Mycological Progress (2019) 18:1057–1069 https://doi.org/10.1007/s11557-019-01503-4 ORIGINAL ARTICLE Two new species of Eutypella and a new combination in the genus Peroneutypa (Diatrypaceae) Mehdi Mehrabi 1 & Bita Asgari 2 & Roghayeh Hemmati 1 Received: 26 April 2019 / Revised: 11 June 2019 / Accepted: 26 June 2019 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract We describe two new species of Eutypella, E. persica from dead branch of Alnus sp. in Guilan province and E. quercina from dead branch of Quercus sp. in East Azerbaijan province of Iran, using morphological and molecular data. Eutypella persica has large stromata with 20–70 perithecia in a valsoid arrangement, sulcate to smooth ostioles, and produces an asexual structure on both natural substrate and culture media. Eutypella quercina, lacking the asexual structure, is characterized by mostly circular stromata with 10–50 perithecia in a valsoid arrangement and smooth ostioles. Phylogenetic relationships of Eutypella with other genera of the Diatrypaceae are here inferred using maximum parsimony and neighbor-joining analyses of the ITS rDNA and partial β-tubulin gene. The new combination, Peroneutypa iranica, is proposed based on morphological features. Keywords Diatrypaceous fungi . Iran . Phylogeny . Taxonomy . Xylariales Introduction The family Diatrypaceae (Xylariales, Sordariomycetes), introduced by Nitschke (1869) with Diatrype Fries as the type genus, includes 16 genera and more than 1500 species (Maharachchikumbura et al. 2015; Dayarathne et al. 2016; Mehrabi et al. 2016; Shang et al. 2017). The perithecial ascomata embedded in a stroma, long stalked asci and allantoid ascospores are characteristic of the family (Glawe and Rogers 1984; Rappaz 1987). The anamorph genera Cytosporina Sacc., Libertella Desm. and Naemospora Sacc. have been linked to the Diatrypaceae (Glawe and Rogers 1984). Members of this family are common saprophytes or are often isolated as pathogens or endophytes and are cosmopolitan, associated with a very wide host range of woody plants (Glawe and Rogers 1984; Rappaz 1987; Trouillas and Section Editor: Marc Stadler * Mehdi Mehrabi mehrabimhd@yahoo.com 1 Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran 2 Department of Botany, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran Gubler 2010; Trouillas et al. 2011; Pitt et al. 2013; de Errasti et al. 2014; Liu et al. 2015). The genus Eutypella (Nitschke) Saccardo, a perithecial ascomycete belonging to the Diatrypaceae, was established by Saccardo (1875), based on E. cerviculata (Fr.) Sacc. as its type species (Saccardo 1882). Eutypella, segregated from Eutypa Tulasne & C. Tulasne by having collectively erumpent clusters of perithecial beaks (Glawe and Rogers 1984; Rappaz 1987; Vasilyeva and Stephenson 2006), is characterized by stromata in valsoid configuration, usually comprising host tissues or a mixture of host and fungal tissues, mostly sulcate, sometimes rounded ostioles, eight-spored asci and allantoid ascospores (Glawe and Rogers 1984; Vasilyeva and Stephenson 2006). According to Rappaz (1987), the number of perithecia per stromata; amyloidity of ascal apical ring; and the size of perithecia, ostioles, asci and ascospores are key characters for the delimitation of Eutypella species. The asexual morphs Libertella and Cytosporina have been assigned to Eutypella (Kirk et al. 2008; Glawe and Rogers 1982; Glawe and Rogers 1984). At present, 249 species of Eutypella are listed in Index Fungorum (2019), but only a few have sequence data. Some species formerly classified in Eutypella have already been transferred to Peroneutypa Berl. based on morphological characteristics and phylogenetic data (Acero et al. 2004; Carmarán et al. 2006; de Almeida et al. 2016; Shang et al. 2017). Peroneutypa was introduced by Berlese (1900) for fungi 1058 Table 1 Isolates used in the phylogenetic analysis Taxon CBS128335 CBS128339 IRAN 2346C JL567 IPV-FW349 BCRC33729 CBS 216.87 CBS 223.87 CBS 273.87 DSIERRA600 ATCC 52655 CBS 240.87 UCD779St IRAN 2281C UCDDCh400 CBS 205.87 IRAN 2347C CBS 212.87 DCASH200 CBS 271.87 ANM 1075 HUEFS 194228 ATCC 52484 IRAN 2280C IRAN 2344C ANM 1947 F-091,966 DHB500 CBS128327 CBS 622.84 CBS 292.87 F-091,961 CBS 210.87 CBS 291.87 CBS 291.87 DCA900 CBS 217.87 CBS 248.87 CBS 219.87 CBS 244.87 Host Ficus carica Vitis vinifera Citrus sp. Vitis vinifera Carpinus betulus ? Fraxinus excelsior Fraxinus excelsior Populus tremula Populus balsamifera subsp. trichocarpa Populus trichocarpa Salix borealis Vitis vinifera Juglans regia Salix lasiolepis Fagus sylvatica Alnus sp. Acer campestre Quercus sp. Betula sp. ? ? Acer sp. Quercus brantii Quercus brantii ? Quercus faginea Vitis vinifera Citrus paradisi Vitis vinifera Fraxinus excelsior Arundo donax Ulmus sp. Salix sp. Salix sp. Vitis vinifera Acer campestre Acer pseudoplatanus