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Clavariaceae, Agaricales) in Poland Andrzej Szczepkowski; Warsaw

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Clavariaceae, Agaricales) in Poland Andrzej Szczepkowski; Warsaw Acta Mycologica Article ID: 5527 DOI: 10.5586/am.5527 ORIGINAL RESEARCH PAPER in ECOLOGY OF FUNGI Publication History Received: 2020-03-10 Morphological and Molecular Evidence for Accepted: 2020-08-04 Published: 2020-12-28 the Occurrence of Hodophilus variabilipes Handling Editor (Clavariaceae, Agaricales) in Poland Andrzej Szczepkowski; Warsaw University of Life Sciences – SGGW, Poland; 1 2 3 Piotr Chachuła ,Marek Halama , Bartosz Pencakowski https://orcid.org/0000-0002- 1Pieniny National Park, Poland 9778-9567 2Museum of Natural History, University of Wrocław, Poland , 3Department of Pharmaceutical Biotechnology, Wrocław Medical University, Poland Authors Contributions PC: feld research, specimen identifcation, photographic documentation, manuscript Abstract writing; MH: research idea, specimen identifcation, Hodophilus variabilipes, a species hitherto unknown in Poland, has been reported phylogenetic analysis, manuscript from a single locality in the Pieniny Mts (S Poland). Te frst studies on H. writing; BP: molecular variabilipes were those of macro- and microscopic features and molecular experiments, phylogenetic characteristics (ITS1-5.8S-ITS2 rDNA sequences). Here, a brief description and analysis, manuscript writing illustration of H. variabilipes based on Polish material are given, and the ecology Funding and characteristics distinguishing H. variabilipes from related species of the genus This research was fnanced with Hodophilus are briefy discussed. authors’ private funds and cofnanced by the statutory funds Keywords of the Museum of Natural History, Camarophyllopsis; Hygrotrama; ITS; phylogeny; DNA barcoding; Pieniny National University of Wrocław. Park Competing Interests No competing interests have been declared. 1. Introduction Copyright Notice Te genus Hodophilus R. Heim ex R. Heim was recently recovered by Birkebak et © The Author(s) 2020. This is an al. (2016) based on the polyphyly of agaricoid members of the family Clavariaceae. open access article distributed It can be distinguished from Camarophyllopsis Herink s. str. and Lamelloclavaria under the terms of the Creative Commons Attribution License, Birkebak & Adamčík by a hymeniderm pileipellis composed of broadly infated which permits redistribution, (globose, obpyriform to sphaero-pendunculate) terminal elements, with commercial and noncommercial, predominantly parietal pigment and the absence of clamps (Arnolds, 1986, 1990; provided that the article is Birkebak et al., 2016; Horak, 1968). Te genus Hodophilus, hitherto unknown in properly cited. Poland, is known to comprise approximately 26 species worldwide (Adamčík, Jančovičová, Looney, Adamčíková, Birkebak, et al., 2017; Adamčík, Jančovičová, Looney, Adamčíková, Grifth, et al., 2017; Adamčík et al., 2016, 2018, 2020; Crous et al., 2017), 15 of which are in Europe (Adamčík et al., 2018, 2020; Adamčík, Jančovičová, Looney, Adamčíková, Birkebak, et al., 2017; Adamčík, Jančovičová, Looney, Adamčíková, Grifth, et al., 2017). During feld mycological studies held in 2014 and subsequently in 2016 in a transitional area of vegetation between rupicolous calcareous grasslands (Origano- Brachypodietum laserpitietosum, Origano-Brachypodietum stachyetosum) and thermophilous beech forest (Carici albae-Fagetum) in the Pieniny Mts, the frst author found pale brown to grayish brown agaricoid fruiting bodies of an omphalioid habit. Laboratory studies (based on microscopy and sequencing) have shown that they are probably one of the most common species of the genus Hodophilus, that is, Hodophilus variabilipes Jančovičová, Adamčík & Looney, a fungus recently described from material from widely scattered localities in several European countries, including the Czech Republic, Denmark, France, the United Kingdom, Slovakia (Malé Karpaty Mts; holotypus), and Sweden (Adamčík, Jančovičová, Looney, Adamčíková, Grifth, et al., 2017). Te primary goals of the present study were to investigate and describe the frst collections of H. variabilipes from Poland using morphological and molecular characteristics. We used phylogenetic analysis of nuclear ribosomal DNA (ITS) Acta Mycologica / 2020 / Volume 55 / Issue 2 / Article 5527 Publisher: Polish Botanical Society 1 Chachuła et al. / Hodophilus variabilipes in Poland sequences to determine the phylogenetic position of the study material. We also aimed to provide new insight into the European distribution and ecology of this species. 