriopsis lichenophila (Speg.) Etayo, Bibliotheca Lichenologica 84: 71. 2002.Typus: Brazil, Apiahy, on Anaptychia cf. podocarpa, 1881, J. Puiggari (holotype LPS1587). Solenopezia [Solenopeziza] tetraspora Henn., in Engler, Pflanzenw. Ost-Afrikas Nachbarg., Teil C: 30. 1895. Typus: Tanzania, on thallus of Physcia integrata Nyl., unknown collection date, Holst n. 795 (type not located). Ascomata perithecioid, superficial, globose to subglobose, yellow to orange, collapsed when dry, with tiny subiculum on the lower part of ascomata, covered by abundant septate hyaline hairs, 150–250 μm diam. Ascomatal wall formed by several layers of cells, yellowish externally, up to 20 μm thick, inner hyaline, 10–12 μm, KOH–. Asci unitunicate, subcylindrical, 4-spored, 70–80 × 7–10 μm (n = 20). Ascospores hyaline, 1-septate, ellipsoid, with pointed and sometimes apiculate ends, slightly constricted at the septa, smooth-walled, without perisporium, (16.8–)19.4–24.4(–26.6) × (5.9–)6.2–7.5(–8.4) μm (n = 50). Conidiomata not observed. Distribution, habitat and host range: This species is known from numerous localities from South America and Africa (Tanzania), growing on several species of Heterodermia s. lat. (Etayo 2002, 2017). However, the record from Tanzania needs revision as we were not able to locate the specimen in the collection of P.C. Hennings stored at B. Specimens examined: Bolivia, Chuquisaca Department, Belisario Boeto Province, close to Padilla between Nuevo Mundo and Santa Rosa, 18º57’12”S, 64º16’37”W, 1790 m a.s.l., transition between Boliviano-Tucumano forests and dry interandean vegetation, on Heterodermia cf. spinigera, 16 Jul. 2015, A. Flakus 26552 (LPB), ibid., on Heterodermia comosa, M. Kukwa 16271 (UGDA L, LPB); Luis Calvo Province, Iñao National Park and Integrated Management Natural Area close to Ticucha, between Tranqua and Monte Agudo, 19º39’50”S, 63º49’14”W, 1022 m a.s.l., disturbed area with shrabs, on lower and upper part of Heterodermia cf. comosa on twigs, 18 Jul. 2015, J. Etayo 32702, 32709 (hb. Etayo, LPB); Santa Cruz Department, Manuel María Caballero Province, Monte Empalme near Siberia, 17º50’05”S, 64º42’09”W, 2439 m a.s.l., partly grazed Yungas cloud forest near stream, on Heterodermia podocarpa, 8 Nov. 2016, A. Flakus 28086 (KRAM L-74693, LPB); Tarija Department, Aniceto Arce Province, Tariquía Flora and Fauna National Reserve between la Cumbre and camamento los Alisos, 22º02’38”S, 64º35’47”W, 2460 m a.s.l., Boliviano-Tucumano forest with Alnus acuminata and Polylepis, on Leucodermia sp., 22 Jul. 2015, A. Flakus 26977 (KRAM L-74690, LPB); ibid., 22º01’02”S, 64º34’51”W, 2135 m a.s.l., on Heterodermia cf. comosa, 22 Jul. 2015, A. Flakus 27056 (KRAM L-74691, LPB); ibid., close to los Alisos camp, 22º01’25”S, 64º34’06”W, 1900 m a.s.l., disturbed BolivianoTucumano forest with Alnus acuminata, Podocarpus and Solanaceae, on Heterodermia sp. growing on twig, 24 Jul. 2015, A. Flakus 27086 (KRAM-L, LPB), M. Kukwa 16600 (UGDA L, LPB); ibid., on Leucodermia fertilis, 24 Jul. 2015, A. Flakus 27087 (KRAM L-74693, LPB); ibid., close to Coyambuyo, between Padcaya and Bermejo, 22º17’23”S, 64º28’50”W, 942 m a.s.l., Tucumano-Boliviano forest with bryophytes, Lauraceae and Meristomataceae, on corticolous Heterodermia cf. spinigera, 26 Jul. 2015, M. Kukwa 16726 (LPB); Burnet O’Connor Province, close to Entre Ríos, new road between Tarija and Entre Ríos, 21º30’47”S, 64º11’49”W, 1338 m a.s.l., disturbed Tucumano-Boliviano forest with shrubs and Tillandsia, on Heterodermia japonica, 28 Jul. 2015, A. Flakus 27360 (KRAM L-74694, LPB), ibid., on Heterodermia sp., M. Kukwa 16812, 16821 (UGDA L, LPB); la Cumbre close to Entre Ríos, old road between Entre Ríos and Tarija, 21º27’48”S, 64º13’24”W, 1943 m, BolivianoTucumano forest with Podocarpus, on Heterodermia comosa, 28 Jul. 2015, A. Flakus 27392 (KRAM L-74695, LPB), ibid., M. Kukwa 16833a (LPB); ibid., on Heterodermia cf. comosa, 28 Jul. 2015, A. Flakus 27383 (LPB); ibid., on Leucodermia sp., 28 Jul. 2015, A. Flakus 27400 (KRAM L-74696, LPB); ibid., 21º27’50”S, 64º12’51”W, 1924 m a.s.l., Boliviano-Tucumano forest with epiphytes exposed NW, on Heterodermia comosa, 30 Jul. 2015, A. Flakus 27650 (KRAM L-74698, LPB); ibid., 21º25’57”S, 64º19’17”W, 2178 m a.s.l., BolivianoTucumano forest close to small river dominated by shrubs, on Leucodermia cf. fertilis, 31 Jul. 2015, A. Flakus 27780 (LPB); ibid., on Leucodermia leucomelos on twigs, J. Etayo 32928 (LPB); ibid., close to los Pinos, 90 km from Tarija on old road between Entre Ríos and Tarija, 21º25’30”S, 64º19’07”W, 2265 m a.s.l., Boliviano-Tucumano forest dominated by shrubs, with Alnus acuminata, Podocarpus and Ericaceae, on Leucodermia leucomelos, 29 Jul. 2015, A. Flakus 27419 Darmostuk et al.: New lineage of hypocrealean fungi 83 Key of the lichenicolous species included in the family Paranectriaceae 1a. Asci with two types of ascospores (macroand microspores) ................................................................................................. 