The Podospora anserina (Rabenh.) Niessl species complex in metropolitan and overseas France with description of a new species, Podospora reunionensis Silar, sp. nov.

MycologieMycologie cryptogamiecryptogamie 2025 ● 46 ● 6 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. Diffusion – Publications scientifiques Muséum national d’Histoire naturelle CP 41 – 57 rue Cuvier F-75231 Paris cedex 05 (France) Tél. : 33 (0)1 40 79 48 05 / Fax : 33 (0)1 40 79 38 40 [email protected] / http://sciencepress.mnhn.fr © Publications scientifiques du Muséum national d’Histoire naturelle, Paris, 2025 ISSN (électronique / electronic) : 1776-100 Cryptogamie, Mycologie est indexé dans / Cryptogamie, Mycologie is indexed in: – Biological Abstracts – Current Contents – Science Citation Index – Publications bibliographiques du CNRS (Pascal) Cryptogamie, Mycologie est distribué en version électronique par / Cryptogamie, Mycologie is distributed electronically by: – BioOne® (http://www.bioone.org/loi/crym) Directeur De la publication / Publication director: Gilles BLOCH Président du Muséum national d’Histoire naturelle réDacteur en chef / editor-in-chief: Philippe SILAR assistant De réDaction / assistant editor: Violette GRUNENBERGER ([email protected]) Mise en page / Page layout: Violette GRUNENBERGER réDacteurs associés / associate editors Slavomír ADAMČÍK Institute of Botany, Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523, Bratislava (Slovakia) Cony DECOCK Mycothèque de l’Université catholique de Louvain, Earth and Life Institute, Microbiology, Université catholique de Louvain, Croix du Sud 3, B-1348 Louvain-la-Neuve (Belgium) Damien ERTZ Meise Botanic Garden, Department Research, Nieuwelaan 38, BE-1860 Meise (Belgium) André FRAITURE Botanic Garden Meise, Domein van Bouchout, B-1860 Meise (Belgium) Kevin D. 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 5/Extract of Figure 5 87 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) © Publications scientifiques du Muséum national d’Histoire naturelle, Paris. www.cryptogamie.com/mycologie The Podospora anserina (Rabenh.) Niessl species complex in metropolitan and overseas France with description of a new species, Podospora reunionensis Silar, sp. nov. Philippe SILAR Valérie GAUTIER Christophe LALANNE Université Paris Cité, CNRS, Laboratoire Interdisciplinaire des Energies de Demain, F-75013 Paris (France) [email protected] (corresponding author) Narumon TANGTHIRASUNUN Department of Biology, School of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520 (Thailand) Marvyn ARTHUR Université Paris Cité, CNRS, Laboratoire Interdisciplinaire des Énergies de Demain, F-75013 Paris (France) Fanny E. HARTMANN Tatiana GIRAUD Écologie Systématique et Évolution, CNRS, Université Paris-Saclay, AgroParisTech, F-91198 Gif-sur-Yvette (France) Submitted on 3 December 2024 | Accepted on 12 April 2025 | Published on 16 December 2025 Silar P., Gautier V., Lalanne C., Tangthirasunun N., Arthur M., Hartmann F. E. & Giraud T. 2025. — The Podospora anserina (Rabenh.) Niessl species complex in metropolitan and overseas France with description of a new species, Podospora reunionensis Silar, sp. nov. Cryptogamie, Mycologie 46 (6): 87-100. https://doi.org/10.5252/cryptogamiemycologie2025v46a6. http://cryptogamie.com/mycologie/46/6 ABSTRACT Podospora anserina (Rabenh.) Niessl is a model fungus that was shown to belong to a complex of seven species with morphologically indistinguishable sexual fruiting bodies. Through the morphological analyses and ITS (Intergenic Transcribed Spacer) sequences of 86 newly-isolated strains, as well as the genome sequences of five strains, we show that members of the P. anserina species complex present different distribution ranges in metropolitan and overseas France. Podospora anserina is present all-over metropolitan France, including Corsica, while P. comata Milovtz. is restricted to the Northern part and P. pauciseta (Ces.) Traverso to the Southern part of continental France. The Guadeloupe hosts P. pseudoanserina C.Boucher, T.S.Nguyen & P.Silar and La Réunion island a species new to science, described here as Podospora reunionensis Silar, sp. nov. This species is closely related to P. comata, but exhibit clear morphological and biological differences. KEY WORDS Podospora anserina species complex, Podosporaceae, Sordariales, geographical structuration, genome sequence, new species. 