Accepted Manuscript Taxonomy and phylogeny of the Auriculariales (Agaricomycetes, Basidiomycota) with stereoid basidiocarps Vera Malysheva, Viacheslav Spirin PII: S1878-6146(17)30051-X DOI: 10.1016/j.funbio.2017.05.001 Reference: FUNBIO 813 To appear in: Fungal Biology Received Date: 27 August 2016 Revised Date: 6 April 2017 Accepted Date: 2 May 2017 Please cite this article as: Malysheva, V., Spirin, V., Taxonomy and phylogeny of the Auriculariales (Agaricomycetes, Basidiomycota) with stereoid basidiocarps, Fungal Biology (2017), doi: 10.1016/ j.funbio.2017.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Taxonomy and phylogeny of the Auriculariales (Agaricomycetes, Basidiomycota) with 2 stereoid basidiocarps 3 4 Vera Malysheva a & Viacheslav Spirin b, c* 5 6 a Komarov Botanical Institute, Russian Academy of Sciences, Popova 2, 197376 St. Petersburg, 7 Russia 8 9 b Botany Unit (Mycology), Finnish Museum of Natural History, P.O. Box 7, FI-00014 University of 10 Helsinki, Finland 11 12 c Natural History Museum, University of Oslo, P.O. Box 1172, Blindern, 0318 Oslo, Norway 13 14 *Corresponding author. Tel.: +358465918235, e-mail: [email protected] (V. Spirin) 15 16 Abstract 17 18 In the present study, we investigate taxonomy of the Auriculariales with effused or cupulate, 19 persistent basidiocarps; generic and species concepts are revised based on morphological and DNA 20 evidences. The genus Eichleriella is reinstated to embrace ten closely related species with ellipsoid- 21 ovoid basidia, and the genus type, E. incarnata , is placed to the synonyms of E. leucophaea . 22 Eichleriella bactriana , E. desertorum and E. sicca are described as new to science. In addition, four 23 species earlier treated as members of Exidiopsis or Heterochaete are combined to the genus. The 24 genus name Heteroradulum (type Radulum kmetii ) is introduced for seven species with large, 25 obconical, stipitate basidia. Of them, H. adnatum and H. semis are described as new. Two new 26 genera, Amphistereum (with two species, type Eichleriella schrenkii ) and Sclerotrema (monotypic, 27 type Exidiopsis griseobrunnea ), are proposed; HirneolinaMANUSCRIPT (monotypic, type H. hirneoloides ) and 28 Tremellochaete (with two species, type Exidia japonica ) are restored as good genera. The type 29 species of Heterochaete , H. andina , is congeneric with Exidiopsis (type E. effusa ). 30 31 Key words. Heterobasidiomycetes; internal transcribed spacer; perennial basidiocarps; wood- 32 inhabiting fungi 33 34 1. Introduction 35 36 The Auriculariales are an order of the Agaricomycetes (Basidiomycota) which has been introduced 37 as a result of morphological, cytological and phylogenetic studies (Bandoni 1984, Weiss & 38 Oberwinkler 2001, Hibbett et al. 2014). Alongside the so-called jelly fungi ( Auricularia, Exidia 39 etc.), it embraces a number of corticioid, hydnoid and poroid genera. Traditional generic division 40 within the order rested on macroscopic (the fructification type) and microscopic (type of basidia, 41 presence of sterile hymenial cells etc.) characters has not been revised so far although recent DNA- 42 based studies questionedACCEPTED a monophyly of some accepted genera (Weiss & Oberwinkler 2001, 43 Sotome et al. 2014). 44 45 The Auriculariales are wood-decomposers inhabiting various hosts, from the tropics to the subartic 46 zone. Some of them are able to survive under extreme climatic conditions, in particular, in arid 47 regions or dry habitats. Two main strategies to persist the drought occur in the order. The first one is 48 represented by genera Auricularia and Exidia : their gelatinous basidiocarps are able to dry out and 49 revive again during the raining season. This feature is not unique to the Auriculariales since 50 fructifications of the same kind are known in the Dacrymycetes and Tremellomycetes . The second ACCEPTED MANUSCRIPT 51 strategy is to develop steady, in some cases perennial fruitbodies, resuming their growth under 52 favourable conditions (the so-called stereoid basidiocarps in a case of non-poroid species). Species 53 with stereoid basidiocarps are widely distributed in many orders of the Agaricomycetes , although 54 they are certainly a minority in the Auriculariales . They have been conventionally addressed to 55 genera Eichleriella and Exidiopsis (Burt 1915, Wells 1961, Raitviir & Wells 1966, Wells & Raitviir 56 1980). No attempts to introduce phylogenetically reliable concepts of these genera have been 57 performed yet. Therefore, the aim of the present study is to re-evaluate species and genus concepts 58 of the stereoid Auriculariales , and define their main phylogenetic lineages. 