Expanding the genus Neobaryopsis (Calcarisporiaceae, Hypocreales): descriptions of two new species and two new combinations based on morphological and molecular data

291 Expanding the genus Neobaryopsis (Calcarisporiaceae, Hypocreales): descriptions of two new species and two new combinations based on morphological and molecular data Valerii Darmostuk1, Javier Etayo2, Pamela Rodriguez-Flakus1, Martin Kukwa3, Adam Flakus1 1 W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krakow, Poland 2 Navarro Villoslada 16, 3° dcha., 31003 Pamplona, Navarra, Spain 3 DepartmentofPlantTaxonomyandNatureConservation,FacultyofBiology,UniversityofGdańsk,WitaStwosza59,PL-80-308Gdańsk,Poland Corresponding author: Valerii Darmostuk ([email protected]) Copyright: © Valerii Darmostuk et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The genus Neobaryopsis (Calcarisporiaceae) comprises lichenicolous fungi with bright-colored perithecia, long, multiseptate, filiform ascospores, and mononematous conidiophores or distinctive synnematous-like asexual morphs. The genus was originally described from species growing on Lobariella spp., but its close morphological similarity to Neobarya long obscured its phylogenetic identity. In this study, we reassess the systematics of Neobarya-like taxa using a combination of morphological and multi-locus phylogenetic analyses (ITS, LSU, tef1, rpb2). Our results confirm that several lichenicolous species historically placed in Neobarya in fact belong to the genus Neobaryopsis, which is closely related to Calcarisporium. We describe two new species (Neobaryopsis eriodermaticola and N. teloschistis) and propose two new combinations (Neobaryopsis ciliaris and N. peltigerae). This work expands the genus Neobaryopsis to five recently accepted species, some of which exhibit strong host specificity and a primarily South American distribution. An identification key for species of Neobaryopsis and Neobarya-like fungi is provided. Our results clarify the systematics of these morphologically similar lichenicolous fungi and provide a framework for future studies on host specificity, biogeography, and evolutionary relationships within Hypocreales. Key words: Ascomycota, cloud forest, diversity, lichenicolous fungi, multi-locus phylogeny, South America, taxonomy Introduction Lichenicolous fungi, a specialized ecological group associated with lichen hosts, represent a remarkably diverse assemblage with a wide range of morphological adaptations. They occur on all continents, but comprehensive studies have been limited to certain regions. In numerous cases, the lack of molecular sequence data for these fungi substantially limits the accuracy of their generic assignment and impedes confident inference of their phylogenetic placement. Recent studies have shown that the lichenicolous lifestyle has evolved independently multiple times throughout fungal evolutionary history. Academic editor: Thorsten Lumbsch Received: 20 August 2025 Accepted: 1 October 2025 Published: 5 November 2025 Citation: Darmostuk V, Etayo J, Rodriguez-Flakus P, Kukwa M, Flakus A (2025) Expanding the genus Neobaryopsis (Calcarisporiaceae, Hypocreales): descriptions of two new species and two new combinations based on morphological and molecular data. MycoKeys 124: 291–308. https://doi.org/10.3897/ mycokeys.124.169315 MycoKeys 124: 291–308 (2025) DOI: 10.3897/mycokeys.124.169315 292 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Moreover, this group also exhibits a high degree of morphological plasticity, making taxonomy based solely on morphology particularly challenging (Suija et al. 2015, 2024; Pino-Bodas et al. 2017; Flakus et al. 2019; Darmostuk and Flakus 2024; Diederich et al. 2024; Etayo et al. 2024). The genus Neobarya, with the type species Neobarya parasitica, was established to accommodate fungicolous species with pyriform, light-colored (white, green, yellow, or orange) perithecia, lacking a distinct subiculum, and linear asci with long, septate, filiform ascospores (Eriksson and Hawksworth 1986). The generic concept was later expanded to include species growing on lichens that share similar morphological and anatomical features, although concerns were raised about the placement of fungicolous and lichenicolous taxa in the same genus (Etayo 2002; Candoussau