88 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) Silar P. et al. RÉSUMÉ Le complexe d’espèces Podospora anserina (Rabenh.) Niessl en France métropolitaine et outre-mer, avec la description d’une nouvelle espèce, Podospora reunionensis Silar, sp. nov. Podospora anserina (Rabenh.) Niessl est un champignon modèle qui appartient à un complexe de sept espèces indistinguables sur le critère de la morphologie de leurs fructifications sexuelles. À travers les analyses de la morphologie et des séquences ITS (Intergenic Transcribed Spacer) de 86 souches nouvellement isolées, ainsi que la détermination des séquences des génomes de cinq souches, nous montrons ici que les membres du complexe ont différentes aires de distribution en France métropolitaine et d’outremer. Podospora anserina est présent partout en France métropolitaine, y compris en Corse ; P. comata Milovtz. est restreinte dans le nord et P. pauciseta (Ces.) Traverso dans le sud de la partie continentale du pays. La Guadeloupe héberge P. pseudoanserina C.Boucher, T.S.Nguyen & P.Silar et l’île de La Réunion une nouvelle espèce décrite ici comme Podospora reunionensis Silar, sp. nov. Cette nouvelle espèce est très proche génétiquement de P. comata, mais montre de claires différences morphologiques et biologiques avec celle-ci. INTRODUCTION Podospora anserina (Rabenh.) Niessl is a model fungus used in several laboratories to study various biological processes such as sexual reproduction, prions, ageing, signal transduction, heterokaryon incompatibility, epigenetics, plant biomass degradation and genome maintenance and evolution (Silar 2020; Bhunjun et al. 2024). Molecular analyses of four small DNA regions of the genome (Rchr3, Rchr4, Rchr6 and the Intergenic Transcribed Spacer-ITS) have shown that the species formerly known as “P. anserina” is actually a species complex with presently seven members that are indistinguishable based on morphological criteria (i.e., their perithecia, asci and ascospores appear identical), but may differ in the distribution ranges (Boucher et al. 2017). The different species can, however, be differentiated by their behavior when cultivated in different media, i.e., the efficiency and timing with which they produce fruiting bodies. In particular, perithecia are distributed onto the thallus differently in different species (Boucher et al. 2017). High quality genome sequences supported the subdivision in different species and the differences in distribution range (Vogan et al. 2019; 2021b; Hartmann et al. 2021; AmentVelásquez et al. 2024). However, the phylogenomic analyses could not resolve the relationships between the different species so far, likely due to their rapid diversification (Boucher et al. 2017; Ament-Velásquez et al. 2024). Some controversies remain regarding the naming of P. anserina and other species of the complex. Indeed, Wang et al. renamed the fungus as Triangularia anserina (Rabenh.) X. Wei Wang & Houbraken and placed it in the Podosporaceae family within the Sordariales order (Wang et al. 2019). However, this suggestion met strong resistance, especially from the scientific community working with P. anserina as a genetic model (Silar 2020; Ament-Velásquez et al. 2020; Bhunjun et al. 2024). The three genera defined by Wang et al. (2019) to accommodate the species of the Podosporaceae (i.e., Podospora, Cladorrhinum Sacc. & Marchal and Triangularia Boedijn) were synonymized (Ament-Velásquez et al. 2020) and a proposal to change the type species of the Podospora genus from P. fimiseda (Ces. & De Not.) Niessl to P. anserina sensu stricto (s.s.), as defined in Boucher et al. (2017), was issued (Vogan et al. 2021a). While the decision regarding the type species chosen for the genus Podospora is pending, it is best to continue to use P. anserina as the name for the fungus (Bhunjun et al. 2024). Species from the P. anserina complex are coprophilous and thus frequently found on various herbivore dung, especially those of large animals, such as cows and horses (Silar 2020). However, they can also be found in soil (Silar 2020), and a strain was even isolated as an endophyte (Matasyoh et al. 2011). At the present time, the distribution of the various species is not well-known. In order to better understand the range distribution of the species complex, we isolated numerous new strains from herbivore dung and also from soil collected in metropolitan and overseas France. To this end, we describe here a method that enables the preferential recovery of Sordariales species from soil samples. Among the 86 strains that we isolated, two strains displayed differences in their Internal Transcribed Spacer (ITS) with those of the seven described species. We sequenced the genomes of these two strains, as well as three other strains. We calculated their average nucleotide identity (ANI) with the known type species of the complex using FungANI (Lalanne & Silar 2025) to assign them to species. Based