Acer pseudoplatanus Prunus spinosa Origin Australia Australia Iran Spain Italy Taiwan Switzerland Switzerland Switzerland USA USA Norway USA Iran USA Switzerland Iran Switzerland USA Switzerland USA Brazil Unknown Iran Iran USA Spain USA Australia Italy Switzerland Spain France Switzerland Switzerland USA France Switzerland Switzerland Switzerland GenBank accession numbers ITS β-Tubulin HQ692573 HQ692619 KR605649 JN975370 AM399021 JX507804 AJ302417 AJ302421 AJ302418 KT425184 KT425235 AJ302420 GQ293907 KJ767718 DQ006946 AJ302437 KR605644 AJ302433 GQ293947 AJ302436 KU320619 KM396615 AJ302441 KM245033 KR605648 KU320613 AJ302444 GQ293925 HQ692590 AJ302446 AJ302458 AJ302447 AJ302448 AJ302449 HM164737 HM164715 AJ302451 DQ006922 AJ302454 AJ302455 HQ692524 HQ692523 KY352428 JN975407 AM920693 AY951682 KT425165 KT425166 KT425168 KT425119 KT425170 KT425167 GQ293980 KY352426 DQ007002 – KY352434 – GQ294003 – – KR363998 – KY352429 KY352430 – – GQ293993 HQ692502 DQ006964 DQ006966 – – DQ006962 HM164771 HM164749 DQ006970 DQ006974 AY684198 DQ006958 Reference Trouillas et al. (2011) Trouillas et al. (2011) Mehrabi et al. (2016), this study Luque et al. (2012) Rocchi et al. (2010) Hsieh et al. (2005), Mirabolfathy et al. (2013) Acero et al. (2004), Trouillas et al. (2015) Acero et al. (2004), Trouillas et al. (2015) Acero et al. (2004), Trouillas et al. (2015) Trouillas et al. (2015) Trouillas et al. (2015) Acero et al. (2004), Trouillas et al. (2015) Trouillas et al. (2010b) Mehrabi et al. (2015), this study Rolshausen et al. (2006) Acero et al. (2004) Mehrabi et al. (2016), this study Acero et al. (2004) Trouillas et al. (2010b) Acero et al. (2004) de Almeida et al. (2016) de Almeida et al. (2016) Acero et al. (2004) Mehrabi et al. (2015), this study Mehrabi et al. (2016), this study de Almeida et al. (2016) Acero et al. (2004) Trouillas etal. (2010b) Trouillas et al. (2011) Acero et al. (2004), Rolshausen et al. (2006) Acero et al. (2004), Rolshausen et al. (2006) Acero et al. (2004) Acero et al. (2004) Acero et al. (2004), Rolshausen et al. (2006) Trouillas and Gubler (2010) Trouillas and Gubler (2010) Acero et al. (2004), Rolshausen et al. (2006) Rolshausen et al. (2006) Acero et al. (2004), Trouillas and Gubler (2004) Acero et al. (2004), Rolshausen et al. (2006) Mycol Progress (2019) 18:1057–1069 Allocryptovalsa cryptovalsoidea (T) Allocryptovalsa rabenhorstii Allocryptovalsa rabenhorstii Anthostoma decipiens Anthostoma decipiens Biscogniauxia latirima Cryptosphaeria eunomia var. eunomia Cryptosphaeria eunomia var. fraxini Cryptosphaeria ligniota Cryptosphaeria multicontinentalis (T) Cryptosphaeria pullmanensis Cryptosphaeria subcutanea Cryptovalsa ampelina Cryptovalsa ampelina Diatrype bullata Diatrype disciformis Diatrype disciformis Diatrype spilomea Diatrype stigma Diatrype undulata Diatrype virescens Diatrypella atlantica (T) Diatrypella frostii Diatrypella iranensis (T) Diatrypella macrospora (T) Diatrypella major Diatrypella quercina Diatrypella verruciformis Diatrypella vulgaris (T) Eutypa armeniacae Eutypa astroidea Eutypa consobrina Eutypa crustata Eutypa laevata Eutypa laevata Eutypa lata Eutypa lata var. aceris Eutypa lejoplaca Eutypa maura Eutypa petrakii var. petrakii Strain Taxon Eutypa petrakii var. petrakii Eutypa sparsa Eutypa tetragona Eutypella australiensis (T) Eutypella caricae Eutypella cearensis (T) Eutypella cerviculata Eutypella cerviculata Eutypella citricola Eutypella citricola Eutypella leprosa Eutypella microtheca (T) Eutypella parasitica Eutypella persica Eutypella prunastri Eutypella quercina Eutypella semicircularis (T) Eutypella vitis Eutypella vitis Halodiatrype avicenniae (T) Halodiatrype salinicola (T) Monosporascus cannonballus Monosporascus cannonballus (T) Peroneutypa alsophila Peroneutypa comosa Peroneutypa curvispora Peroneutypa diminutispora (T) Peroneutypa kochiana Peroneutypa scoparia Peroneutypa scoparia Quaternaria quaternata Quaternaria quaternata Xylaria berteroi Strain CBS 245.87 3802-3b CBS 284.87 CNP03 CBS 274.87 HUEFS 131070 CBS 221.87 M68 CBS128330 IRAN 2349C CBS 276.87 CBS128336 TO1/1 IRAN 2540C CBS 277.87 IRAN 2543C MP 4996 ATCC 64171 MSUELM13 MFLUCC15–0953 MFLUCC15–1277 CMM3646 ATCC 26931 CBS 250.87 BAFC 393 HUEFS 136877 HUEFS 192196 F-092,373 DFMAL100 IRAN 2345C CBS 278.87 IRAN 2348C YMJ 95101511 Host Salix borealis Populus sp. Sarothamnus scoparius Acacia longifolia subsp. sophorae Ficus carica Unidentified plant Alnus glutinosa Alnus glutinosa Vitis vinifera Salix sp. Tilia sp. Citrus paradisi ? Alnus sp. Prunus avium Quercus sp. Alnus acuminata Vitis labrusca Vitis vinifera Avicennia sp. Submerged marine wood Boerhavia sp. ? Arthrocnemum fruticosum ? Unidentified plant Unidentified plant Atriplex halimus Robinia pseudoacacia Gleditsia sp. Fagus sylvatica Fagus sp. ? Origin Norway Switzerland France Australia France Brazil