et al. 2007; Etayo and Sancho 2008). Lawrey et al. (2015) obtained molecular data for the generic type Neobarya parasitica and for the lichenicolous species Neobarya usneae. Their results showed that these two species belong to different families within Hypocreales, Clavicipitaceae and Hypocreaceae, respectively. Based on a combination of morphological features and phylogenetic evidence, the authors established a new lichenicolous genus, Lichenobarya, with Lichenobarya usneae as the type species. Subsequently, Flakus et al. (2019) described another monotypic lichenicolous genus, Neobaryopsis, for Neobarya-like specimens growing on Lobariella spp. in Bolivia. This genus was defined by its narrowly pyriform, yellowish to orange ascomata; long, filiform ascospores; and a synnematous-like asexual morph with a white stipe and a hyaline, pink to yellowish-orange conidial mass. Recent phylogenetic analyses have shown that Neobaryopsis forms a well-supported clade sister to the anamorphic genus Calcarisporium, suggesting its affinity with the family Calcarisporiaceae, whereas Neobarya belongs to Clavicipitaceae (Flakus et al. 2019; Darmostuk et al. 2025). Four additional Neobarya species growing on lichen hosts (N. ciliaris, N. darwiniana, N. lichenophila, and N. peltigerae) showed morphological similarity to Neobaryopsis, but for a long time molecular data for these taxa were lacking (Etayo 2002; Candoussau et al. 2007). In this study, we clarify the phylogenetic placement of Neobarya ciliaris and N. peltigerae, and we describe two additional species that belong to the genus Neobaryopsis. Material and methods Taxon sampling and morphological studies This study is based on freshly collected material of lichenicolous species, complemented by herbarium specimens deposited at KRAM, LPB, UGDA, and the personal herbarium of J. Etayo (hb. Etayo). Morphological and anatomical characters were examined using standard stereo and compound microscopes (Nikon SMZ 800 and 80i DIC; Leica S9i and S9D). Ascomata sections were prepared manually using a razor blade or with a freezing sliding microtome Microm HM 430 (Thermo Fisher Scientific, USA) combined with a BFS-MP freezing stage and a BFS-3MP controller. Sections and squash mounts were examined in distilled water, 10% KOH (K), or lactophenol cotton blue (LPCB; Fluka, no. 61335-100ML). Photomicrographs of anatomical characters were obtained under transmitted differential interference contrast (DIC) microscopy. 293 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis All measurements were made in distilled water or LPCB. Measurements (if n > 10) are given as (min.–)x ––SD–x –+SD(–max.), where x is the arithmetic mean and SD is the standard deviation. DNA extraction, PCR amplification, and DNA sequencing Lichen thalli with ascomata of lichenicolous fungi were stored at –20 °C until processing. 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. DNA was extracted from 4 to 8 clean ascomata or hymenia, depending on each specimen, using the QIAamp DNA Investigator Kit (Qiagen, Germany) following the manufacturer’s instructions. Four loci — nuclear rDNA internal transcribed spacer (ITS), nuclear rDNA large subunit (LSU), a fragment of the DNA-directed RNA polymerase II subunit two gene (rpb2), and a fragment of the region coding for protein synthesis elongation factor 1 alpha (tef1) – were generated. The primers used and polymerase chain reaction (PCR) conditions are listed in Table 1. Amplification was performed in a total reaction volume of 25 μl, consisting of 3 μl (5 μl for protein-coding regions) of genomic DNA template, 1 μl (3 μl for protein-coding regions) of each forward and reverse primer, 13 μl of DreamTaq PCR Master Mix (2×) (Thermo Fisher Scientific, USA), and 7 μl (1 μl for protein-coding regions) of double-distilled, deionized water. PCR products were sequenced in both directions by Macrogen (Amsterdam, the Netherlands). The newly generated sequences were carefully checked, assembled, and edited manually using Geneious Pro 8.0 (Biomatters Ltd.) and were deposited in GenBank. Detailed information on the sequences used in this study is provided in Table 2. Phylogenetic analyses All generated sequences were first subjected to BLAST nucleotide searches (Altschul et al. 1990) to discard