on the ITS sequences of the 86 strains of the complex that we isolated, we found that: 1) P. anserina s.s. is present all-over metropolitan France, including the Corsica island, while P. comata Milovtz. appears more specific to the Northern part of France and P. pauciseta to the Southern part; 2) P. pseudoanserina C.Boucher, T.S.Nguyen & P.Silar is present in the Guadeloupe island from the French Antilles; and 3) a species new to science is present in the La Réunion Island from the Indian ocean (Fig. 1). This latter species is described here as Podospora reunionensis Silar, sp. nov. Unlike the other species from the complex that cannot be differentiated by morphology, this species produced plumpier ascospores carrying a much longer primary appendage and a bigger and much more prominent secondary appendage(s) at the base of the pedicel than the other members of the complex. MOTS CLÉS Podospora anserina complexe d’espèces, Podosporaceae, Sordariales, structuration géographique, séquence génomique, espèce nouvelle. 89 The Podospora anserina species complex with description of a new species, Podospora reunionensis sp. nov. CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) MATERIAL AND METHODS RecoveRy of new stRains fRom the P. anserina species complex Strains were recovered from dung as described (Silar 2020). To isolate Sordariales strains from soils, M0 plates (0.25 g/L KH 2 PO 4 , 0.3 g/L K 2 HPO 4 , 0.25 g/L MgSO 4 -7H 2 O, 0.5 g/L urea, 0.05 mg/L thiamine, 0.25 µg/L biotin, 2.5 mg/L citric acid, 2.5 mg/L ZnSO4, 0.5 mg/L CuSO4, 125 µg/L MnSO4, 25 µg/L boric acid, 25 µg/L sodium molybdate, 25 µg/L iron alum, 10 g/L agar) were supplemented with 0.5 g of shredded miscanthus by adding the complex biomass onto the plates after the agar rigidified. These were then inoculated with c. 0.5 mL of soil. For each soil sample, three plates were prepared. One was left at room temperature, a second one was incubated at 65°C for 30 minutes and then transferred to room temperature; the third one was incubated overnight at 37°C and then transferred to room temperature. The three plates were then left at room temperature in the presence of light for up to two months. Note that the plates were not treated with antibiotics or chemicals to prevent the growth of bacteria or the presence of small animals (such as mites and collembolans), because the stress brought by their presence and/or grazing may promote fruiting body development of Sordariales (Silar, unpublished observations). The plates were regularly checked (once or twice weekly) for the presence of perithecia typical of Sordariales fungi. If such fruiting bodies developed, Languedoc-Roussillon Midi Pyrénées Aquitaine Limousin Poitou-Charentes Guyane Martinique Guadeloupe Mayotte Réunion Auvergne-Rhône-Alpes Provence-Alpes Côte-d’Azur Corse Bourgogne Franche-Comté Centre Val de Loire Ile-de-France Alsace Champagne-Ardenne Lorraine Nord-Pas-de-Calais Picardie Pays de la Loire Normandie Bretagne Podospora anserina Podospora pauciseta Podospora pseudoanserina Podospora reunionensis Silar, sp. nov.Podospora comata fig. 1. — Map of France with stars pointing to locations where the strains of the present study originated from. 90 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) Silar P. et al. fig. 2. — FungANI analyses of the Podospora Rabenh. strains PSN1303, PSN1705, PSN1871 and PSN1899. The Average Nucleotide Identities (ANIs) are at the top, and at the bottom are given the ANIs obtained when comparing the genomes in the two directions (reference and tested genome IDs are given below the graphic), as well as the percentages of zero hits, the estimated size of the region specific for each genome and the numbers of BLAST made during the analysis. The graphic represents the number of BLAST detecting sequences with the color-coded percentages and in the boxes are the actual number of BLAST with such hit percentages. In all cases, ANIs were higher than 99.5% and the strains had less than 1.5% of specific sequences. These were scattered all over the genomes. For more information see (Lalanne & Silar 2025). PSN1303 vs P. anserina S PSN1871 vs P. pseudoanserina CBS 253.71 PSN1899 vs P. pseudoanserina CBS 253.71 PSN1705 vs P. anserina S 91 The Podospora anserina species complex with description of a new species, Podospora reunionensis sp. nov. CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) ascospores ejected from them were collected onto projection plates as described previously (Silar 2020), except that the plates were supplemented with three antibiotics (chloramphenicol 25 µg/mL, tetracycline 50 µg/mL and kanamycin 50 µg/mL) to prevent bacterial