Switzerland Latvia Australia Iran Switzerland Australia Slovenia Iran Switzerland Iran Panama USA USA Thailand Thailand Brazil Unknown France Argentina Brazil Brazil Spain France Iran Switzerland Iran Taiwan GenBank accession numbers ITS β-Tubulin AJ302456 AY684220 DQ006923 HM581945 AJ302460 KM396639 AJ302468 JF340269 HQ692579 KR605647 AJ302463 HQ692569 AM295770 KX828144 AJ302464 KX828139 JQ517314 AJ302466 DQ006943 KX573916 KX573915 JX971617 FJ430598 AJ302467 KF964568 KM396641 KM396647 AJ302462 GQ293962 KR605646 AJ302469 KR605645 KC473562 – AY684201 DQ006960 HQ692479 – – – – HQ692512 KY352439 – HQ692533 KY352451 – KY352449 – – DQ006999 KX573931 KX573932 – – – – – – – GQ294029 KY352452 – KY352464 KC473561 Reference Acero et al. (2004) Trouillas and Gubler (2004) Rolshausen et al. (2006) Trouillas et al. (2010a), Trouillas et al. (2011) Acero et al. (2004) de Almeida et al. (2016) Acero et al. (2004) Arhipova et al. (2012) Trouillas et al. (2011) Mehrabi et al. (2016), this study Acero et al. (2004) Trouillas et al. (2011) Piškur et al. (2007) This study Acero et al. (2004) This study Chacón et al. (2013) Acero et al. (2004) Rolshausen et al. (2006) Dayarathne et al. (2016) Dayarathne et al. (2016) Unpublished Unpublished Acero et al. (2004) Grassi et al. (2014) de Almeida et al. (2016) de Almeida et al. (2016) Acero et al. (2004) Trouillas et al. (2010b) Mehrabi et al. (2016), this study Acero et al. (2004) Mehrabi et al. (2016), this study Fu et al. (2013) Mycol Progress (2019) 18:1057–1069 Table 1 (continued) Sequences with bold numbers are generated in this study; others are from GenBank. (T) = ex-type strain. ATCC, American Type Culture Collection, Manassas, USA; BCRC, Bioresource Collection and Research Center, Food Industry Research and Development Institute, China; CBS, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CMM, Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes,” Federal Rural University of Pernambuco, Brazil; IRAN…C, Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; Others are not registered abbreviations 1059 1060 Mycol Progress (2019) 18:1057–1069 Diatrypella macrospora IRAN 2344C (T) Diatrypella iranensis IRAN 2280C (T) Diatrypella quercina F-091,966 Diatrype bullata UCDDCh400 100/100 54/-Clade A Diatrype spilomea CBS 212.87 Diatrype disciformis CBS 205.87 74/75 Diatrype disciformis IRAN 2347C Diatrype virescens ANM 1075 100/100 Diatrype undulata CBS 271.87 Diatrype stigma DCASH200 100/100 100/100 Cryptosphaeria eunomia var. eunomia CBS 216.87 Clade B Cryptosphaeria eunomia var. fraxini CBS 223.87 100/100 Eutypa armeniacae CBS 622.84 58/100 57/98 Eutypa lata DCA900 Eutypa laevata CBS 291.87 Eutypa laevata CBS 291.87 95/99 Clade C Eutypa lata var. aceri CBS 217.87 72/62 Eutypa petrakii var. petrakii CBS 244.87 96/72 63/-- Eutypa petrakii var. petrakii CBS 245.87 Eutypella caricae CBS 274.87 Diatrypella atlantica HUEFS 194228 (T) 100/60 Diatrypella frostii ATCC 52484 93/100 Diatrypella major ANM 1947 Clade D Diatrypella vulgaris CBS128327 (T) 93/100 Diatrypella verruciformis DHB500 Eutypella prunastri CBS 277.87 Eutypa lejoplaca CBS 248.87 Eutypa maura CBS 219.87 Eutypa sparsa 3802-3b Clade E 89/94 Eutypa crustata CBS 210.87 Eutypa astroidea CBS 292.87 92/93 Eutypa consobrina F-091,961 Eutypa tetragona CBS 284.87 100/100 Cryptosphaeria ligniota CBS 273.87 99/100 Cryptosphaeria multicontinentalis DSIERRA600 (T) Clade F Cryptosphaeria subcutanea CBS 240.87 100/100 Cryptosphaeria pullmanensis ATCC 52655 100/100 Anthostoma decipiens JL567 Clade G Anthostoma decipiens IPV-FW349 Eutypella cerviculata M68 100/100 97/100 82/-- 75/71 Eutypella cerviculata CBS 221.87 99/81 96/97 Eutypella quercina IRAN 2543C 98/100 Eutypella semicircularis MP 4996 (T) Clade H Eutypella persica IRAN 2540C 51/-Eutypella cearensis HUEFS 131070 (T) Eutypella parasitica TO1/1 Halodiatrype salinicola MFLUCC15–1277 (T) 100/100 Clade I Halodiatrype avcenniae MFLUCC15–0953 (T) Peroneutypa diminutispora HUEFS 192196 (T) 100/98 100/100 94/100 53/-Peroneutypa comosa BAFC 393 Peroneutypa kochiana F-092,373 Clade J 98/100 Peroneutypa scoparia IRAN 2345C Peroneutypa scoparia DFMAL100 Peroneutypa alsophila CBS 250.87 100/100 Peroneutypa curvispora HUEFS 136877 100/100 Allocryptovalsa rabenhorstii IRAN 2346C 100/100 Allocryptovalsa rabenhorstii CBS128339 100/100 Allocryptovalsa cryptovalsoidea CBS128335 (T) 99/100 62/-Eutypella microtheca CBS128336 (T) Eutypella vitis MSUELM13 Clade K Eutypella vitis ATCC 64171 92/94 Eutypella citricola CBS128330 94/73 99/100 Eutypella citricola IRAN 2349C Eutypella leprosa CBS 276.87 Eutypella australiensis CNP03 (T) 100/100 Cryptovalsa ampelina UCD779St Clade L Cryptovalsa ampelina IRAN 2281C 99/100 100/100 Quaternaria quaternata CBS 278.87 Clade M Quaternaria quaternata IRAN 2348C 100/100 Monosporascus cannonballus CMM3646 Clade N Monosporascus cannonballus