A new heat-resistant species of Leiothecium Samson & Mouch. (Eurotiales, Ascomycota) from soil of Argentina

MycologieMycologie cryptogamiecryptogamie 2025 ● 46 ● 4 Cryptogamie, Mycologie est une revue en flux continu publiée par les Publications scientifiques du Muséum, Paris Cryptogamie, Mycologie is a fast track journal published by the Museum Science Press, Paris Les Publications scientifiques du Muséum publient aussi / The Museum Science Press also publish: Adansonia, Geodiversitas, Zoosystema, Anthropozoologica, European Journal of Taxonomy, Naturae, Comptes Rendus Palevol, Cryptogamie sous-sections Algologie, Bryologie. 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HYDE School of Science, Mae Fah Luang University, 333 M. 1 T.Tasud Muang District, Chiang Rai 57100 (Thailand) Valérie HOFSTETTER Station de recherche Agroscope Changins-Wädenswil, Dépt. Protection des plantes, Mycologie, CH-1260 Nyon 1 (Switzerland) Sinang HONGSANAN College of Life Science and Oceanography, Shenzhen University, 1068, Nanhai Avenue, Nanshan, ShenZhen 518055 (China) Egon HORAK Schlossfeld 17, A-6020 Innsbruck (Austria) Jing LUO Department of Plant Biology & Pathology, Rutgers University New Brunswick, NJ 08901 (United States) Ruvishika S. JAYAWARDENA Center of Excellence in Fungal Research, Mae Fah Luang University, 333 M. 1 T.Tasud Muang District, Chiang Rai 57100 (Thailand) Chen JIE Instituto de Ecología, Xalapa 91070, Veracruz (México) Sajeewa S.N. MAHARCHCHIKUMBURA Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University (Oman) Pierre-Arthur MOREAU UE 7144. Faculté des Sciences pharmaceutiques et biologiques. Université Lille Nord de France. F-59006 Lille (France) Tian QING Center of Excellence in Fungal Research, Mae Fah Luang University 333 M. 1 T.Tasud Muang District, Chiang Rai 57100 (Thailand) Sylvie RAPIOR Laboratoire de Botanique, Phytochimie et Mycologie / UMR -CNRS 5175 CEFE, Faculté de Pharmacie, 15, avenue Charles-Flahault, Université Montpellier I, BP 14491, 34093 Montpellier Cedex 5 (France) Franck RICHARD Université de Montpellier II, CEFE/CNRS Campus du CNRS, 1919, route de Mende, 34293 Montpellier Cedex 5 (France) Naritsada THONGKLANG Center of Excellence in Fungal Research, Mae Fah Luang University, 333 M. 1 T.Tasud Muang District, Chiang Rai 57100 (Thailand) Xiang-Hua WANG CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Lanhei Road 132, Kunming 650201, P. R. (China) couverture / cover: Extrait de la Figure 4/Extract of Figure 4 51 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) © Publications scientifiques du Muséum national d’Histoire naturelle, Paris. www.cryptogamie.com/mycologie A new heat-resistant species of Leiothecium Samson & Mouch. (Eurotiales, Ascomycota) from soil of Argentina Stella Maris ROMERO Instituto Multidisciplinario de Biología Vegetal (IMBIV), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-Universidad Nacional de Córdoba), Avenida Vélez Sarsfield 1611, 5000 Córdoba (Argentina) smromer[email protected] (corresponding author) Ernesto RODRÍGUEZ ANDRADE Tecnológico Nacional de México/Instituto Tecnológico Superior de Ciudad de Hidalgo, Avenida Ing. Carlos Rojas Gutiérrez 2120. CP 61100 Ciudad Hidalgo, Michoacán (México) [email protected] Andrea Irene ROMERO Instituto de Micología y Botánica (InMiBo), Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET-Universidad de Buenos Aires, Intendente Güiraldes 2160, Pab. II Ciudad Universitaria, C1428EGA Buenos Aires (Argentina) [email protected] Leonardo David AMARILLA Cátedra de Diversidad Biológica III, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Avenida Vélez Sarsfield 299, 5000 Córdoba (Argentina) and Instituto Multidisciplinario de Biología Vegetal (IMBIV), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-Universidad Nacional de Córdoba), Avenida Vélez Sarsfield 1611, 5000 Córdoba (Argentina) [email protected] Ricardo M. COMERIO Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Anguil “Ing. Agr. G. Covas” Ruta Nacional Nº 5, km 580, CC 11 6326 Anguil, La Pampa (Argentina) [email protected] Submitted on 11 April 2024 | Accepted on 27 January 2025 | Published on 29 September 2025 Romero S. M., Rodríguez Andrade E., Amarilla L. D. & Comerio R. M. 2025. — A new heat-resistant species of Leiothecium Samson & Mouch. (Eurotiales, Ascomycota) from soil of Argentina. Cryptogamie, Mycologie 46 (4): 51-60. https://doi.org/10.5252/cryptogamie-mycologie2025v46a4. http://cryptogamie.com/mycologie/46/4 ABSTRACT Leiothecium Samson & Mouch. is a genus enclosed into the family Aspergillaceae Link (Eurotiales) including L. ellipsoideum Samson & Mouch. and L. cristatum Y. Marín, Stchigel & Cano, species exhibiting ascospore walls somewhat or irregularly reticulated and prominent crests. During a research on heat-resistant fungi from soil of the semi-arid region of Northern Argentina, several Leiothecium spp. isolates were obtained; morphological and molecular studies were carried out. Phylogenetics showed strong support for the monophyly of the Leiothecium clade and L. ellipsoideum as the sister taxon. Some isolates were identified as Leiothecium ellipsoideum, and a new species provided with KEY WORDS Aspergillaceae, Argentina, reticulated ascospores, cleistothecial ascomata, semi-arid region, new species. 52 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) Romero S. M. et al. reticulate-foveate ascospore ornamentation without prominent crests was described: Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. Molecular analysis and scanning electron microscopy (SEM) images support the description of L. dictyophorum S.M.Romero & Comerio, sp. nov. as a new species. RÉSUMÉ Une nouvelle espèce de Leiothecium Samson & Mouch. résistante à la chaleur (Eurotiales, Ascomycota) issue du sol d’Argentine. Leiothecium Samson & Mouch. est un genre inclus dans la famille des Aspergillaceae Link (Eurotiales) qui comprend L. ellipsoideum Samson & Mouch. et L. cristatum Y.Marín, Stchigel & Cano, des espèces qui présentent des parois d’ascospores quelque peu ou irrégulièrement réticulées et des crêtes proéminentes. Lors d’une recherche sur les champignons résistants à la chaleur du sol de la région semi-aride du nord de l’Argentine, plusieurs isolats de Leiothecium spp. ont été obtenus; des études morphologiques et moléculaires ont été réalisées. La phylogénétique a montré un fort soutien à la monophylie du clade de Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. et de L. ellipsoideum comme taxon frère. Certains isolats ont été identifiés comme Leiothecium ellipsoideum, et une nouvelle espèce dotée d’une ornementation d’ascospores réticulées-fovéates sans crêtes proéminentes a été décrite : Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. L’analyse moléculaire et les images de microscopie électronique à balayage (MEB) soutiennent la description de L. dictyophorum S.M.Romero & Comerio, sp. nov. en tant que nouvelle espèce. INTRODUCTION The genus Leiothecium Samson & Mouch. was described in the course of a study on thermoresistant soil fungi (Samson & Mouchacca 1975). In that work, a single species, Leiothecium ellipsoideum Samson & Mouch., was described from a soil sample collected between rocks in Mystras, Greece. Almost thirty-five years later, Marin-Felix et al. (2014) described Leiothecium cristatum Y.Marín, Stchigel & Cano, from a rainforest soil sample collected in Iguazú National Park, Misiones province, Argentina. Leiothecium is one of the fifteen genera accommodated in the family Aspergillaceae Link in Eurotiales (Houbraken et al. 2020); species in the genus presents dark cleistothecial ascomata, ascus with evanescent walls, and net-ornamented ascospores. Leiothecium includes, up to now, the only two species previously mentioned. During broad research on heat-resistant fungi from the semiarid region of Northern Argentina, more than two hundred isolates were obtained from soil. Most isolates were