potential contamination by unrelated fungi. Alignments for each region were generated using MAFFT (Katoh and Standley 2013), as implemented on the GUIDANCE2 web server (Penn et al. 2010). The single-locus phylogenies for all loci were generated (results not shown) to detect incongruences in topology. PartitionFinder 2 (Lanfear et al. 2017) was used to select the best partition scheme for our dataset and substitution Table 1. Loci, primers, and polymerase chain reaction (PCR) conditions used in this study. Loci PCR primers Sequence (5′–3′) PCR Cycles References ITS ITS1F CTT GGT CAT TTA GAG GAA GTA A 94 °C: 5 min (94 °C: 30 s, 54 °C: 30 s, 72 °C: 30 s) × 4 cycles; (94 °C: 30 s, 48 °C: 30s, 72 °C: 1 min) × 32 cycles. A final extension of 72 °C: 10 min White et al. (1990); Gardes and Bruns (1993) ITS4 TCC TCC GCT TAT TGA TAT GC LSU LROR GTA CCC GCT GAA CTT AAG C 94 °C: 5 min (94 °C: 30 s, 54 °C: 30 s, 72 °C: 30 s) × 4 cycles; (94 °C: 30 s, 48 °C: 30s, 72 °C: 1 min) × 32 cycles. A final extension of 72 °C: 10 min Rehner and Samuels (1994); Vilgalys and Hester (1990) LR5 TCC TGA GGG AAA CTT CG tef1 EF1-983F GCY CCY GGH CAY CGT GAY TTY AT (95 °C: 30 s, 55 °C: 50 s, 72 °C: 1 min) × 35 cycles. A final extension of 72 °C: 10 min Rehner and Buckley (2005) EF1-2228R AT GAC ACC RAC RGC RAC RGT YTG rpb2 fRPB2-5F GAY GAY MGW GAT CAY TTY YGG 94 °C: 90 s (94 °C: 30 s, 55 °C: 90 s, 68 °C: 2 min) × 40 cycles. A final extension of 68 °C: 5 min Liu et al. (1999) fRPB2-7CR CCC ATR GCT TGY TTR CCC AT 294 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis models for each partition. A single substitution model was selected for each region (K80+G for ITS1 and ITS2, TIM+I+G for 5.8S, LSU, the first codon position of tef1 and rpb2, F81+I for the second codon position of tef1 and rpb2, TVM+G for the third codon position of tef1, and HKY for the third codon position of rpb2) under a greedy search algorithm and the Akaike information criterion (AIC) (Lanfear et al. 2012). For tef1 and rpb2, each codon position was analyzed as a distinct partition: the first, second, and third codon positions. The coding domain sequence (CDS) of the protein-coding regions was detected using the Augustus web tool (Stanke et al. 2008). The dataset included 21 specimens Table 2. List of specimens, with host/substrate, strain/voucher, country of origin, and GenBank accession numbers used in phylogenetic analyses. Species Strain / Voucher Host / substrate Country GenBank accession numbers ITS LSU tef1 rpb2 Albomorchellophila morchellae KUNCC 21-10005 T Morchella sp. China OP580900 OP580862 OP585424 – Albomorchellophila morchellae KUNCC 21-10100 Morchella sp. China OR420019 OR420021 OR420838 – Calcarisporium arbuscula CBS 111.57 Russula fellea United Kingdom MH857665 MH869205 – – Calcarisporium arbuscula CBS 518.66 Boletus the Netherlands MH858872 MH870517 – – Calcarisporium arbuscula CBS 900.68 T decaying agaric Germany MH859249 KX442598 KX442596 KX442597 Calcarisporium cordycipiticola CGMCC 3.17904 fruiting body of Cordyceps militaris China KT945001 KX442604 KX442605 KX442607 Calcarisporium cordycipiticola CGMCC 3.17905 T fruiting body of Cordyceps militaris China KT944999 KX442599 –KX442594 Calcarisporium cordycipiticola CGMCC 3.17906 fruiting body of Cordyceps militaris China – KX442592 KX442591 KX442590 Calcarisporium guizhouense DY05042 Cordyceps sp. China PP809658 PP809662 PP823899 – Calcarisporium guizhouense DY05041 T Cordyceps sp. China PP124948 PP133530 PP146564 – Calcarisporium xylariicola HMAS 276836 T Xylaria sp. Italy KX442603 KX442601 KX442595 KX442600 Calcarisporium yuanyangense YFCC 22099256 T Ophiocordyceps nutans China OQ954173 OQ954171 OQ981389 OQ981390 Neobaryopsis andensis Etayo 20-11 (LPB) Lobariella pallida Bolivia MT153958 MT153987 – – Neobaryopsis andensis Flakus 25967.1 (KRAM L-71220.1) T Lobariella pallida Bolivia MT153956 MT153985 PP583634 – Neobaryopsis andensis Flakus 25967.2 (KRAM L-71220.2) Lobariella pallida Bolivia MT153957 MT153986 – – Neobaryopsis ciliaris Kukwa 15174b (LPB) Leucodermia leucomelos Bolivia PX418470 PX418477 PX438769 PX438774 Neobaryopsis ciliaris Etayo 34451 (LPB) Leucodermia boryi Bolivia PX418469 PX418476 PX438768 – Neobaryopsis eriodermaticola Kukwa s.n. (KRAM L-75209) T Erioderma sp. Bolivia PX418471 PX418478 – – Neobaryopsis