2 1b. Asci with one type of ascospores ........................................................................................................................................... 4 2a. Macrospores with pointed ends, mostly > 50 μm in length, ascomata with dense arachnoid tomentum at the lower part ................................................................................................................................................................................ 3 2b. Macrospores with rounded ends, mostly 40–60 μm in length, ascomata rarely with arachnoid tomentum at the lower part .................................................................................................................................................Ovicuculispora parmeliae 3a. Macrospores with hair-like surface, (53.0–)56.4–66.2(–75.8) μm in length .........................Ovicuculispora cf. macrospora 3b. Macrospores with smooth surface, 67–105 μm in length ..........................................................Ovicuculispora macrospora 4a. Ascospores filiform, (8–)10–15-septate .................................................................................................................................. 5 4b. Ascospores ellipsoid to ovoid, 1–3-septate to muriform ......................................................................................................... 8 5a. Ascomata solitary, lemon yellow, ascospores (125–)140–155(–180) × (2.8–)3.4–4.4(–4.6) μm, on Tylophoron ...................................................................................................................................Rossmaniella tylophori 5b. Ascomata in group of 3–12, pale to bright orange, on other hosts .......................................................................................... 6 6a. Ascomata entirely covered by whitish tomentum, ascospores (12–)14–16-septate, individual cells (6.4–)8.0–12.2 (–12.8) μm, on Coenogonium ........................................................................................................ Rossmaniella coenogonii 6b. Ascomata covered by yellowish tomentum in the lower half, ascospores 8–12-septate, individual cells > 12 μm, on various hosts except Coenogonium ......................................................................................................................................... 7 7a. Ascomata in group of 3–5(–7), without distinct hairs near the ostiolate part, ascospores (160–)165–175(–180) × (2.4–)2.5–2.8(–3.0) μm, individual cells (13.2–)15.2–21.0(–23.8) μm ................................................ Rossmaniella cryptica 7b. Ascomata in group (5–)7–12, with distinct hairs near the ostiolate part, ascospores (165–)180–200(–210) × (2.8–)3.0– 3.4(–3.8) μm, individual cells (11.0–)11.5–13.7(–14.5) μm ................................................................. Rossmaniella filispora 8a. Ascospores muriform .............................................................................................................................................................. 9 8b. Ascospores 1–3-septate .........................................................................................................................................................11 9a. Ascomata 200–400 μm, asci < 140 μm in length, ascospores 25–45 μm in length ............................................................. 10 9b. Ascomata 400–750 μm, asci 140–220 μm in length, ascospores (45–)54–75(–92) μm in length ...........Paranectria alstrupii 10a. Ascomata globose, 300–400 μm, asci 2–4-spored, ascospores (10–)13–18(–23) μm in width ...........Paranectria superba 10b. Ascomata ovoid to pyriform, 140–260(–300) μm, asci (4–)8-spored, ascospores (7–)8–12(–14) μm in width .................................................................................................................... Ciliomyces oropensis 11a. Asci 4-spored, ascospores 1-septate, on Physciaceae ........................................................... Sphaeronectria lichenophila 11b. Asci 8-spored, ascospores 3-septate, on Ephebe ................................................................................... Paranectria