ATCC 26931 (T) Biscogniauxia latirima BCRC33729 Xylaria berteroi YMJ 95101511 85/-61/-- 100/100 50.0 Mycol Progress (2019) 18:1057–1069 Consensus tree inferred from maximum parsimony analysis of the Diatrypaceae, based on combined dataset of ITS rDNA/β-tubulin. Bootstrap support values for maximum parsimony (MP, left) and neighbor joining (NJ, right) > 50% (1000 replicates) are shown above the nodes. Dashes replace non-significant values (< 50%). Two new proposed species are shown in bold. The tree is rooted to Biscogniauxia latirima and Xylaria berteri (Xylariaceae). (T) = ex-type strain ƒFig. 1 having perithecia with long prominent necks, small, clavate asci with truncated apices and allantoid ascospores (Saccardo 1905; Carmarán et al. 2006). Rappaz (1987) proposed P. bellula (Desm.) Berl. as the type species of Peroneutypa. Carmarán et al. (2006) then reinstated Peroneutypa based on morphological characteristics and phylogenetic data. Phylogenetic analyses have supported maintaining Peroneutypa as an independent genus within the Diatrypaceae (Acero et al. 2004; Trouillas et al. 2011; de Almeida et al. 2016; Dayarathne et al. 2016; Shang et al. 2017). Within the family Diatrypaceae, species delimitation is difficult due to the morphological similarity among species (Glawe and Rogers 1984; Rappaz 1987). Therefore, molecular analyses have been recently applied to elucidate the taxonomy and phylogeny of the Diatrypaceae, mostly based on ITS and β-tubulin sequence data (Acero et al. 2004; Carmarán et al. 2009; Trouillas et al. 2010a, 2010b, 2011; Luque et al. 2012; Chacón et al. 2013; Mehrabi et al. 2015; Dayarathne et al. 2016; de Almeida et al. 2016; Mehrabi et al. 2016; Shang et al. 2017). In an investigation of the family Diatrypaceae in Iran, 2013–2016, we found two specimens of Eutypella on dead branches of Alnus sp. and Quercus sp. Based on morphological and molecular data, combined sequence dataset of ITS rDNA, and partial β-tubulin gene, two new species of Eutypella are described. Peroneutypa iranica (Petr.) Mehrabi is also suggested as a new combination based on morphological examination of type material. 1061 used to check the amyloidity of asci after a 5% KOH pretreatment. Averages and standard deviations were calculated manually or using DinoCapture software. Photographs were taken under a Dino Capture 2.0 image software installed on a Olympus BH-2 microscope (Olympus, Tokyo, Japan). Macroscopic observations were carried out using an Olympus SZH stereo microscope. Holotypes are preserved at the Fungus Reference Collection (IRAN…F) of Herbarium Ministerii Iranici Agriculturae IRAN, Iranian Research Institute of Plant Protection (Tehran). Ex-type cultures are deposited at the Iranian Fungal Culture Collection (IRAN…C) of the IRAN Herbarium. DNA extraction, amplification, and sequencing DNA extraction was performed according to Liu et al. (2000) with an initial step of grinding the mycelia in liquid nitrogen. Potato dextrose broth (PDB) was used to produce mycelium for DNA extraction. The ITS region (ITS1-5.8S-ITS2) was amplified using primers ITS1 and ITS4 as described by White et al. (1990). Amplification of partial β-tubulin gene was achieved using primers Bt2a and Bt2b (Glass and Donaldson 1995). The PCR reaction (25 μL) contained 1 μL of each primer (10 pmol/μL, Takapouzist Inc.), 1.0 μL genomic DNA (30 ng/μL), 2.5 μL 10× high yield PCR buffer (Jena Bioscience, Germany), 0.3 μL Taq polymerase (5 units/ μL, Jena Bioscience, Germany), 1 μL MgCl2 (25 mM), 0.5 μL dNTP (10 mM), and 17.7 μL sterile distilled water. PCR amplification of both regions was carried out according to Mehrabi et al. (2016). The PCR products were purified using a multiscreen filter plate (Millipore Corp., Bedford, MA, USA) in Macrogen Company, South Korea. The purified DNA samples were then submitted for sequencing to a capillary sequencing machine (ABI Prism 3730XL, Applied Biosystem, Foster City, CA) of the same company. Materials and methods Sequences alignment and phylogenetic analyses Isolation and identification Two specimens of Eutypella were collected from East Azerbaijan and Guilan provinces of Iran. Type material for the new combination was retrieved from the Herbarium Ministerii Iranici Agriculturae “IRAN,” Iranian Research Institute of Plant Protection (Tehran). Pure cultures were obtained by a single-ascospore isolation as described by Trouillas et al. (2010b). Colony growth and characteristics were determined on Potato Dextrose Agar (PDA, Merck, Germany) incubated at 24 °C in the dark for 30 days. Colony colors were determined with the color charts of Rayner (1970). Measurements of microscopic features were taken from material mounted in cotton blue. The ascus dimensions include the spore-bearing part. Melzer’s reagent was New sequences generated in this study were checked