eriodermaticola Etayo 33243 (LPB) Erioderma sp. Bolivia PX418472 PX418479 PX438770 – Neobaryopsis peltigerae Stöckli s.n. (KRAM L-75209) Peltigera sp. Switzerland PX418473 PX418480 PX438771 PX438775 Neobaryopsis peltigerae Suija s.n. (TUF095161) Peltigera sp. Finland PX418483 – – – Neobaryopsis peltigerae Darmostuk 1695 (KRAM L-75217) Peltigera sp. Norway PX418474 PX418481 PX438772 PX438776 Neobaryopsis teloschistis Kukwa 16418 (UGDA L) T Teloschistes flavicans Bolivia PX418475 PX418482 PX438773 PX438777 Verticimonosporium diffractum CBS 310.72 T Cocos nucifera, decaying leaf Papua New Guinea MH860483 MH872200 – – Verticimonosporium ellipticum CBS 100388 T Palmae, decaying petiole Peru MH862700 MH874307 – – Sequences obtained in this study are shown in bold. Abbreviations: T: type specimen or ex-type strain; –: indicates unavailable data or sequence. 295 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis belonging to 12 species of the genera Calcarisporium, Neobaryopsis, and Verticimonosporium (Calcarisporiaceae), as well as two specimens of Albomorchellophila morchellae, which were selected as the outgroup. Maximum likelihood (ML) analyses were carried out using a heuristic search as implemented in IQ-TREE 2.1.2 on CIPRES Science Gateway (Ronquist et al. 2012), and 1000 ultrafast bootstrap replicates were selected to estimate branch support (Minh et al. 2020). A Bayesian inference (BI) phylogenetic tree was generated in MrBayes 3.2.6 on CIPRES Science Gateway (Ronquist et al. 2012) using the partitions and substitution models obtained by PartitionFinder 2. The posterior probabilities were calculated by sampling trees using the Markov chain Monte Carlo (MCMC) approach. Two independent parallel runs were randomly started, each with four incrementally heated chains (the temperature parameter for MCMC chains was 0.15). The MCMC was allowed to run for 100 million generations, sampling every 1000th tree and discarding the first 25% of the sampled trees as a burn-in factor. The analysis was stopped when the standard deviation of split frequencies dropped below 0.01. The resulting ML and BI phylogenetic trees were visualized in FigTree 1.4.4 (http://tree.bio.ed.ac. uk/software/figtree/) and Inkscape 0.92 (https://inkscape.org/). The final tree topology was based on the 50% majority-rule consensus tree from the BI analysis, with Bayesian posterior probabilities (BPP) and maximum likelihood bootstrap (MLB) values indicated above branches. Branches with Bayesian posterior probability values >0.97 and maximum likelihood bootstrap values >70 were considered supported. Results Phylogenetic analyses In this study, 25 new sequences from six specimens of lichenicolous fungi were generated. The multi-gene dataset of the family Calcarisporiaceae included 3221 characters (568 of ITS, 909 of LSU, 910 of tef1, and 834 of rpb2), of which 893 were parsimony-informative sites, 119 were singleton sites, and 1975 were constant sites. The phylogenetic tree showed a similar topology from ML and BI analyses; therefore, the BI tree was selected to represent and discuss the phylogenetic relationships among taxa (Fig. 1). In our phylogenetic analyses, two species of the genus Verticimonosporium clustered in a well-supported clade (1/100) and showed a sister relationship to the remaining genera of Calcarisporiaceae. The five species of the anamorphic genus Calcarisporium included in the analyses clustered together in a moderately supported clade (0.98/75). The species Neobarya ciliaris and N. peltigerae, along with two undescribed lichenicolous species (growing on Erioderma sp. and Teloschistes flavicans) and Neobaryopsis andensis, formed a highly supported clade (1/99), which showed a sister relationship to the genus Calcarisporium. This clade comprises four highly supported subclades, corresponding to specimens from the same lichen hosts, as well as a singleton specimen on Teloschistes flavicans. However, the internal relationships within the Neobaryopsis clade remain unresolved. Two specimens of Neobaryopsis eriodermaticola clustered in a well-supported clade (1/100) and showed an un- 296 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Figure 1. Phylogenetic relationships within the family Calcarisporiaceae inferred from a Bayesian Inference analysis (50% majority-rule consensus tree) of a combined ITS, LSU, tef1, and rpb2 data set. Two specimens of Albomorchellophila morchellae were used as the outgroup. The numbers above branches indicate Bayesian posterior probabilities (BPP) > 0.97 and maximum likelihood bootstrap (MLB) support values > 70% respectively (BPP/ MLB). Sequences from type material are indicated by superscript T. Calcarisporium Neobaryopsis outgroup Verticimonosporium 0.02 Albomorchellophila morchellae KUNCC 21.10100 Albomorchellophila morchellae KUNCC 21.10005T Verticimonosporium diffractum CBS 310.72T Verticimonosporium ellipticum CBS 100388T Neobaryopsis ciliaris Kukwa 15174b Neobaryopsis ciliaris Etayo 34451 Neobaryopsis peltigerae Stöckli s.n. Neobaryopsis peltigerae Darmostuk 1695 Neobaryopsis peltigerae Suija s.n. Neobaryopsis eriodermaticola Etayo 32243 Neobaryopsis eriodermaticola Kukwa s.n.T Neobaryopsis teloschistis Kukwa 16418T Neobaryopsis andensis Etayo 20-11 Neobaryopsis andensis Flakus 25967.2 Neobaryopsis andensis Flakus 25967.1T Calcarisporium arbuscula CBS 900.68T Calcarisporium arbuscula CBS 518.66 Calcarisporium yuanyangense YFCC 22099256T Calcarisporium guizhouense DY05041T Calcarisporium guizhouense DY05042 Calcarisporium xylariicola HMAS 276836T Calcarisporium cordycipiticola CGMCC 3.17905T Calcarisporium cordycipiticola CGMCC 3.17906 Calcarisporium cordycipiticola CGMCC 3.17904 1/100 1/99 1/100 1/100 1/100 0.99/87 -/72 -/61 0.98/75 0.98/97 1/99 1/100 0.99/97 1/100 1/100 1/100 1/99 0.97/83 1/100 -/54 supported sister relationship to the clade comprising N. andensis and N. teloschistis. The type species of Neobarya, N. parasitica, belongs to Clavicipitaceae and is phylogenetically unrelated to the genus Neobaryopsis. Taxonomy Neobaryopsis Flakus, Etayo, Kukwa & Rodr. Flakus Neobaryopsis Flakus, Etayo, Kukwa & Rodr. Flakus, in Flakus, Etayo, Miadlikowska, Lutzoni, Kukwa, Matura & Rodriguez-Flakus, Plant and Fungal Systematics 64(2): 307 (2019) Type species. Neobaryopsis andensis Flakus, Etayo, Kukwa & Rodr. Flakus. 297 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Neobaryopsis ciliaris (Etayo) Darmostuk, Etayo, Kukwa & Flakus, comb. nov. MycoBank No: 859618 Neobarya ciliaris Etayo, Biblioth. Lichenol. 84: 74 (2002). Basionym. Typus. Colombia • Nariño, Pasto, corregimiento El Encano, vereda Sta. Isabel, 30-S lago La Cocha (Guamués), 2700 m, en cilios de Leucodermia lutescens, July 1998, J. Etayo 17386, J. Muñoz & B. Ramírez (holotype COL!, isotype hb. Etayo!). For detailed description see (Etayo 2002). Notes. This species was described from a few localities in Colombia growing on Heterodermia s. lat. (including Leucodermia spp.) (Etayo 2002), with subsequent records from Bolivia and Ecuador (Etayo 2017; this study). Neobaryopsis ciliaris typically grows on the cilia of the host, though in some specimens it was found on the lower surface of the thalli. Specimens examined. bolivia • Dept. Chuquisaca, Prov. Belisario Boeto, close to Padilla between Nuevo Mundo and Santa Rosa, 18°57'12"S, 64°16'37"W, 1790 m, transition between Boliviano-Tucumano forests and dry inter-Andean vegetation, on cilia of Leucodermia leucomelos, 16 Jul. 2015, J. Etayo 29440 (hb. Etayo, LPB) • Prov. Hernando Siles, 15 km west of Monte Agudo, 19°48'57"S, 64°06'00"W, 1815 m, disturbed Tucumano-Boliviano forest, on cilia of Leucodermia leucomelos, 20 Jul. 2015, J. Etayo 30322 (hb. Etayo, LPB) • Dept. Cochabamba, Prov. Carrasco, near Río Batea Mayu close to Monte Punku, lower montane Yungas cloud forest, 17°32'27"S, 65°16'14"W, 2553 m, on corticolous Leucodermia leucomelos, 28 Nov. 2014, M. Kukwa 15158b (LPB) • ibid., 17°31'33"S, 65°16'21"W, 2430 m, lower montane Yungas cloud forest, on thallus of Heterodermia sp. on trunk, 28 Nov. 2014, J. Etayo 34196 (hb. Etayo) • ibid., near Río Lopez Mendoza, 17°30'25"S, 65°16'51"W, 2248 m, lower montane Yungas cloud forest, on thallus of Heterodermia japonica on trunk, 27 Nov. 2014, J. Etayo 33933 (LPB, hb. Etayo) • ibid., Wayra Mayu close to Monte Punku, 17°33'30"S, 65°16'08"W, 2750 m, on corticolous Leucodermia leucomelos, 28 Nov. 2014, M. Kukwa 15174b (LPB) • valle de Zongo, bosque nublado, near metal bridge, 16°07'41"S, 68°05'55"W, 2450 m, on cilia of Heterodermia sp., 29 May 2011, J. Etayo 26735 (LPB) • Prov. Tiraque, Parque