related to Aspergillus sect. Fumigati, but thirty-four isolates represented several interesting taxa (Romero et al. 2020). Nine isolates from different soil samples resembled L. ellipsoideum, but in some of them, morphological differences in the ascospores ornamentation were noted. The objective of this work was to present and describe a new Leiothecium species based on phylogenetics and morphological data. MATERIAL AND METHODS IsolatIon Fifty 200 g soil samples were collected in the summer of 2009 and winter of 2011 from several sites in Catamarca and La Rioja provinces, Northern Argentina. A detailed description of the sampling area was done in Romero et al. (2020). About 200 g of soil per sample were collected using a sterile stainless-steel scoop and transferred aseptically into sterile self-sealing plastic bags of appropriate capacity. The samples were transported and stored at room temperature until processing in the laboratory. From each sample, c. 5 g of soil was placed into a conical flask, mixed with 100 mL of sterile melted Malt Extract Agar (MEA, Oxoid CM0059B, United Kingdom) plus 50 ppm of chloramphenicol, and heated at 75°C for 30 min. The suspension was plated into sterile Petri dishes (9.0 cm diam.) and, once gelled, incubated at 30°C for periods of less than thirty days (Samson et al. 2019). All colonies were transferred to individual Petri dishes (5.0 cm diam.) containing MEA to obtain pure cultures. The fungal strains were deposited in the culture collection of the Herbarium, Universidad de Buenos Aires, Argentina (BAFC) and the culture collection of the Faculty of Medicine, Universitat Rovira i Virgili, Reus, Catalonia, Spain (FMR). PhenotyPIc characterIzatIon Genus characterization was conducted according to Guarro et al. (2012). To carry out species identification, inoculations were made from spore suspensions prepared with agar 0.2 % w/v and Tween 80 0.05 % w/v solution. The isolates were inoculated on each plate in a three-point pattern using a micropipette with an inoculum size of 1 μl per spot. Leiothecium isolates utilized in this study are listed in Table 1. For standard macro-morphological observations cultures on MEA, Oatmeal Agar (OA), Creatine Sucrose Agar (CREA) and Potato Dextrose Agar (PDA) were performed according to Samson et al. (2019); all plates were incubated at 25°C in the dark, and examined at 7 and 14 days. Extra cultures on MEA were carried out and incubated at 15, 35 and 40°C for MOTS CLÉS Aspergillaceae, Argentine, ascospores réticulées, ascomates cleistothéciques, région semi-aride, espèce nouvelle. 53 A new heat-resistant species of Leiothecium from soil of Argentina CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) 7 days; fungal strains were also inoculated on Czapek Yeast Agar 20% Sucrose (CY20S) and Malt Extract Yeast Extract 50% Glucose Agar (MY50G) and incubated at 25°C for 7 days according to Pitt & Hocking (2009). The microscopic mounts to determine micro-morphological features, from MEA colonies, were made in lactic acid 85% w/w and lactofuchsin (Carmichael 1955; Samson et al. 2019). Preparations were observed through a Zeiss Axioscop microscope. Mature ascomata were crushed, coated with gold, and observed and photographed with a Zeiss Supra 40 (extra high tension = 3 Kv; working distance = 3.7-6 mm) scanning electron microscope. Dna extractIon, amPlIfIcatIon, anD sequencIng Total DNA was extracted directly from colonies on PDA after 7-10 days incubation at 25°C in darkness, following the Fast DNA kit protocol (Bio 101, Inc., Vista, Canada, United States) with the homogenization step repeated three times with a FastPrep FP120 instrument (Thermo Savant, Holbrook, New York, United States). After each homogenization, the sample was kept in ice for 10 min. DNA was quantified with GeneQuant pro (Amersham Pharmacia Biotech, Cambridge, England) (Marimon et al. 2006). Extracted DNA was used to amplify a fragment of beta-tubulin (BenA) (T10/Bt2b