affinis (KRAM L-74697, LPB); ibid., 21º24’50”S, 64º18’33”W, 2149 m a.s.l., on corticolous Leucodermia leucomelos, 29 Jul. 2015, M. Kukwa 16865b (LPB); ibid., close to Soledad, old road between Entrerios and Chuquiaca, 21º39’45”S, 64º07’22”W, 1750 m, Boliviano-Tucumano forest with shrubs and Alnus acuminata, on Leucodermia leucomelos, 31 Jul. 2015, J. Etayo XI-7 (LPB) Notes: Etayo (2002) discussed the morphological similarity of the ascomata of Sphaeronectria lichenophila to the genus Paranectria and pointed out the differences between this species and other fungi of Nectriopsis. These differences are supported by resulting phylogenetic analyses which showed a close relationship between Sphaeronectria and Paranectria rather than Nectriopsis. DISCUSSION Phylogenetic relationships in the order Hypocreales Hypocreales is an order within the Sordariomycetes that includes a diverse range of species with many potential applications (Hyde et al. 2020, Hou et al. 2023). The main research efforts have focused on species with practical applications, while groups without such importance have received little attention. Consequently, the taxonomic system of Hypocreales seems to be incomplete (Hyde et al. 2020). For instance, the connection between their asexual and sexual stages is often unclear, most researchers have focused primarily on the phylogeny within individual families, rather than extensively exploring the relationships between the families themselves and more comprehensive studies Persoonia – Volume 54, 202584 have shown contrasting results regarding the phylogenetic relationships within the order Hypocreales (e.g. Chaverri et al. 2011, Hirooka et al. 2012, Quandt et al. 2014, Giraldo et al. 2015, Lombard et al. 2015, 2016, Crous et al. 2021, Hou et al. 2023, Li et al. 2023, Perera et al. 2023, Sun et al. 2023, Xiao et al. 2023, Yu et al. 2024). Therefore, our study has considerably broadened the sampling, including several poorly known lichenicolous taxa to reconstruct a comprehensive multi-locus phylogeny of Hypocreales. As a result, this study has successfully established a phylogenetic framework for Hypocreales, revealing a new lineage associated with lichens. To accommodate the genera Ovicuculispora, Paranectria, as well as Nectriopsis lichenophila and Nectria byssophila-like species, this lineage is described as a new family named Paranectriaceae. Several studies reported similar topologies for the family Bionectriaceae and related families, which were previously considered part of a broader concept of Bionectriaceae (Maharachchikumbura et al. 2016, Hongsanan et al. 2017, Hyde et al. 2020, Perera et al. 2023). The family Tilachlidiaceae consistently exhibits a highly supported sister relationship with the Bionectriaceae clade in multiple studies (Maharachchikumbura et al. 2016, Hongsanan et al. 2017, Hyde et al. 2020). Perera et al. (2023) introduced a new family Stromatonectriaceae, which showed a well-supported sister relationship with Tilachlidiaceae. The resulting comprehensive phylogeny of the order further confirms the relationship between these families (Stromatonectriaceae and Tilachlidiaceae) rather than exclusively with Bionectriaceae. The family Myrotheciomycetaceae, consisting of a few hyphomycetous genera, has only recently been described although its phylogenetic relationships were still unclear (Crous et al. 2018). Our phylogenetic analyses have revealed that this family forms a well-supported clade related to the clade comprised of Bionectriaceae, Flammocladiellaceae, Stromatonectriaceae, Tilachlidiaceae and Xanthonectriaceae. However, the relationship of the family Myrotheciomycetaceae with other families of Hypocreales is still being elucidated, but our analyses and previous studies suggest its association with the Bionectriaceae and related families (Hou et al. 2023, Perera et al. 2023). The phylogenetic placement of the family Flammocladiellaceae has been unclear, but some studies suggested possible phylogenetic relationships to Cordycipitaceae, Ophiocordycipitaceae, or even Clavicipitaceae (Maharachchikumbura et al. 2016, Crous et al. 2018). In other studies, it appeared as a sister clade to Sarocladiaceae, Tilachlidiaceae or Myrotheciomycetaceae (Hyde et al. 2020, Li et al. 2024). Our study found that Flammocladiellaceae formed a sister clade to the family Ijuhyaceae with a strong support, which is congruent the results found by Perera et al. (2023). Those two families share similar morphological features, such as globose pale yellow to orange