Nacional Carrasco, Camino de las Nubes-Cotany Alto road, 17°17'28"S, 65°44'05"W, 4146 m, open high Andean vegetation, on cilia of corticolous Leucodermia leucomelos, 2 Dec. 2014, J. Etayo 29629 (LPB) • Dept. La Paz, Prov. Bautista-Saavedra, Área Natural de Manejo Integrado Nacional Apolobamba, near la Curva, W oriented valley close to Charazani, 15°06'30"S, 69°01'50"W, 3550 m, open area with schrubs and Polylepis near the river, on cilia of Leucodermia boryi on twigs, 14 Nov. 2014, J. Etayo 35009 (hb. Etayo, LPB) • Prov. Franz Tamayo, Área Natural de Manejo Integrado Nacional Apolobamba, below Pelechuco, 14°49'08"S, 69°03'50"W, 3560 m, open area with shrubs and Polylepis trees, on cilia of Leucodermia boryi on tree, 20 Nov. 2014, J. Etayo 34451 (hb. Etayo, LPB) • Parque Nacional y Área Natural de Manejo Integrado Madidi, below Keara Bajo, 14°41'47"S, 69°04'10"W, 3160 m, open area with shrubs and scattered trees, on cilia of Leucodermia vulgaris on trunk, 18 Nov. 2014, J. Etayo 34418 (hb. Etayo, LPB) • Dept. Santa Cruz, Prov. Caballero, El Camino de las Orquídeas, 17°50'27"S, 64°41'59"W, 2510 m, Yungas cloud forest, on cilia of Heterodermia podocarpa, 17 Aug. 2012, J. Etayo 28802 (hb. Etayo, LPB). 298 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Neobaryopsis eriodermaticola Darmostuk, Etayo, Kukwa & Flakus, sp. nov. MycoBank No: 859616 Fig. 2 Typus. bolivia • Dept. Cochabamba, Prov. Chapare, Parque Nacional Carrasco, Sillar road close to Villa Tunari, 17°06'58"S, 65°41'19"W, 1020 m, Sub-Andean Amazon forest close to plantation, on apothecia of Erioderma sp., 3 Dec. 2014, M. Kukwa s.n. & A. Flakus (holotype KRAM L-75209, isotype LPB) Etymology. Named after the host lichen genus, Erioderma. Description. Ascomata perithecioid, in small groups, superficial, with loose white arachnoid subiculum, without stromata, pyriform to elongate pyriform, collapsing by lateral pinching when dry, (250–)310–370(–410) μm high, (160–)210–250(–300) μm wide (n = 15), smooth, pale orange to orange. Perithecial wall 25–40 µm thick, not changing color in K, composed of two regions: external, pale yellow region with thick-walled, isodiametric cells, 2–4 μm diam., and inner region with hyaline, thin-walled, flattened cells, 5–8 × 2.5–3.5 μm. Periphyses 0–1 septate, c. 7–10 × 1.5–3 µm. Asci cylindrical to narrowly cylindrical, 8-spored, (120–)135–160(–185) × (4.0–)4.2–4.8(–5.3) Figure 2. Neobaryopsis eriodermaticola (holotype, except B. and F. from Etayo 33243). A, B. Ascomata and white hyphomycetous colony on the host thallus; C. Squashed ascoma; D. Section of the ascoma; E. Asci; F. Conidia; G. Ascus apex (in LPCB); H. Ascus with ascospores. Scale bars: 250 µm (A, B), 50 µm (C, D), 25 µm (E), 10 µm (F, H), 5 µm (G). 299 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis µm (n = 12), apex thickened with a conspicuous cap c. 3.5–4 µm high. Ascospores thread-like, multiseptate (septa hardly visible on young ascospores), not constricted at the septum, hyaline, with rounded apical parts, smoothwalled, twisted in the ascus, (80–)95–110(–120) × (1.8–)2.0–2.2(–2.4) µm (n = 20). Asexual stage hyphomycetous, present near the lower part of perithecia, colonies effuse. Conidiophores short, unbranched, 0–2-septate, hyaline. Conidiogenous cells terminal, hyaline, thin-walled, smooth, cylindrical to slightly tapering, phialidic, 25–40 × 1.5–2 μm. Conidia ellipsoidal to cylindrical, sometimes slightly constricted in the middle, hyaline, 0-septate, smooth, slightly truncated, (7.4–)8.0–9.4(–9.6) × (2.6–)2.8–3.8(–4.2) µm (n = 25). Host, distribution, and ecology. Neobaryopsis eriodermaticola is currently known from a few localities in the montane Yungas cloud forest in Bolivia (3200–3500 m) and the Sub-Andean Amazon forest, where it grows on apothecia or rarely on thallus of Erioderma spp. Notes. Neobaryopsis eriodermaticola is morphologically similar to N. peltigerae, which grows on Peltigera spp. (see notes below). Neobaryopsis peltigerae differs from the new species in possessing subglobose to slightly clavate terminal cells at the perithecial apex, a feature absent in N. eriodermaticola. Additionally, N. peltigerae has shorter ascospores, 35–75 µm (vs. (80–)95– 110(–120) µm in N. eriodermaticola), and different lichen hosts (Candoussau et al. 2007). The anamorphic