primers; Glass & Donaldson 1995), a fragment of the RNA polymerase II subunit 2 gene (rpb2) (RPB2-5F/RPB2-7cR primers; Liu et al. 1999), a fragment of 28S nrRNA gene (LSU) (LR0R/LR5 primers: Rehner & Samuels 1994; Vilgalys & Hester 1990), and the internal transcribed spacer (ITS) (ITS5/ITS4 primers; White et al. 1990). The PCR amplifications were made in a total volume of 25 μL containing 5 μL 10x PCR Buffer (Invitrogen, CA, United States), 0.2 μM dNTPs, 0.5 μM of each primer, 1 U Taq DNA polymerase, and 1-10 ng nuclear DNA. PCR conditions were set as follows: for BenA and ITS regions, initial denaturation at 95°C for 5 min, followed by 35 cycles of (denaturation during 30 s at 95°C, annealing during 1 min at 55°C, and extension during 90 s at 72°C), and a final extension step at 72°C for 10 min. For rpb2 and LSU regions, initial denaturation at 95°C for 5 min, followed by 35 cycles of (45 s at 95°C, 1 min at 56°C, and 90 s at 72°C) and a final extension step at 72°C for 10 min. Single-band PCR products were purified from agarose gels and sequenced at Macrogen Europe, which uses large-scale sequencing developed by “Applied Biosystem”, which works using the Sanger method (Macrogen Inc., Madrid, Spain). Sequence assembly and editing were carried out using SeqMan software v.7.0 (DNAStar Lasergene, Madison, WI, United States). PhylogenetIc analysIs We used a total of 10 taxa including: two representative taxa of Monascus, Monascus purpureus Went and Monascus argentinensis Stchigel & Guarro, one representative taxa of L. cristatum, two representative taxa of L. ellipsoideum, and four representative taxa of Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov.; Xeromyces bisporus L.R.Fraser, was used as outgroup. We generate twenty-one new sequences which sixteen correspond to Leiothecium S.M.Romero & Comerio, sp. nov., four correspond to L. ellipsoideum and one corresponds to L. cristatum. GenBank accession numbers for the newly generated sequences in this study and others corresponding to reference or ex-type strains are listed in Table 1. DNA sequences were initially aligned with Muscle ver. 3.6 (Edgar 2004a, b), followed by manual alignment in the data editor of BioEdit ver. 7.0.1 (Hall 1999). The alignment was table 1. — Data used for phylogenetic analysis in this study and their corresponding GenBank accession numbers. Type species are denoted by (T). Sequences derived from this study are shown in bold. (a), Marin-Felix et al. 2014; (b), Vu et al. 2019; (c), Barbosa et al. 2017; (d), Houbraken & Samson 2011; (e), Gueidan et al. 2008; (f), Pettersson et al. 2011; (g), Peterson 2008; (h), Abarenkov et al. 2024. Species name Strain number GenBank accession number BenA rpb2ITS LSU Leiothecium cristatum Y.Marín, Stchigel & Cano (T) FMR 11998T = CBS 134260T = NBRC 109843T PP187391 HF954976.1(a) NR_145180.1(a) MH877551.1(b) Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. (T) BACF 53468 = BAFCcult 4595 = FMR 15063 PP149064 PP149071 PQ359515 PQ359520 Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. BAFCcult 4592 = FMR 15065 PP149067 PP149074 PQ359516 PQ359521 Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. BAFCcult 4608 = FMR 15066 PP149065 PP149072 PQ359517 PQ359522 Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. BAFCcult 4597 = FMR 15067 PP149066 PP149073 PQ359518 PQ359523 Leiothecium ellipsoideum Samson & Mouch. (T) CBS 607.74T KY709178.1(c) JN121541(d) NR_144922.1(a) NG_057811.1(e) Leiothecium ellipsoideum Samson & Mouch. BAFCcult 4598 = FMR 15064 PP149063 PP149070 PQ359519 PQ359524 Monascus argentinensis Stchigel & Guarro CBS 109402 KY709174.1(c) JN121423.1(d) JF922046.1(f) KY645974.1(d) Monascus purpureus Went NRRL 1596 EU014110.1(g) EF669718.1(g) OQ694412.1(h) NG_069605.1(b) Xeromyces bisporus L.R.Fraser CBS 236.71 KY709179.1(c) JN121466.1(c) MH860089.1(b) NG_057813.1(e) 54 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) Romero S. M. et al. performed separately for each