with FinchTV v. 1.4.0 (Geospiza Inc.). The alignments were obtained using ClustalX version 2.0 (Thompson et al. 1997). Sequences of the ITS rDNA and β-tubulin were analyzed individually and in combination. Phylogenetic analyses were performed with neighbor joining (NJ) and maximum parsimony (MP) as implemented in PAUP* v4.0b10 (Swofford 2003). MP analysis was performed using the heuristic search option with tree bisection and reconnection (TBR) as the branch swapping algorithm and 1000 random sequence additions. Characters were equally weighted, and gaps were treated as missing data. Branches of zero length were collapsed and all parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated by 1000 bootstrap 1062 Mycol Progress (2019) 18:1057–1069 Mycol Progress (2019) 18:1057–1069 Eutypella persica (IRAN 16758F, holotype). a Stromata on dead branch of Alnus sp. b Ostioles. c, d Transverse section through stromata. e, f Longitudinal section through stromata. g Pycnidia on substrate with a light yellow stratum of spores. h Transverse section through pycnidia. i, j Peridium. k, l Asci. m, n Conidiophores and conidiogenous cells. o Ascospores. p Conidia. Bars = 5 mm (a), 500 μm (b), 1 mm (c–h), 20 μm (i, j), and 10 μm (k–p) ƒFig. 2 replications (Hillis and Bull 1993). Measures calculated for parsimony included tree length (TL), consistency index (CI), retention index (RI), homoplasy index (HI), and rescaled consistence index (RC). The NJ analysis was performed using Kimura-2-parameter nucleotide substitution model (Kimura 1980). All characters were unordered and of equal weight. Bootstrap values were obtained from 1000 NJ bootstrap replicates. Trees were drawn with FigTree v. 1.4.0 (Rambaut 2012). To determine whether the sequences for the two regions could be combined in one dataset, the partition homogeneity test (PHT) was applied from PAUP v4.0b10 (Swofford 2003). The sequences generated in this study were deposited in GenBank (Table 1). The finalized alignment and tree were deposited in TreeBASE, submission ID: 24352 (TreeBASE 2019). 1063 457 were constant, 104 parsimony uninformative, and 512 parsimony informative. Parsimony analysis resulted in 100 most parsimonious trees of 2671 steps with a CI of 0.435, RI of 0.718, HI of 0.565, and RC of 0.312. Analysis of the combined dataset provided higher support than the individual datasets and resolved the relationships among the Diatrypaceae. Members of the family Diatrypaceae, included in our phylogenetic analysis of the combined ITS rDNA and β-tubulin (Fig. 1), were divided into 15 well-supported clades (Fig. 1), giving a similar result to previous studies (Acero et al. 2004; Trouillas et al. 2011; de Almeida et al. 2016; Dayarathne et al. 2016; Shang et al. 2017; Senwanna et al. 2017). This analysis strongly supports the position of Eutypella persica and E. quercina as new species within the family Diatrypaceae, concordant with morphological traits. Our new Eutypella species were grouped together with high bootstrap support (98/100%) in a clade containing authentic strains of E. cerviculata and E. semicircularis S. Chacón & M. Piepenbr., occupying a sister relationship to a clade of Anthostoma decipiens (DC.) Nitschke with 97/100% bootstrap support. Taxonomy Results Phylogeny To clarify the relationships of the newly described species within the genus, we conducted phylogenetic analyses using sequences of the ITS region (466–630 bp) and β-tubulin (366–430 bp) individually (not shown) and combined (Fig. 1). The sequences generated in this study were aligned against all sequences of well-documented Eutypella species and other members of the Diatrypaceae including Allocryptovalsa Senwanna, Phookamsak & K.D. Hyde, Anthostoma Nitschke (Diatrypaceae), Cryptosphaeria Ces. & De Not., Cryptovalsa Ces. & De Not., Diatrype Fr., Diatrypella (Ces. & De Not.) De Not., Eutypa Tul. & C. Tul., Halodiatrype Dayar. & K.D. Hyde, Monosporascus Pollack & Uecker, Peroneutypa Berl., and Quaternaria Tul. & C. Tul. mostly from Acero et al. (2004), Trouillas et al. (2010a, 2010b, 2011, 2015), Mehrabi et al. (2015, 2016), and de Almeida et al. (2016) (Table 1). The tree was rooted with Biscogniauxia latirima Y.M. Ju & J.D. and Xylaria berteroi (Mont.) Cooke ex J.D. Rogers & Y.M. Ju, Xylariaceae (Fig. 1). A partition homogeneity test in PAUP 4.0b10 (Swofford 2003) did not show any significant divergence (P = 0.05), indicating that the individual datasets were congruent and produced trees with similar topology. Therefore, the two datasets were combined in a single analysis. The combined dataset of ITS rDNA and β-tubulin contained 1073 positions, of which Eutypella persica Mehrabi, Asgari & Hemmati, sp. nov. Fig. 2 MycoBank: MB 824056 Etymology. persicus, denoting the country of origin. Diagnosis: The species is distinguished by large stromata with groups