ascomata and ellipsoid 1–3(–5)-septate ascospores, which are quite different from representatives of Cordycipitaceae (Crous et al. 2015, Lechat & Fournier 2018). According to our phylogeny, the family Valsonectriaceae forms a distinct lineage, while Hou et al. (2023) and Yu et al. (2024) indicated it as a well-supported clade sister to the Bionectriaceae and related families. Another inconsistency with the phylogenetic results of Hou et al. (2023) is the phylogenetic placement of Pseudoniessliaceae, while the authors find it as a sister clade to the Chrysonectriaceae, Nectriaceae and Neoacremoniaceaea, but our analyses reveal it to be as a sister clade to the Clavicipitaceae, but without statistical support. The family Nectriaceae is indeed one of the most extensively studied groups within the order Hypocreales. It was broadly sampled and included in several multi-gene phylogenetic studies, leading to the development of a robust taxonomy for the family (Lombard et al. 2014, 2015, Crous et al. 2021). However, the relationship of the Nectriaceae to other groups within Hypocreales has shown some inconsistencies across different studies. Phylogenetic analyses conducted by Maharachchikumbura et al. (2016) and Hongsanan et al. (2017) suggested that the Nectriaceae forms a sister clade to the Stachybotriaceae, but with low statistical support in both studies. In more recent studies, Nectriaceae was found to be the sister group to a few sequences of Niesslia species (Niessliaceae) or as a sister to the Chrysonectriaceae and Neoacremoniaceae (Perera et al. 2023, Hou et al. 2023). In our phylogeny, Nectriaceae is monophyletic with high support and similarly to the results by Hou et al. (2023) is closely related to Neoacremoniaceae. Sedecimiella taiwanensis, the type species for Sedecimella, was described based on limited material, and LSU (HM451496) as well as nuSSU (HM451495) sequences are available (Pang et al. 2010). This species was transferred to the genus Neoacremonium (Neoacremoniaceae) and suggested as the first sexual species in this genus (Hou et al. 2023). Simultaneously, other authors described three new asexual Sedecimiella species and together with the genus Heteroacremonium, established the new family Sedecimiellaceae (Li et al. 2023). Our phylogenetic results showed that both families, Neoacremoniaceae and Sedecimiellaceae, formed well-supported clades with strongly supported sister relationships. However, further investigation, including more sequences of both families, especially for sexual taxa, is required to evaluate their circumscriptions. The families Hypocreaceae, Cordycipitaceae, Clavicipitaceae, and Ophiocordycipithaceae form distinct clades within the order Hypocreales, as supported by multiple previous studies (Maharachchikumbura et al. 2016, Hongsanan et al. 2017, Hyde et al. 2020, Perera et al. 2023), as well as in our phylogeny. However, the specific relationships between these families and the circumscriptions are not yet clear. In addition, some species or genera have undergone recent changes regarding their placements, moving from one family to another. These taxonomic changes are often a result of sampling bias, and in response to this, several new lineages at the family level (Polychephalomycetacea, Pseudoniessliaceae, Pseudodiploosporeaceae) have been described recently (Hou et al. 2023, Sun et al. 2023, Xiao et al. 2023). The family Calcarisporaceae was established to accommodate the species of the genus Calcarisporium, which was previously classified as Hypocreales insertae sedis (Sun et al. 2017). The authors demonstrated the relationship of the new family with the Cordycipitaceae, Clavicipitaceae, and Ophiocordycipithaceae subclade, which has been further supported by subsequent research (Hyde et al. 2020, Hou et al. 2023, Perera et al. 2023,). Based on our phylogenetic analyses Calcarisporaceae showed a strongly supported sister relationship to the family Albomorchellophilaceae as it was reported by Yu et al. (2024). Darmostuk et al.: New lineage of hypocrealean fungi 85 Another recently described family within Hypocreales is Cocoonihabitaceae, which currently includes only one known species, Cocoonihabitus sinensis (Zhuang & Zeng 2017). However, only a few sequences of this species are available and therefore the phylogenetic relationships of Cocoonihabitaceae are not resolved well. Previous studies showed the low or moderately supported sister relationship of Cocoonihabitaceae with families Hypocreaceae, Cordycipitaceae or Pseudoniessliaceae (Zhuang & Zeng 2017, Hou et al. 