marker. To address the level of congruence among data partitions and their influence on combined datasets, the incongruence length difference (ILD) test (Farris et al. 1994) was performed. The ILD test with 1000 permutations and a significance level of P <0.01 was conducted in TNT (ILD.run script). Phylogenetic hypotheses were congruent between partitions according to the ILD test (P = 0.14). Based on the results of these incongruence tests, we determined that the different marker matrices can be combined. Maximum parsimony (MP) analysis was done with TNT ver. 1.1 (Goloboff et al. 2008). “Traditional” heuristic searches were carried out with a total of 1000 random addition sequences and submitted to tree-bisection reconnection (TBR) branch swapping, holding 50 trees, followed by a more extensive TBR holding 50 000 trees. The most parsimonious trees found were collected and a strict consensus was calculated using the “Nelsen” option in TNT. Support was estimated by bootstrap as implemented in TNT, resampling 1000 times with TBR set to 100 replications holding 20 trees, followed by a more extensive TBR holding 5000 trees, and saving the consensus for each resampling matrix. Bayesian inference analyses were employed to infer the phylogeny using each dataset separately and subsequently using a combined dataset that included all sequences. Bayesian inference was performed using MrBayes ver. 3.2.7 (Ronquist et al. 2012). Models for each molecular dataset, as well as the combined dataset, were evaluated using ModelTest version 3.06 (Posada & Crandall 1998) to identify the best fit according to the Akaike Information Criterion (Akaike 1974). Bayesian analyses were run with 20 million Metropoliscoupled Markov chain Monte Carlo (MCMCMC) generations with four chains, sampling trees every 100th generation. Stationarity was determined based on the convergence of likelihood scores using TRACER ver. 1.5 (Rambaut & Drummond 2009), and sample points generated before stationarity were eliminated as burn-in (25%). The posterior probabilities (PP) of the clades were determined by a 50% majority-rule consensus of the trees retained. RESULTS PhylogenetIc analyses The alignment of the 10 accession dataset consisted of 2729 aligned positions (Table 2). Summary of the four datasets of DNA regions and the combined dataset, along with an overview of the parameters used for phylogenetic estimation and the statistical results of parsimony analyses, including the consistency index (CI), retention index (RI), number of parsimony-informative sites, tree length, best-fit models of evolution, and estimated log-likelihood are provided in Table 2. The strict consensus tree resulting from parsimony analyses of each dataset and the combined dataset (not shown) presented the same topology as the 50% majority rule consensus tree obtained from Bayesian analyses of the same datasets. For each dataset, both individually and combined, the Bayesian analysis shows that the average standard deviation of split frequencies reaches 0.005 after 20 million generations, and the four runs converged with a potential scale reduction factor (PSRF) tending to 1.001. The 50% majority-rule consensus tree from the Bayesian analysis of the combined dataset is shown in Fig. 1, whereas the trees derived from each individual dataset are shown in Fig. 4. Both parsimony and Bayesian analyses based on the combined dataset showed similar topologies throughout the general phylogeny, as BAFCcult 4597 BAFCcult 4592 BAFCcult 4595T BAFCcult 4608 L. ellipsoideum Samson& Mouch. BAFCcult 4598 L. ellipsoideum Samson& Mouch. CBS 607.74T Leiothecium cristatum Y.Marín, Stchigel& Cano FMR 11998T Monascus purpureus Went NRRL1596 M. argentinensis Stchigel& GuarroCBS 109402 Xeromyces bisporus L.R.Fraser CBS 236.71 100 100 100 66 0.89 1 1 0.99 86 0.93 80 1 0.5 fig. 1. — Fifty percent majority-rule Bayesian consensus of the molecular phylogenetic analyses of the combined dataset. Support