2. Material and Methods 2.1. Morphological Studies Field photographs of fresh basidiomata were taken with the aid of a Panasonic Lumix DMC-FZ50 camera equipped with a Raynox DCR-250 Super Macro. Characteristics that change over time (color, smell, and texture of basidiomata) were noted in the feld. Te macroscopic description is based on both the study of fresh material and the analysis of photos. Microcharacters were observed with a light microscope equipped with a digital camera. All microscopic structures were observed in the dried material. Freehand sections of rehydrated basidiomata pieces were examined in squash preparations in 5% NH3·H2O, 5% KOH, 1% Phloxine B in 5% NH3·H2O, and Melzer’s reagent. Image-grabbing and biometric analyses were performed using the NIS-Elements D 3.1 imaging sofware. Te dimensions of the microcharacters are presented as follows: (minimum) 10–90 percentile values (maximum), average ± standard deviation. For basidiospores, randomly selected mature spores were measured without the hilar appendix. Te length of the basidia was measured excluding the sterigmata. Te following abbreviations are used: L = number of lamellae reaching the stipe, l = number of lamellulae between each pair of lamellae, and Q = the length-width ratio of basidiospores (mean value). Morphological terminology reported by Vellinga (Vellinga, 1988, 1990) and Arnolds (1990) was used. Te nomenclature of vascular plants followed the checklist provided by Mirek et al. (2002) and that of plant communities followed the checklist provided by Pancer-Koteja et al. (2004). Te nomenclature of fungi was based on the Index Fungorum (http://www.indexfungorum.org/). Te studied collections were deposited in the Museum of Natural History, University of Wrocław, Poland (WRSL). 2.2. Molecular Procedures 2.2.1. DNA Extraction, PCR Amplifcation, and DNA Sequencing Te ITS rDNA region (containing ITS1-5.8S-ITS2 sequences), which is commonly used for the separation and identifcation of agaric species (e.g., Adamčík, Jančovičová, Looney, Adamčíková, Grifth, et al., 2017; Borovička et al., 2012; Miller & Buyck, 2002; Schoch et al., 2012; Zhang et al., 2004), was used to confrm the taxonomic afliation of the collected material and to infer the evolutionary relationships between H. variablipes and other representative taxa in this study. Genomic DNA isolates were extracted from two dried specimens (ChP-2014-0007, ChP-2016-0004) using the CTAB procedure described by Murray and Tompson (1980). Spectrometric DNA concentration measurements were conducted using a NanoDrop ND 2000C (Termo Scientifc). Ten, DNA isolates were purifed with a Syngen DNA clean-up Kit (Syngen Biotech) to obtain good quality templates, and the DNA concentration was measured again. ITS rDNA regions were amplifed using the primers ITS1F and ITS4 or ITS1 and ITS4-BR (Gardes & Bruns, 1993; White et al., 1990). PCR was performed in a 20-µL volume with Q5 High-Fidelity DNA Polymerase (New England BioLabs) according to the manufacturer’s protocol. Sanger sequencing was carried out with the BrilliantDye Terminator v3.1 Kit (NimaGen). Reactions were set with fourfold dilution of the reaction premix and addition of BrilliantDye Terminator 5X Sequencing Bufer (NimaGen) according to the manufacturer’s instructions. Sequencing products were separated by capillary electrophoresis on an Applied Biosystems 310 Genetic Analyzer (Termo Fisher Scientifc). Two sequences were generated for this molecular and phylogenetic analysis, along with additional sequences obtained from NCBI GenBank (https://www.ncbi.nlm.nih.gov/). Acta Mycologica / 2020 / Volume 55 / Issue 2 / Article 5527 Publisher: Polish Botanical Society 2 Chachuła et al. / Hodophilus variabilipes in Poland 2.2.2. Sequence Alignment and Phylogenetic Analysis Sequence Scanner Sofware ver. 2.0 was used for basecalling, and peak quality was also checked using the Sanger Quality Check App (Termo Fisher Scientifc). Sequences were manually assembled in BioEdit Sequence Alignment Editor (Hall, 1999) and deposited in GenBank. All obtained sequences were compared with sequences stored in the GenBank database using the BLAST algorithm (Altschul, 1990). Based on the BLASTN results, sequences from foreign collections were selected according to the outcomes of recent phylogenetic studies on the most closely related members of Hodophilus (Adamčík, Jančovičová, Looney, Adamčíková, Grifth, et al. 2017). ITSx (Bengtsson-Palme, 2013), as implemented on the PlutoF web-based workbench (Abarenkov et al., 2010), was then used to cut of fanking gene fragments from all sequences assembled in a data matrix. Prior to the phylogenetic analyses, the ITS1-5.8S-ITS2 DNA sequences were aligned using MAFFT (Katoh et al., 2002, 2005) on MyHits gateway (http://myhits.isb- sib.ch/), with default conditions for gap openings and gap extension penalties and subsequently adjusted by eye in MEGA ver. 7.0 (Tamura et al., 2013). Te alignment was then entered into the program Gblocks ver. 0.91b (Castresana, 2000; Talavera & Castresana, 2007) to objectively eliminate poorly aligned positions and divergent regions
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