of 20–70 perithecia arranged in a valsoid configuration, light brown to brown entostroma, and sulcate or smooth ostioles. Asci 8-spored, inamyloid, sporiferous parts 33–45 × 4.5–6.5 μm. Ascospores 5–7(–8) × 1.5–2.5 μm. Pycnidia immersed in the bark, hymenium labyrinthiform, with conidiogenous cells 7–16 × 1–2 μm and filiform conidia 17–23 × 0.9–1.2 μm. Stromata immersed in the bark of dead branches, 2–7 mm diam., erumpent, aggregated, circular to irregular in shape, blackening the periderm, surface black, rugose due to the necks of perithecia, surrounded by a black line in the host tissue, with groups of 20–70 (av. = 48) perithecia; entostroma light brown to brown. Perithecia 300–600 μm diam., compressed, black, monostichous to distichous, globose to subglobose, necks of the perithecia arranged in a valsoid configuration; ostioles black, opening separately, sulcate or smooth, periphysate, 50–100 μm diam. Peridium 30–50 μm thick, comprising several layers of cells of textura angularis; inner layer cells hyaline, outer layer cells brown to dark brown. Hamathecium composed of filiform, septate, hyaline paraphyses. Asci 33–45 × 4.5–6.5 μm (av. = 35 × 5.2 μm, n = 30), unitunicate, cylindrical, 8-spored, with rounded apex and stipe up to 25 μm long, apical rings inamyloid. Ascospores 5– 7(–8) × 1.5–2.5 μm (av. = 6 × 1.7 μm, n = 30), uniseriate to 1064 Mycol Progress (2019) 18:1057–1069 Mycol Progress (2019) 18:1057–1069 Eutypella quercina (IRAN 16761F, holotype). a Stromata on dead branches of Quercus sp. b, c Ostioles. d–f Transverse section through stromata. g, h Longitudinal section through stromata. i, j Peridium. k, l Asci. m Ascospores. Bars = 5 mm (a), 500 μm (b, c), 1 mm (d–h), 20 μm (i, j), and 10 μm (k–m) ƒFig. 3 irregularly arranged, sometimes agglomerated at the base of ascus, allantoid, hyaline, smooth, aseptate, usually with two oil droplets. Pycnidia immersed in the bark of dead branches, mostly at the edge of stromata, 1–3.5 mm diam., rare, irregular, delimited by a black line in host tissue; hymenium labyrinthiform, yellow to black, with light yellow stratum of conidiospores. Conidiophores 10–30(−40) × 1–2 μm, branched, hyaline, cylindrical. Conidiogenous cells 7–16 × 1–2 μm, proliferating percurrently, becoming inconspicuously annellate after repeated conidiogenesis, cylindrical, straight. Conidia 17–23 × 0.9–1.2 μm (av. = 18.8 × 1 μm, n = 30), hyaline, smooth, filiform, slightly curved to lunate, occasionally straight, aseptate. Cultural characteristics: Colonies on PDA reaching 75 mm after 9 days at 24 °C, circular to slightly irregular, cottony with moderately dense, fluffy aerial mycelium, at first white, becoming buff (45); reverse of the same color. On PDA, pycnidia produced after 3–4 weeks with the same morphology described on natural substrate. Material examined: IRAN, GUILAN: Rezvanshahr, on dead branches of Alnus sp., August 5, 2014, M. Mehrabi (IRAN 16758F, holotype), ex-type culture, IRAN 2540C. GenBank: ITS = KX828144; β-tubulin = KY352451. Notes: Eutypella persica is close to E. quercina by having erumpent stromata with groups of more than 10 perithecia, light brown to brown entostroma, and similar asci and ascospores size. However, it is distinguished from E. quercina by having larger stromata with more perithecia on average, sulcate ostioles, asci with shorter stipes and producing asexual structure on both natural substrates and culture media. Our phylogenetic analyses also support E. persica as distinct from E. quercina. Other similar species to E. persica are E. cerviculata (the type species), E. durieui (Mont.) Berl., and E. semicircularis. Eutypella cerviculata is distinguished from E. persica by having amyloid asci and more sulcate ostioles, 200–300 μm diam. vs. 50–100 μm (Rappaz 1987). Eutypella durieui is differentiated from E. persica by occurring on Quercus spp. and having amyloid asci and ostioles measuring 250–350 μm diam. (Rappaz 1987). Eutypella semicircularis differs from E. persica in having urn-shape and wider asci (10–15 vs. 5– 7 μm) with longer basal parts (up to 130 μm) and semicircular ascospores. A comparison of the ITS region DNA sequence data between E. persica, E. cerviculata and E. semicircularis also revealed base pair differences of 5% and 4% respectively which support establishment of E. persica as a new taxon. 