values above the branches represent Bayesian posterior probabilities (PP>0.5). Support values below the branches represent bootstrap values (BS>50). Scale bar: 0.5 substitutions per site. 55 A new heat-resistant species of Leiothecium from soil of Argentina CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) fig. 2. — Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov.: A, B, colony on MEA, OA, 14 days, 25 at c. 1ºC; C, ascomata from aerial mycelium; D, ascomata immersed in the culture media; E, initials; F, ascoma Textura angularis wall; G-I, ascospores; J, K, chlamydospores; L, broken ascoma showing asci and ascospores (SEM); M, N, ascospores (SEM). Scale bars: E, H, J, 10 μm; F, 20 μm; I, K, L, 5 μm; M, N, 1 μm. AB C D EF G H L MN IJK 56 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) Romero S. M. et al. well as consistent relationships between, Leiothecium clade and Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. clade (Fig. 1). Both parsimony and Bayesian analyses showed strong support for the monophyly of the Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. clade and L. ellipsoideum as the sister taxa. Thus, we present only the consensus tree resulting from Bayesian analyses and on their branches are included posterior probabilities (PP) and bootstrap support (BS). TAXONOMY Order EUROTIALES G.W.Martin ex Benny & Kimbr. Family asPergIllaceae Link Genus Leiothecium Samson & Mouch. Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. (Fig. 2) DIagnosIs. — Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov.; ex affinitate L. ellipsoidei Samson & Mouch. et L. cristati Y.Marín, Stchigel & Cano, ab utroque ascosporarum ornamento reticulato-foveato sine manifestis cristis distinctum. Leiothecium dictyophorum S.M. Romero & Comerio sp. nov.; of the affinity of L. ellipsoideum Samson & Mouch. and L. cristatum Y. Marín, Stchigel & Cano, distinct from both by the ascospores’ ornament reticulate-foveate without prominent crests. tyPe materIal. — Argentina • Catamarca, RN 60 km 1016; 29°30’4”S, 65°37’57”W; 237 m alt.; 22.VIII.2011; S.M. Romero leg.; Holotype: BAFC 53468 (dried culture); BAFCcult 4595 = FMR≈15063, culture ex type; GenBank BenA sequence PP149064, rpb2 sequence PP149071. aDDItIonal sPecImens stuDIeD. — Argentina • Catamarca; RN 60 km 1102; 28°55’15”S, 66°08’46”W; 338 m alt.; 5.I.2009; BAFCcult 4599 = FMR 16764 • RN 38 km 563; 28°35’5”S, 65°52’27”W; 522 m alt.; 12.I.2009; BAFCcult 4592 = FMR 15065 • RN 60 km 1016; 29°30’4”S, 65°37’57”W; 237 m alt.; 2.I.2010; BAFCcult 4608 = FMR 15066 • RN 38 km 563; 28°35’5”S, 65°52’27”W; 522 m alt.; 23.VIII.2011; BAFCcult 4597 = FMR 15067, FMR 16766, BAFCcult 4596 = FMR 16767. mycobank. — MB857727. etymology. — Dictyon (δικτυον, net) and phorum (φορος, bearing) were combined to coin dictyophorum (adj.): net-bearing; regarding the distinctly net-like ornamentation of ascospores. latIn DescrIPtIon Coloniae in agaro malto confecto, 25°C, post 7 dies, 75-79 mm diam., fuscae, albis annulis mycelialibus praeditae; pars aversa fusca. In agaro farina avenae confecto, 25°C, post 7 dies, 60-64 mm diam., fuscae, quasi continuum stratum ascomatum efficientes, mycelium aerium vix effectum. Hyphae hyalinae, laeves, 3-11 μm diam. Cleistothecia globosa, glabra, atro-brunnea, 40-125 μm diam., inter mycelium aerium disposita vel in substratum semiimmersa; parietes cleistotheciorum persistentes, 5-8 μm lati, texturam angularem praebentes, e cellulis atro-brunneis crassitunicatis 15-25 μm diam. compositi. Asci globosi vel subglobosi, 13-15 × 14-18 μm, octospori, evanescentes. Ascosporae unicelulares, ellipsoideae, hyalinae, ornamentum reticulatum-foveatum ostendentes, 5.5-7 × 7-9 μm. Chlamydosporae subglobosae vel ellipsoideae, table 2. — Summary of the four datasets of DNA regions, along with an overview of the parameters used for phylogenetic estimation and the statistical results of parsimony analyses. To establish the nucleotide similarity percentage, a