2023, Perera et al. 2023 Yu et al. 2024). In our multigene phylogenetic analyses, we find Cocoonihabitaceae to be a sister clade comprised of Ophiocordycipithaceae and Polychephalomycetaceae with strong statistical support. The phylogenetic position of the genus Trichonectria was unstable in different studies, and the genus was classified as Hypocreales incertae sedis (Perera et al. 2023) or clustered inside the Niessliaceae clade (Hou et al. 2023). Our phylogenetic analyses reveal that the asexual genus Cylindromonium, with sexual Trichonectria, forms a well-supported clade sister to the newly described family Paranectriaceae. Cylindromonium-Trichonectria clade is not formally described here due to the absence of sequences from the type species Trichonectria hirta and will be treated by us in the future. Our research focused on filling the sampling gaps within hypocrealen fungi represented by lichenicolous species, which are highly specialized and their practical applications are largely unknown. Previously, none of treated here species were included in larger phylogenetic studies and this is the first study that addresses their phylogenetic position based on freshly collected specimens. Therefore, this investigation enhances our comprehension of the evolutionary histories of Hypocreales and provides a solid phylogenetic backbone for improving the systematics of this order. Phylogenetic relationships within the family Paranectriaceae The newly described family Paranectriaceae consists of species characterized by yellow to orange superficial ascomata, scattered or grouped, with distinct whitish tomentum, KOH not reacting ascomata wall and 1-septate to multiseptate or muriform ascospores. The family members are lichenicolous and can infect many unrelated lichen hosts without showing a strict host specificity. Due to the limited molecular data available, the genera Ovicuculispora and Paranectria were classified as members of Bionectriaceae based on morphological features and the lack of KOH reaction in the ascomata (Rossman et al. 1999, Diederich et al. 2018). However, this relationship was never confirmed through further phylogenetic analyses (Perera et al. 2023). The species Ovicuculispora parmeliae was sequenced by Sikaroodi et al. (2001) and the authors demonstrated the phylogenetic placement of this species in Hypocreales without affiliation to the family level. Subsequent phylogenetic studies of Hypocreales did not include this species and therefore its phylogenetic position was unresolved. Later, Telfer et al. (2015) in their biodiversity inventory based on a DNA barcoding approach obtained a sequence of the ITS region for Ovicuculispora parmeliae. This genus is characterized by huge morphological variability especially in terms of macrospore sizes and their ornamentation. This variability together with the wide host spectrum and quite limited molecular data provoked a lot of discussion about the circumscription of this species (Flakus et al. 2006, Etayo 2010, Etayo & van den Boom 2013, Zhurbenko 2014, Tadome & Ohmura 2021, Zhurbenko 2023). During this study, we were able to generate new sequence data from seven specimens of Ovicuculispora parmeliae from South America and Eurasia, which all cluster in a well-supported monophyletic clade (Fig. 2). These specimens showed considerable morphological variation, but because of a lack of strong phylogenetic signal distinguishing them as separate species was impossible at this moment. More specimens from other regions and different hosts are needed to evaluate the morphological variability of this species and the species concept. Another two specimens of Ovicuculispora cf. macrospora were resolved as a highly supported clade sister to O. parmeliae. Those specimens are characterized by slight morphological differences from the protologue of O. macrospora (Etayo 2012). We suggest that those differences can be an intraspecific variation, but more samples are needed to test this hypothesis. The genus Paranectria was included in the Bionectriaceae by Rossman et. al. (1999) based on morphological features. So far, only one sequence of the generic type, Paranectria affinis, was available in public repositories (MZ159749). In our study, six specimens of Paranectria oropenis and two specimens of P. affinis were sequenced and included in the phylogenetic analyses. The resulting phylogeny showed the genus Paranectia to be polyphyletic due to the separate position of the generic type and P. oropensis. These results are also congruent with the morphological differences in