1065 Eutypella atropae (Durieu & Mont.) Sacc. (on Atropa frutescens L.), E. theobromicola Wakef. (on Theobroma cacao L.), E. parasitica R.W. Davidson & R.C. Lorenz (on Acer rubrum L.), E. prunastri (Pers.) Sacc. (on Prunus avium L.), E. padina (Nitschke) Nannf. (on Prunus padus L.), and E. sorbi (Alb. & Schwein.) Sacc. (on Sorbus aucuparia L.) are other species that morphologically resemble the new species, E. persica (Rappaz 1987). All these species except E. parasitica are distinguished from E. persica by having smaller asci and wider ostioles. Eutypella parasitica is mainly characterized by brown and large ascospores, 6.8–11.2 × 2.2– 2.5(–2.8) μm. Eutypella quercina Mehrabi, Asgari & Hemmati, sp. nov. Fig. 3. MycoBank: MB 824057 Etymology. Named after the host genus from which the holotype was collected. Diagnosis: The species is distinguished by stromata with groups of 10–55 perithecia in a valsoid arrangement, white to light brown entostroma and smooth ostioles. Asci 8-spored, inamyloid, sporiferous parts 34–47 × 4.5–6 μm. Ascospores 5–7(−8) × 1.5–2.3 μm. Asexual structure undetermined. Stromata immersed in the bark, 1.5–5 mm diam., erumpent, scattered, mostly circular to irregular in shape, surface black, delineated by a black line in the host tissue, with groups of 10–55 (av. = 22) perithecia; entostroma white to light brown. Perithecia 300–650 μm diam., black, monostichous, globose to ovoid, compressed, in valsoid arrangement; ostioles black, prominent, opening separately, not sulcate, periphysate, 50–120 μm diam. Peridium 30–60 μm thick, comprising several layers of cells of textura angularis; inner layer cells hyaline, outer layer cells brown to dark. Hamathecium composed of filiform, septate, hyaline paraphyses. Asci 34–47 × 4.5–6 μm (av = 37 × 5.1 μm, n = 30), unitunicate, subcylindrical, 8-spored, stipes up to 40 μm long, apical rings inamyloid. Ascospores 5–7(−8) × 1.5–2.3 μm (av. = 6.2 × 1.9 μm, n = 30), uniseriate to irregularly arranged, allantoid, subhyaline, smooth, yellowish in mass, aseptate, usually with two oil droplets. Asexual structure not seen. Cultural characteristics: Colonies on PDA reaching 80 mm after 9 days at 24 °C, irregular, cottony with moderately dense, fluffy aerial mycelium, at first white, primrose (66) at reverse, becoming citrine (13) with smoke gray (105) at central part, buff (45) at reverse. Material examined: IRAN, EASTAZERBAIJAN: Aghoyeh, on dead branches of Quercus sp., July 11, 2015, M. Mehrabi (IRAN 16761F, holotype), ex-type culture, IRAN 2543C. GenBank: ITS = KX828139; β-tubulin = KY352449. Notes: Our phylogenetic analyses indicated that E. quercina is closely related to E. semicircularis and E. cerviculata (Fig. 1). Eutypella quercina resembles E. semicircularis and E. cerviculata by stromata 1066 Mycol Progress (2019) 18:1057–1069 Mycol Progress (2019) 18:1057–1069 1067 Peroneutypa iranica (IRAN 1333F, holotype). a Herbarium material. b Stromata on branch of Wisteria sinensis. c, d Ostioles. e, f Transverse section through stromata. g, h Longitudinal section through stromata. i, j Peridium. k–m. Asci. n Ascospores. Bars = 1 mm (b, e–h), 500 μm (c, d), 20 μm (i, j), and 10 μm (k–n) Peroneutypa iranica differs from all these species by having sulcate ostioles and by the size of asci and ascospores. characteristics (i.e., color, size, and number of perithecia) and ascospore size. However, E. semicircularis, occurring on Alnus acuminata Kunth, is differentiated from E. quercina by having sulcate ostioles, urn-shaped asci with longer stipe (up to 130 μm) and wider spore-bearing part (10–15 vs. 5– 7 μm), and strongly curved ascospores (Chacón et al. 2013). Eutypella cerviculata also differs from E. quercina by having sulcate and wider ostioles and amyloid asci (Rappaz 1987). We compared our new taxon with E. semicircularis and E. cerviculata based on base pair differences, and there are 2% and 4% differences, respectively, in ITS region. There are no β-tubulin sequence data in GenBank for these two species to compare their phylogenetic relationship to our new species. Members of the family Diatrypaceae, included in our phylogenetic analysis of the combined ITS rDNA and β-tubulin (Fig. 1), were divided into 14 well-supported clades (Fig. 1), giving a similar result to previous studies (Acero et al. 2004; Trouillas et al. 2011; de Almeida et al. 2016; Dayarathne et al. 2016; Shang et al. 2017; Senwanna et al. 2017). The results obtained by this study support the polyphyly of the genus Eutypella as is its members fell into four separate clades (D, E, H, K). Two authentic strains of the type species of Eutypella, E. cerviculata, together with ex-type strains of the newly described species, E. quercina and E. persica, together with E. semicircularis were grouped to form the highly supported clade ‘H’ (98/100%). This topology strongly supports the inclusion of our new species within the genus Eutypella. Eutypella cearensis and E. parasitica occupied a basal position to the clade ‘H’. The former species is differentiated from our two new species by having asci with smaller spore-bearing part (17–32 μm length) and longer stipe (de Almeida et al. 2016). Two authentic strains of Anthostoma decipiens, the type species of Anthostoma, were included in the clade ‘G’, occupying a sister relationship to the clade ‘H’ with high bootstrap support (97/100%). Nitschke (1867) erected the genus Anthostoma based on A. decipiens (DC.) Nitschke. This genus comprises 180 species (Index Fungorum 2019), from which some have been transferred to the Xylariaceae under the genera Lopadostoma (Nitschke) Traverso, Anthostomella and Biscogniauxia Kuntze. Rappaz (1992, 1993) examined A. decipiens based on its sexual and asexual morphs and concluded that this fungus belongs to the Diatrypaceae as a distinct genus close to Eutypella. Later, Læssøe and Spooner (1994) combined A. decipiens in Cryptosphaeria. Our phylogenetic analysis is concordant with Rappaz (1992, 1993) and other authors who accepted Anthostoma as a separate genus within the Diatrypaceae (Dayarathne et al. 2016; Shang et al. 2017). According to Dayarathne et al. (2016), this genus needs epitypifying. Clade ‘K’, consisting of Eutypella sensu lato and species of Allocryptovalsa Senwanna, Phookamsak & K.D. Hyde, is a well-supported clade, taking a basal position to the main clade accommodating all other Eutypella species (Fig. 1). Allocryptovalsa was recently described by Senwanna et al. (2017), based on A. polyspora C. Senwanna, Phookamsak & K.D. Hyde as its type species. The genus Allocryptovalsa is mainly distinguished from Eutypella by having polysporous asci (Senwanna et al. 2017). Although E. microtheca Trouillas, W.M. Pitt & Gubler is grouped within the strongly supported clade (100/100) including Allocryptovalsa, it differs from Allocryptovalsa species by possessing 8-spored asci. ƒFig. 4 Peroneutypa iranica (Petr.) Mehrabi, comb. nov. Fig. 4. MycoBank: MB 819437 Basionym: Eutypella iranica Petr., Sydowia 18: 366 (1965). Stromata immersed in the bark, 0.5–2 mm diam., erumpent, scattered, surrounded by a thin, black line in the host tissue, with groups of 3–16 perithecia, only neck visible protruding through host cortex, entostroma whitish to light brown. Perithecia 300–500 μm diam., black, monostichous, globose, in valsoid arrangement; ostioles erumpent, emerging in groups, sulcate, periphysate, 40–120 μm diam. Peridium 30–55 μm thick, comprising several layers of cells of textura angularis; inner layer cells hyaline, outer layer cells brown to dark. Hamathecium composed of filiform, septate, hyaline paraphyses. Asci 10–16 × 3–4.2 μm (av = 13.5 × 3.6 μm, n = 30), unitunicate, urn-shaped, 8-spored, stipes up to 14 μm long, apical rings amyloid. Ascospores 3–4 × 1–1.2 μm (av = 3.6 × 1 μm, n = 30), allantoid, hyaline, smooth, aseptate, usually with 1–2 oil droplets. Asexual structure not seen. Material examined: IRAN, GUILAN: Bandar-e Anzali, on branch of Wisteria sinensis, December 20, 1963, E. Nesbat (IRAN 1333F, holotype). Notes: The ex-type culture of this species could not be purified. Attempts to extract DNA from the holotype specimen were also not successful. Therefore, this species was not included in our phylogenetic analysis. However, morphological features of its asci and ascospores finely match with that of Eutypella iranica (Petrak 1965). The new combination, Peroneutypa iranica, is proposed here based on morphological features of the genus Peroneutypa Berl. that is mainly characterized by urn-shaped and small asci and allantoid ascospores (Carmarán et al. 2006). Shang et al. (2018) provided a key to identification of all 13 species of the genus Peroneutypa. Discussion 1068 Other members of the clade ‘K’, i.e., E. citricola Speg., E. vitis (Schwein.) Ellis & Everh., E. leprosa (Pers.) Berl. and E. australiensis Trouillas, Sosnowski & Gubler form unresolved subclades, distinctly far from the type species, E. cerviculata. This suggests the belonging of all these Eutypella species to some novel genera as previously discussed by some authors (Trouillas et al. 2011; Dayarathne et al. 2016; de Almeida et al. 2016; Shang et al. 2017). Taxonomic position of the genera Eutypella, Eutypa, Diatrypella, and Diatrype within the family Diatrypaceae is unresolved based on this study and previous analyses (Acero et al. 2004; Trouillas et al. 2011; de Almeida et al. 2016). Insufficient sequence data together with lack of the ex-type strains of many important genera such as Quaternaria, Peroneutypa, and Cryptovalsa are two major challenges in phylogenetic studies of the Diatrypaceae. In this study, we provide comprehensive phylogeny of Eutypella performed to date. To evaluate the taxonomic structure of the family Diatrypaceae, a larger number of taxa must be examined using a broader molecular array. Acknowledgments The Research Institute of Modern Biological Techniques (University of Zanjan) is acknowledged for providing the laboratory equipment and facilities. The authors thank Dr. C.C. Carmarán, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires for her valuable comments on a new combination proposed in this paper. 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