BLAST (Highly similar sequences -megablastfrom NCBI) was performed, using sequences obtained from type strains of Leiothecium ellipsoideum Samson & Mouch. and L.dictyophorum S.M.Romero & Comerio, sp. nov. BenA rpb2LSU ITS ALL Number of taxa 10 10 10 10 10 Average sequence length 358 848 849 540 — % of nucleotide similarity 96 98.9 99.7 99.5 — Aligned sequence length 437 850 854 588 2729 N° sites parsimony informative 101 123 22 38 284 Consistency index 0.84 0.74 0.71 0.7 0.76 Retention index 0.82 0.69 0.64 0.65 0.78 Tree length (steps) 299 373 79 163 917 Best-fit model of evolution, Akaike information criterion (AIC) K2 + G K2G T92 + G K2 + G K2 + G Estimated log-likelihood –1767.2 –2747.6 –1610.8 –1568.4 –7915.3 DIchotomous key for the known Leiothecium samson & mouch. sPecIes 1. Ascospores reticulate, without prominent crests ............... L. dictyophorum S.M.Romero & Comerio, sp. nov. 1’. Ascospore wall reticulated, with prominent crests ........................................................................................ 2 2. Ascospores presenting several crests .......................................................... L. ellipsoideum Samson & Mouch. 2’. Ascospores with only two crests .......................................................... L. cristatum Y.Marín, Stchigel & Cano 57 A new heat-resistant species of Leiothecium from soil of Argentina CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (4) hyalinae, plerumque terminales umbonataeque, nonnumquam intercalares, etiam endogenae, crassitunicatae, 6-18 × 5-12.5 μm diam. englIsh DescrIPtIon Colonies on MEA, 25ºC, 7 days, 75-79 mm diam., dark, with aerial white mycelium in concentric circles; reverse dark (Fig. 2A). At 35°C, covering the whole plate before 7 days; reverse dark due to numerous ascomata. At 15°C, 7 days, 0-8 mm diam.; at 40°C, 7 days, 21-31 mm diam. On PDA, 25°C, covering the whole plate at 7 days, dark, with scarce aerial white mycelium, reverse dark. On OA, 25°C, 7 days, 60-64 mm, dark colonies forming an almost continuous layer of ascomata, aerial mycelium scarce (Fig. 2B). At 35°C, spreading all over the plate before 7 days; development in subtle circles, aerial mycelium less floccose than in MEA. On CY20S, 25°C, 7 days, 83 mm diam. On MY50G, 25°C, 7 days, 42 mm diam. Mycelium composed of hyaline, smooth-walled, hyphae, 3-11 μm wide. Cleistothecial ascomata, globose, glabrous, dark-brown 40-125 μm diam., both intermixed within the white mycelium and semi-submerged in agar (Fig. 2C, D); with a persistent wall, textura angularis, 5-8 μm thick, composed of dark brown thick-walled cells of 15-25 μm diam. (Fig. 2F, G). Asci globose to subglobose, 13-15 × 14-18 μm, eight-spored, evanescent. Ascospores one-celled, ellipsoidal, hyaline, 5.5-7 × 7-9 μm, loosely reticulated (Fig. 2I); SEM shows a reticulated-foveate ornamentation in mature ascospores. Chlamydospores present, subglobose to ellipsoidal, hyaline, usually terminal but sometimes intercalary, endogenously generated present as well, smoothand thickwalled, 6-18 × 5-12.5 μm. notes Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. displayed a distinctive ascospore ornamentation which set it morphologically apart from L. ellipsoideum and L. cristatum, consisting in a reticulate-foveate-net-ascospores without prominent crests (Fig. 2N). Some ascospores of L. dictyophorum S.M.Romero & Comerio, sp. nov. have shown areas where the net was not completely developed, as it is shown at the central region of the ascospore illustrated in the Fig. 2M. Probably, this particular feature, present in several ascospores, reflects some unripeness degree. Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. ascospores has not exhibited prominent A CD B fig. 3. — Ascospores (SEM): A, B, Leiothecium dictyophorum S.M.Romero & Comerio, sp. nov. : A, BAFCcult 4608; B, BAFCcult 4599; C, D, Leiothecium ellipsoideum Samson & Mouch.: C, BAFCcult 4598; D, BAFCcult 4604. Scale bars: 1 μm.