ascospores between Paranectria affinis (3-septate ascospores) and other lichenicolous species of the genus, e.g. P. alstrupii, P. oropensis and P. superba (muriform ascospores). Thereby, we reinstated the old genus Ciliomyces, which currently included one species. Ciliomyces oropensis (=Paranectria oropensis) also presents morphological variation and a broad host spectrum. However, the taxonomical value of ascospores variation found in this species has been questioned, as many authors considered it to be dependent on the maturity stage of collected specimens (Diederich 2003, Brackel 2008, Hafellner & Obermayer 2009, Navarro-Rosinés & Llimona 2018). We included in our phylogenetic analyses six specimens of Ciliomyces oropensis collected from different regions and on various hosts, and they all were clustered in a well-supported clade (Fig. 2). Nectriopsis lichenophila was previously placed in Bionectriaceae, but in our phylogenetic analyses, it formed a well-supported monophyletic clade in the family Paranectriaceae. As a result we proposed a new monotypic genus Sphaeronectria to accommodate N. lichenophila. The four sequenced specimens of N. lichenophila included in this study showed phylogenetic differences, but their morphological features are identical. We suggest that these phylogenetic differences reflect intraspecific variability across studied loci. However, the genus Nectriopsis including more than 70 fungicolous and lichenicolous species which are characterized by pale, superficial ascomata, not reacting in KOH (or being rarely violaceous), thin-walled setae and 1-septate ascospores (Samuels 1988, Rossman et al. 1999, Diederich et al. 2018). The case of Nectriopsis lichenophila suggests that Nectriopsis needs a critical taxonomic revision with additional sampling because its morphology may represent several homoplasies. The new genus Rossmaniella is placed in a new family Persoonia – Volume 54, 202586 Paranectriaceae. The genus is characterized by the yellowish to bright orange, ovoid to obpyriform ascomata, and clustered in groups, with a well-developed tomentum at the lower part of ascomata and filiform ascospores. At least part of previous records applying the name Nectria byssophila on different lichen hosts refers to the species of the genus Rossmaniella (Etayo & Sancho 2008, Etayo 2003, 2017). We obtained the sequences from eight specimens of the genus Rossmaniella and they represented four species clustered in a wellsupported clade in our phylogeny. Lichenicolous species in other lineages of Hypocreales Lichenicolous fungi represent a highly diverse ecological group of fungi related to lichens and comprising more than 2300 described species (Diederich et al. 2018). The lichenicolous lifestyle evolved multiple times in fungi, most of them belonging to Ascomycota. The Hypocreales is a group of fungi that is particularly rich in lichenicolous fungi, with a total of 180 species described (Diederich et al. 2018). However, phylogenetic studies have given little attention to hypocrealean lichenicolous species, resulting in DNA sequences being available for only about 10 % (18 species) of the taxa (Crous et al. 2019, Flakus et al. 2019a, Berger et al. 2020, Darmostuk 2021, Berger & Zimmermann 2022, Crous et al. 2023a, b, Ohmaki et al. 2023). Traditionally, lichenicolous fungi were described based on morphological data, the host specificity, and frequently included in genera described for fungi with other lifestyles. That situation was widely extended in the order Hypocreales. Therefore, the circumscription of many lichenicolous genera remain to tentative taxonomical changes when more lichenicolous taxa are included in phylogenetic analyses. One such example is Neobarya (Clavicipitaceae), which was established to accommodate host-specific parasites of fungi or lichens (Candoussau et al. 2007). Lawrey et al. (2015) conducted the first phylogenetic assessment (based on ITS and LSU region) of several Neobarya species, demonstrating that the lichenicolous Neobarya usneae (Etayo 2002) was not phylogenetically related to the generic type (Neobarya parasitica). As a result, the new lichenicolous genus, Lichenobarya, was described and placed in Hypocreaceae. However, the phylogenetic position of other lichenicolous species within the genus Neobarya (N. ciliaris, N. darwiniana, N. lichenophila and N. peltigerae) remains unclear due to a lack of molecular data. Another example is Neobaryopsis a new monotypic genus described to accommodate a Neobarya-like species growing on Lobariella (Flakus et al. 2019a). It was