stage of Neobaryopsis eriodermaticola also has smaller conidia, (7.4–)8.0–9.4(–9.6) × (2.6–)2.8–3.8(–4.2) µm, compared to those reported from cultures of N. peltigerae (11–14 × 3–7 µm). Our phylogenetic results indicate that the new species is sister to Neobaryopsis andensis and N. teloschistis, although this relationship lacks significant statistical support (Fig. 1). Neobaryopsis andensis can be distinguished by its larger ascomata, 500–700 µm high (vs. (250–)310–370(–410) μm high in N. eriodermaticola), longer asci and ascospores, smaller conidia, 4–7 × 2–2.5 μm (vs (7.4–)8.0–9.4(–9.6) × (2.6–)2.8–3.8(–4.2) µm in N. eriodermaticola), as well as different lichen hosts (Flakus et al. 2019). Specimens examined. bolivia • Dept. Cochabamba, Prov. Tiraque, Parque Nacional Carrasco, Camino de los Nubes, Antenas Sillar-Villa Tunari old road, 17°12'32"S, 65°41'52"W, 3520 m, upper montane Youngas cloud forest, on thallus and especially apothecia of Erioderma sp. on twigs, 30 Nov. 2014, J. Etayo 33243 (hb. Etayo, LPB) • Dept. La Paz, Prov. Nor Yungas, Parque Nacional y Área Natural de Manejo Integrado Cotapata of Unduavi by Sillu Tincara pre-Columbian route, transition Páramo Yungeño – Yungas montane cloud forest, 16°17'22"S, 67°53'29"W, 3518 m, on Erioderma leylandii, 25 May 2011, J. Etayo 28016 (LPB) • ibid., bosque nublado yungas, sendero que parte de la Estación de servicio, 16°17'09"S, 67°51'00"W, 3220–3250 m, on Erioderma leylandii on twigs, 24 May 2011, J. Etayo 27692 (hb. Etayo). Neobaryopsis peltigerae (Lowen, Boqueras & Gómez-Bolea) Darmostuk, comb. nov. MycoBank No: 859619 Neobarya peltigerae Lowen, Boqueras & Gómez-Bolea, Sydowia 59(2): 206 (2007). Basionym. 306 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Diederich P, Ertz D, Braun U (2024) Flora of Lichenicolous Fungi (Vol. 2), Hyphomycetes. National Museum of Natural History, Luxembourg, 544 pp. Divakar PK, Crespo A, Wedin M, Leavitt SD, Hawksworth DL, Myllys L, McCune B, Randlane T, Bjerke JW, Ohmura Y, Schmitt I, Boluda CG, Alors D, Roca-Valiente B, Del-Prado R, Ruibal C, Buaruang K, Núñez-Zapata J, Amo de Paz G, Rico VJ, Molina MC, Elix JA, Esslinger TL, Tronstad IKK, Lindgren H, Ertz D, Gueidan C, Saag L, Mark K, Singh G, Dal Grande F, Parnmen S, Beck A, Benatti MN, Blanchon D, Candan M, Clerc P, Goward T, Grube M, Hodkinson BP, Hur J-S, Kantvilas G, Kirika PM, Lendemer J, Mattsson J-E, Messuti MI, Miadlikowska J, Nelsen M, Ohlson JI, Pérez-Ortega S, Saag A, Sipman HJM, Sohrabi M, Thell A, Thor G, Truong C, Yahr R, Upreti DK, Cubas P, Lumbsch HT (2015) Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi. The New Phytologist 208: 1217–1226. https://doi. org/10.1111/nph.13553 Eriksson OE, Hawksworth DL (1986) Notes on Ascomycete systematics. Nos 1-224. Systema Ascomycetum 5: 113–174. Etayo J (2002) Aportación al conocimiento de los hongos liquenícolas de Colombia. Bibliotheca Lichenologica 84: 1–154. Etayo J (2017) Hongos liquenícolas de Ecuador. Opera Lilloana 50: 1–550. Etayo J, Sancho LG (2008) Hongos liquenícolas del Sur de Sudamérica, especialmente de Isla Navarino (Chile). Bibliotheca Lichenologica 98: 1–302. Etayo J, Darmostuk V, Kukwa M, Flakus A (2024) High diversity of Trichonectria (Hypocreales) inhabiting Usnea in Bolivia. Plant and Fungal Systematics 69: 109–124. https://doi.org/10.35535/pfsyst-2024-0011 Ferdinandsen C, Winge O (1909) Mycological Notes II. Botanisk Tidsskrift 29: 305–319. Flakus A, Etayo J, Miadlikowska J, Lutzoni F, Kukwa M, Matura N, Rodriguez-Flakus P (2019) Biodiversity assessment of ascomycetes inhabiting Lobariella lichens in Andean cloud forests led to one new family, three new genera and 13 new species of lichenicolous fungi. Plant and Fungal Systematics 64: 283–344. https://doi. org/10.2478/pfs-2019-0022 Francisco CS, Ma X, Zwyssig MM, McDonald BA, Palma-Guerrero J (2019) Morphological changes in response to environmental stresses in the fungal plant pathogen Zymoseptoria tritici. Scientific Reports 9: e9642. https://doi.org/10.1038/s41598-019-45994-3 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x Katoh K, Standley DM (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010 Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PartitionFinder: Combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution 29: 