59 60 61 2. Material and methods 62 63 Fungal collections . Type specimens and collections from herbaria H, LE, S, O, CFMR, TAAM, K, 64 FH, NY, PRM, CWU, TNS, PC, GB, TENN were studied. Herbarium acronyms are given 65 according to Thiers (2016). Morphological routine of this study follows Miettinen et al. (2012). In 66 all cases, 20 tramal hyphae, 20 basidia and 20–30 basidiospores per specimen were measured. For 67 presenting measurements, 5% extreme values from both ends of variation are given in parentheses. 68 The following abbreviations are used in descriptions below: L – mean spore length, W – mean 69 spore width, Q – L/W ratio, n – number of measurements per specimens; CB (+) means a presence 70 of moderate cyanophilous reaction in Cotton Blue. Specimens sequenced for this study are marked 71 by asterisk (*). 72 73 DNA extraction, PCR and sequencing . In total, 51 dried specimens from Europe, Africa, Asia and 74 North America were selected for molecular sampling (Table 1). DNA was extracted from small 75 fragments of dried basidiocarps. The procedure of DNA extraction completely corresponded to the 76 manufacturer’s protocol of the NucleoSpin Plant II Kit (Macherey-Nagel GmbH & Co. KG). 77 The following primers were used for both amplificationMANUSCRIPT and sequencing: ITS1F-ITS4 78 (http://www.biology.duke.edu/fungi/mycolab/primers. htm) for ITS region; primers JS1 (Landvik 79 1996) and LR5 (http://www.biology.duke.edu/fungi/mycolab/primers.htm) for LSU region. PCR 80 products were purified using the Fermentas Genomic DNA Purification Kit (Thermo Scientific, 81 Thermo Fisher Scientific Inc., MA, USA). 82 83 Sequencing was performed with an ABI model 3130 Genetic Analyzer (Applied Biosystems, CA, 84 USA) using the BigDye Terminator Cycle Sequencing Ready Reaction Kit. Raw data were 85 processed using MEGA 6 (Tamura et al. 2013). 86 87 Sequence alignment and phylogenetic analyses . In addition to 96 newly produced sequences (48 88 nrITS and 48 nrLSU), 23 nrITS and 35 nrLSU (including outgroups) sequences were retrieved from 89 GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) (Table 1). 90 Both nrITS and nrLSU sequences were aligned using the MAFFT online version 7 (Katoh & Toh 91 2008), with the Q-INS-i strategy (http://mafft.cbrc.jp/alignment/server/). For nrITS + nrLSU 92 phylogeny, ambiguouslyACCEPTED aligned regions in ITS dataset were identified and excluded from the 93 alignment for the subsequent analyses using TrimAl software (Capella-Gutierrez et al. 2009). In a 94 few cases, alignments were adjusted manually using MEGA 6. Two different datasets were 95 assembled for the phylogenetic analyses – nrITS (for Eichleriella and Heteroradulum spp.) and 96 nrITS + nrLSU (for major clades of the Auriculariales ). Alignments are deposited in TreeBASE: 97 S20135 (nrITS dataset) and S20134 (nrITS + nrLSU dataset). 98 99 Phylogenetic reconstructions have been performed for all datasets with Maximum Likelihood (ML), 100 Bayesian Inference (BI) and Maximum Parsimony (MP) analyses. Auricularia mesenterica ACCEPTED MANUSCRIPT 101 (voucher FO 25132) and Sistotrema brinkmannii (isolate 236) were used as outgroups (Table 1). 102 Before the analyses, the best-fit substitution models for the alignment were estimated for each 103 dataset based on Akaike Information Criterion (AIC) using FindModel web server 104 (http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). In both BI and ML analyses 105 for nrITS and nrITS + nrLSU datasets the model employed was GTR+G. ML analysis was run in 106 the PhyML server, v. 3.0 (http://www.atgc-montpellier.fr/phyml/), with one hundred rapid bootstrap 107 replicates. BI analysis was performed using MrBayes 3.1 (Ronquist & Huelsenbeck 2003) software 108 for two independent runs, each with 10 million generations with sampling every 100 generations, 109 under described model and four chains. MP analysis was performed using PAUP*4.0.b10 110 (Swofford 2002). One hundred heuristic searches were conducted by stepwise addition with random 111 sequence addition and tree bisection-reconnection (TBR) branchswapping algorithm. One tree was 112 held at each step during stepwise addition and the number of trees retained was limited to 100. 113 Parsimony bootstrap analysis was performed with 1000 replicates. Gaps were treated as missing 114 characters. The evolutionary pairwise distance between ITS sequences was assessed using MEGA 6 115 with GTR model. 116 117