suggested that this genus belongs to Cordycipitaceae, marking the first record of a lichenicolous lifestyle in this family. Our phylogenetic results revealed that lichenicolous taxa belong at least to seven lineages of Hypocreales, i.e. the families Bionectriaceae (Lasionectria lecanodes and Pronectria robergei), Nectriaceae (Microcera physciae and Roselliniella spp.), Hypocreaceae (Lichenobarya usneae), Calcarisporaceae (Neobaryopsis andensis), Paranectriaceae (Ciliomyces oropensis, Paranectria affinis, Ovicuculispora spp., Rossmaniella spp., Sphaeronectria lichenophila), Niessliaceae (Niesslia cladoniicola) as well as the Cylindromonium-Trichonectria clade. However, many lichenicolous fungi still require further study to enhance our understanding of the evolution of this lifestyle in Hypocreales. Additionally, data on facultatively lichenicolous, such as Sarocladium strictum (Sarocladiaceae), are necessary to have a more complete picture of the changes in the lifestyle in Hypocreales (Hawksworth 1979, Diederich et al. 2018). The Cylindromonium-Trichonectria clade included several sequences of the asexual genus Cylindromonium (generic type C. eugenicola) and sexual genus Trichonectria (generic type T. hirta) and forming a well-supported clade in our phylogeny. The relationship between both genera has been long discussed in the literature. An acremonium-like asexual morph has been suggested for several Trichonectria species, but has never been confirmed by molecular data (Lowen & Hawksworth 1986, Lowen 1989, 1995). Lowen (1995) and later Glenn et al. (1997) proposed that C. rhabdosporum (as Acremonium rhabdosporum) corresponds to the asexual morph of Trichonectria rubefaciens (as Nectria rubefaciens) and this statement was used for a long time to indicate the asexual-sexual relationship in Trichonectria. Recently, Ohmaki et al. (2023) described Cylindromonium dirinariae and confirmed the relationship between a trichonectria-like sexual morph and a Cylindromonium asexual morph by multigene phylogenetic analyses. The phylogenetic affinities of both genera have been challenging, e.g. Cylindromonium was classified as a member of Nectriaceae based on phylogenetic analyses (Crous et al. 2019), but Trichonectria was suggested to belong to the Bionectriaceae based on morphological data, although it was placed as Hypocrelaes incertae sedis (Rossman et al. 1999, Perera et al. 2023). In addition, the relationship between Cylindromonium and Trichonectria raised a nomenclatural problem. However, Cylindromonium eugeniicola, a generic type of Cylindromonium, is associated with leaf litter and is not closely related (Figs 1, 2) to lichenicolous species of this genus (Crous et al. 2023b). The generic type of Trichonectria is T. hirta, known from several collections across Europe but for which no molecular data is available. As T. hirta is characterized by quite different morphology, e.g. multiseptate vs 1-septate ascospores from other species classified currently in the genus, the rest of lichenicolous species may belong to another genus. The fresh collections, culture and molecular data for this species are crucial to further clarify the genus circumscription and phylogenetic position of the other species placed in Trichonectria. The genus Roselliniella is characterized by erumpent, brownish perithecioid ascomata, thin-walled asci without apical apparatus, filamentous interascal filaments and brown, aseptate ascospores (Hoffman & Hafellner 2000). The genus originally was classified as a member of the order Sordariales by morphological data (Hoffmann & Hafellner 2000). Later, Hawksworth et al. (2010) obtained the LSU sequences data for two species of Roselliniella and resolved the genus in the order Hypocreales rather than Sordariales, but without indicating the family placement. Our phylogenetic results indicated that Roselliniella is closely related to Nectria and would belong to Nectriaceae. These results do not fit with the features of the family Nectriaceae, which is characterized by unilocular, white, yellow, orange-red or purple ascomata and phialidic asexual morphs (Lombard et al. 2015). Therefore, it is crucial to evaluate the phylogenetic placement of the genus Roselliniella by conducting a wide sampling of taxa, including the generic type. Darmostuk et al.: New lineage of hypocrealean fungi 87 The recently described genus Neobaryopsis is characterized by very particular narrowly pyriform, yellowish to orange ascomata developing on a reduced white arachnoid subiculum, and large, multiseptate needlelike ascospores