1695–1701. https://doi.org/10.1093/molbev/mss020 Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34: 772–773. https://doi. org/10.1093/molbev/msw260 Lawrey JD, Etayo J, Dal-Forno M, Driscoll KE, Diederich P (2015) Molecular data support establishment of a new genus for the lichenicolous species Neobarya usneae (Hypocreales). The Bryologist 118: 83–92. https://doi.org/10.1639/0007-2745-118.1.083 307 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Lin X, Alspaugh JA, Liu H, Harris S (2015) Fungal morphogenesis. Cold Spring Harbor Perspectives in Medicine 5: a019679. https://doi.org/10.1101/cshperspect.a019679 Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092 Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, Lanfear R (2020) IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Molecular Biology and Evolution 37: 1530–1534. https://doi. org/10.1093/molbev/msaa015 Mombert A, Hairaud M, Boittin F, Priou J-P, Gardiennet A, Van Vooren N (2023) Ascomycètes non lichénisés nouveaux pour la France. Notules n° 4. Ascomycete.Org : Revue Internationale pour la Taxinomie des Ascomycota 15: 41–46. https://doi. org/10.25664/ART-0368 Nadal M, Garcia-Pedrajas MD, Gold SE (2008) Dimorphism in fungal plant pathogens. FEMS Microbiology Letters 284(2): 127–134. https://doi.org/10.1111/j.15746968.2008.01173.x Penn O, Privman E, Ashkenazy H, Landan G, Graur D, Pupko T (2010) GUIDANCE: A web server for assessing alignment confidence scores. Nucleic Acids Research 38: W23– W28. https://doi.org/10.1093/nar/gkq443 Pino-Bodas R, Zhurbenko MP, Stenroos S (2017) Phylogenetic placement within Lecanoromycetes of lichenicolous fungi associated with Cladonia and some other genera. Persoonia 39: 91–117. https://doi.org/10.3767/persoonia.2017.39.05 Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences: Evidence for cryptic diversification and links to Cordyceps teleomorphs. Mycologia 97: 84–98. https://doi.org/10.3852/mycologia.97.1.84 Rehner SA, Samuels GJ (1994) Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Research 98: 625– 634. https://doi.org/10.1016/S0953-7562(09)80409-7 Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029 Stanke M, Diekhans M, Baertsch R, Haussler D (2008) Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics (Oxford, England) 24: 637–644. https://doi.org/10.1093/bioinformatics/btn013 Stenroos S, Velmala S, Pykälä J, Ahti T (2016) Lichens of Finland. Norrlinia 30: 1–896. Suija A, Ertz D, Lawrey JD, Diederich P (2015) Multiple origin of the lichenicolous life habit in Helotiales, based on nuclear ribosomal sequences. Fungal Diversity 70: 55–72. https://doi.org/10.1007/s13225-014-0287-4 Suija A, Berger F, Arsenault A, Baines P, Zimmermann E, Etayo J (2024) New species, a taxonomic combination, and records of lichenicolous Abrothallus (Abrothallales, Dothideomycetes): Species on Peltigeraceae and Pannariaceae. Mycological Progress 23: 1–58. https://doi.org/10.1007/s11557-024-01994-w Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990 Wang L, Lin X (2012) Morphogenesis in fungal pathogenicity: Shape, size, and surface. PLoS Pathogens 8: e1003027. https://doi.org/10.1371/journal.ppat.1003027 308 MycoKeys 124: 291–308 (2025), DOI: 10.3897/mycokeys.124.169315 Valerii Darmostuk et al.: Expanding the genus Neobaryopsis Westberg M, Arup U, Ekman S, Isaksson R, Johannesson H, Vicente R, Svensson M (2022) Additions to the flora of lichens and lichenicolous fungi in Fennoscandia. Graphis Scripta 34: 12–21. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols, a Guide to Methods and Applications. Academic Press Inc., San Diego, CA, and London, 315–322. https://doi.org/10.1016/B978-0-12-3721808.50042-1 Zhurbenko MP (2009) New and interesting lichenicolous hypocrealean fungi from the Northern Hemisphere. Sydowia 61: 177–188. Zhurbenko MP, Kobzeva AA (2014) Lichenicolous fungi from Northwest Caucasus, Russia. Herzogia 27: 377–396. https://doi.org/10.13158/heia.27.2.2014.377 Zimmermann E, Berger F (2018) Ein Beitrag zur Kenntnis der Lichenicolen Mycobiota Österreichs. Funde aus Tirol I. Herzogia 31: 732–762. https://doi.org/10.13158/ heia.31.1.2018.732 Zimmermann E, Feusi S (2018) Bemerkenswerte Funde lichenicoler Pilze anlässlich der Bryolich Jahresversammlung 2017 in der Lenk (Schweiz, Berner Oberland). Meylania 61: 38–46.