contaminations. Ascospores were then transferred onto G medium (ammonium acetate 4.4 g/L, bactopeptone 15 g/L and agar 13 g/L) supplemented with 5 g/L of yeast extract and three antibiotics (chloramphenicol 25 µg/mL, tetracycline 50 µg/mL and kanamycin 50 µg/mL). A 30-minutes heat-shock at 65°C was immediately applied to promote ascospore germination. If this failed, an overnight heat-shock at 37°C was then applied to a second batch of freshly collected ascospores. Note that in the case of strains of the P. anserina species complex, the presence of yeast extract in G medium and heat-shock are dispensable, while it is often mandatory for most other species of Sordariales. For species the P. anserina complex, the four ascospores of several asci were collected PSN1303 M2 M0 + miscanthus Perithecia on M0 + miscanthus Rosettes of asci ascus ascospores PSN1705 PSN1871 PSN1899 fig. 3. — Main morphological features of PSN1303, PSN1705, PSN1871 and PSN1899. Scale bars: Perithecia on M0 + miscanthus, rosettes of asci, 250 µm; ascus, ascospores, 50 µm. 92 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) Silar P. et al. vs P. anserina S vs P. pauciseta CBS 237.71 vs P. comata T vs P. pseudocomata CBS 415.72 vs P. bella-mahoneyi CBS 112042 vs P. pseudoanserina CBS 253.71 vs P. pseudopauciseta CBS 253.71 fig. 4. — FungANI analysis of the genome of Podospora reunionensis Silar, sp. nov. strain PSN1158 with those of all type strains of the P. anserina (Rabenh.) Niessl species complex. The layouts are the same as on Figure 2. 93 The Podospora anserina species complex with description of a new species, Podospora reunionensis sp. nov. CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) separately; when possible, one ascus with four germinated ascospores was then selected, and the four thalli obtained from the four ascospores of this F0 generation were stored separately at –80°C (Silar 2020). DNA was extracted using a quick method adapted from plant (Bellstedt et al. 2010) and yeast (Liu et al. 2011) DNA extraction protocol. A 5 mm × 5 mm × 5 mm plug of agar taken from a plate onto which the fungus grew was put in a 1.5 mL Eppendorf tube along with 200 µL of a DNA extraction buffer that was made as follows. Firstly, 8 mL of 0.2 M stock solution of anhydrous sodium in water was freshly mixed with 17 mL of 0.2 M stock solution of sodium bicarbonate in water into 100 mL final volume of sterile water to make a 0.05 M carbonate buffer with pH = 9.6. The extraction buffer was then produced by mixing 96 mL of the carbonate/bicarbonate solution with 4 mL of 0.5 M of polyvinylpyrrolidone, 200 mg of bovine serum albumin fraction V (Sigma-Aldrich cat#A4503) and 50 µL of tween 20. The mycelium plug was then broken at speed 4.0 for 20 s in a TeSeE Precess 24 (Bio-Rad, Hercules, CA, United States), and the tubes were incubated at 95°C for 15 minutes. The tube was gently shaken with a finger and transferred on melting ice for one minute. The tube was then vortexed for 10 seconds and centrifuged at c. 20 000 g for 10-20 seconds in an Eppendorf centrifuge machine. The ITS (Intergenic Transcribed Spacer from the rDNA cluster) was amplified with the ITS5 and ITS4 or ITS1 and ITS5 primer pairs directly on 5 µL of supernatant. The PCR products were sent to Genewiz from AZENTA (Takeley, UK) for sequencing with ITS1 (or ITS5) and ITS4. Note that for Sordariales, ITS5 and ITS4 amplifications and sequencing with ITS5 gave more reliable results than ITS1 and ITS4 amplifications and ITS1 sequencing, respectively. Genome sequencinG of stRains fRom the P. anserina species complex. We sequenced the genome of mat1-1 (aka mat+) and mat1-2 (aka mat-) homokaryotic isolates. To obtain them, a self-fertile dikaryotic F0 thallus was used to produce homokaryotic selfsterile F1 progenies. These were confronted to each other to A1 D GH IJ EF A2 B1 M2 + Guibourtia demeusii M2 + miscanthusM2 B2 C1 C2 fig. 5. — Morphology of Podospora reunionensis Silar, sp. nov: A, mycelium morphology after eight days of incubation on M2; B, M0 + Guibourtia demeusii L. wood shavings; C, M0 + miscanthus; on the right are enlargements to see the presence/absence and positions of the perithecia; D, Perithecia on M0 + miscanthus; E, rosettes of asci; F, peridium showing a textura intricata; G, ascus; H, ascospores; I, cladorrhinum-like anamorph; J, spermatia. Scale bars: A-C, 1 cm; D, E, 250 µm; F-H, 50 µm; I, J, 10 µm. 100 CRYPTOGAMIE, MYCOLOGIE • 2025 • 46 (6) Silar P. et al. bhunJun c. s., chen y. J., phukhamsakDa c., boekhout t., GRoenewalD J. Z., mckenZie e. h. c., fRancisco e. c., fRisvaD J. c., GRoenewalD m., huRDeal v. 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