and short synnematous conidiomata and was originally placed on Cordycipitaceae (Flakus et al. 2019a). This is incongruent with our results , based on a larger number of loci, which supported that Neobaryopsis andensis belong to Calcarisporiaceae, closely related to the asexual genus Calcarisporium. Therefore, we suggest, that Neobaryopsis belongs to the family Calcarisporaceae instead of Cordycipitaceae. Neobaryopsis belongs to the so-called group of Neobarya-like lichenicolous fungi to which Neobarya, Leptobarya and Lichenobarya also belong (Candoussau et al. 2007; Lawrey et al. 2015, Flakus et al. 2019a). Despite the high level of morphological similarity among those genera, they belong to different phylogenetic lineages of the order Hypocreales, i.e. Lichenobarya to Hypocreaceae, Neobarya to Clavicepitaceae, Neobaryopsis to Calcarisporaceae and the new Rossmaniella to Paranectriaceae. This pattern can indicate that Neobarya-like habits have evolved separately in Hypocreales. However, future studies including a wider sampling of the abovementioned genera and other neobaryalike genera will improve our knowledge on the evolution of cryptic speciation in Hypocreales. Tropical ecosystems harbour a significant fraction of the known mycobiota, including the highest diversity of lichen species (Hawksworth 2012). However, the number of known lichenicolous fungi in these regions has remained relatively small. This has led to the discovery of numerous lichenicolous species from South America in previous years years (e.g., Flakus et al. 2014, 2019a, Etayo et al. 2015, Etayo 2010, 2017, Darmostuk & Flakus 2024) The tropical cloud forests of Bolivia, one of the world’s biodiversity hotspots, appear to be extremely diverse and particularly rich in undescribed species, especially within the order Hypocreales (Flakus et al. 2019a, b, Crous et al. 2023a, b, Etayo et al. 2024). In this study, we demonstrated that extensive sampling of tropical fungi can help clarify their phylogenetic position and influence order-wide systematics. We provide new distribution and phylogenetic data for the monotypic genus Sphaeronectria, an exclusively tropical genus, as well as Rossmaniella and Ovicuculisopora, which are widely distributed in tropical regions. The lichenicolous lifestyle has evolved multiple times from different ancestors throughout the evolutionary history of Ascomycota (e.g., Divakar et al. 2015, Suija et al. 2015). Our phylogenetic results suggest that lichenicolous taxa belong at least to seven lineages of Hypocreales, revealing relationships with species with different lifestyles. However, some lichenicolous species within the Hypocreales are related to saprotrophs, known from plant debris (Lasionectria and Pronectria in Bionectriaceae, Niesslia in Niessliaceae). Additionally, other taxa, such as Lichenobarya in Hypocreaceae and Neobaryopsis in Calcarisporaceae, are nested within clades comprised of fungicolous species. Furthermore, a few lichenicolous species are related to insect pathogens, such as Microcera in Nectriaceae. Further evolutionary studies of Hypocreales should include data on lichenicolous species, as they are widely distributed within the order, and this lifestyle is supposed to have undergone gains or losses multiple times throughout evolutionary history. ACKNOWLEDGEMENTS We are greatly indebted to our colleagues and all staff of the Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, La Paz, for their generous long-term cooperation and the curator of herbarium K, UPS, and M. Zhurbenko for the loan of specimens. We would also like to thank SERNAP (http:// sernap.gob.bo), and the staff of all protected areas for granting permits for scientific studies, as well as their assistance and logistical support during the field work. Special thanks to N. Matura (Kraków) and A. Beresova (Bratislava) for their valuable help at the molecular lab. This research was financially supported by the National Science Centre (NCN) in Poland (DEC-2013/11/D/NZ8/ 03274), by SYNTHESYS+ (ES-TAF-687) and CAM Atracción de Talento program (2020-T1/AMB-19852). VD, PRF and AF received additional support under statutory funds from the W. Szafer Institute of Botany, Polish Academy of Sciences. Declaration on conflict of interest The authors declare that there is no conflict of interest. REFERENCES Altschul SF, Gish W, Miller W, et al. (1990). Basic local alignment search tool. Journal of Molecular Biology 215: 403–410. https:// doi.org/10.1016/S0022-2836(05)80360-2 Barr ME (1990). 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