Mycosphere 6 (3): 385–400(2015) www.mycosphere.org Copyright © 2015 ISSN 2077 7019 Article Mycosphere Online Edition Doi 10.5943/mycosphere/6/3/12 Towards a backbone tree for Seimatosporium, with S. physocarpi sp. nov. Chada Norphanphoun1,2, Sajeewa S. N. Maharachchikumbura3, Dinushani A. Daranagama2,5,Timur S. Bulgakov6, Darbhe J. Bhat7,8, Ali H. Bahkali9, Kevin D. Hyde1,2 ,4,8 1 Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, China 2 Institute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand 3 Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Xiaohe District, Guiyang City, Guizhou Province 550006, People’s Republic of China 4 World Agroforestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, China 5 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No 3 1st West Beichen Road, Chaoyang District Beijing 100101, People’s Republic of China 6 Academy of Biology and Biotechnology, Southern Federal University, Rostov-on-Don 344090, Rostov region, Russia 7 Formerly Department of Botany, Goa University, Goa, India 8 No. 128/1–J, Azad Housing Society, Curca, Goa Velha, India 9 Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451,Kingdom of Saudi Arabia Norphanphoun C, Maharachchikumbura SSN, Daranagama A, Bulgakov TS, Bhat DJ, Bahkali AH, Hyde KD 2015 – Towards a backbone tree for Seimatosporium, with S. physocarpi sp. nov. Mycosphere 6(3), 385–400, doi 10.5943/mycosphere/6/3/12 Abstract The genus Seimatosporium is saprobic or pathogenic on plants, and are ‘pestalotioid fungi’. The genus presently belongs in Discosiaceae (Amphisphaeriales) and includes 78 species epithets. In this study, we observed three specimens of Seimatosporium from Russia and they are characterized by morphological and sequence data. We analyzed combined ITS and LSU gene sequence data of 42 species representing the genera Discostroma (7), Sarcostroma (2) and Seimatosporium (32, including the three new strains) with Pseudopestalotiopsis theae as the outgroup taxon. One isolate from dead branches of Physocarpus opulifolius is unique and is introduced as Seimatosporium physocarpi sp. nov., in this paper. It can be distinguished from similar and related species by phenotypic conidial characters and phylogenetic analyses. A specimen from Rosa kalmiussica Chrshan. & Lasebna (often included in Rosa canina sensu lato) is designated as an epitype for S. rosae, the type of the genus. In addition, a collection of S. lichenicola is described, illustrated and compared with other species in the genus. Key words – Discosiaceae – new species – phylogeny – epitype – combine gene – taxonomy. Introduction The genus Seimatosporium was introduced by Corda (1833), with S. rosae Corda as the type species. This genus is presently placed in the family the Discosiaceae, Amphisphaeriales (Senanayake et al. 2015). The sexual morph of Seimatosporium has been identified as Discostroma Submitted 29 February 2015, Accepted 31 May 2015, Published online 30 June 2015 Corresponding Author: Kevin D. Hyde – e-mail – kdhyde3@gmail.com 385 (Hyde et al. 2011) and are known as ‘pestalotioid fungi’ (Nag Raj 1993). Seimatosporium is a relatively well-known saprobic and plant pathogenic coelomycetous taxon (Sutton 1980, Tanaka et al. 2011). Based on their conidial features, Nag Raj (1993) provided an account of Seimatosporium species and rearranged them into five natural groupings under extant generic names. These are Seimatosporium sensu stricto (S. botan Sat. Hatak. & Y. Harada), Sporocadus (with S. lichenicola (Corda) Shoemaker & E. Müll., the generic type), Sarcostroma (with S. foliicola (Berk.) Shoemaker, as type of the genus), Diploceras (with S. hypericinum (Ces.) B. Sutton, as type of the genus), and Vermisporium (with S. leptospermi R.G. Bagn. & Sheridan). Tanaka et al. (2011) restudied Diploceras, Sporocadus, Sarcostroma, Diploceras, and Vermisporium, which lack sexual morphs, and concluded that they should be placed under the genus Seimatosporium, based on a monophyletic grouping in LSU and ITS phylogenetic analyses. This finding was similar to the large diversity of Seimatosporium species as treated in Sutton (1980). According to Index Fungorum (2015) Seimatosporium comprises 78 species. The characteristic features of the genus are conidia with pigmented median cells, with or without an apical appendage, and single, branched or unbranched basal appendage (Nag Raj 1993). In the present study, we report on three specimens of Seimatosporium, collected in Russia. With evidence from morphological and phylogenetic analyses we introduce a new species S. physocarpi; designate an epitype for S. rosae, and provide a detailed description for S. lichenicola which is the asexual morph of Discostroma fuscellum. Material & Methods Sample collection and specimen examination Samples were collected during May 2014 from the Rostov region of European Russia and returned to the laboratory in small paper bags. Specimens were examined under a Motic SMZ 168 dissecting microscope for fungal fruiting bodies. Hand sections of the fruiting structures were mounted in water for microscopic studies. The fungi were examined under a Nikon Ni compound microscope and photographed using a Canon EOS 600D digital camera fitted to the microscope. The images were processed with Adobe Photoshop CS5 Extended version 12.0 software (Adobe Systems Inc., The United States) and the Tarosoft (R) Image Frame Work program v. 0.9.7 was used for measurements. Single spore isolation was followed the method detailed in Chomnunti et al. (2014) and the germinating spores were transferred aseptically to malt extract agar (MEA) plates and incubated at 18 °C. Colony characters were observed and measured after a week and one month. The herbarium materials are deposited in the Mae Fah Luang University Herbarium, Chiang Rai, Thailand (MFLU) and New Zealand Fungal Herbarium, New Zealand (PDD) and living cultures deposited at Mae Fah Luang University Culture Collection (MFLUCC) and Fungal Biodiversity Centre (CBS). Facesoffungi numbers and Index Fungorum numbers are provided as detailed in Jayasiri et al. (2015) and Index Fungorum (2015). DNA extraction, PCR amplification and sequencing Genomic DNA were extracted from the growing mycelium on PDA, following the methods of Telle & Thines (2011). Polymerase chain reactions (PCR) were carried out using primer pairs of ITS5 and ITS4 to amplify the internal transcribed spacers (ITS) (White et al. 1990), and large subunit rDNA (LSU) was amplified using primer pairs of LROR and LR5 (Vilgalys & Hester 1990). The amplification reaction was performed in a 50 μl reaction volume containing, 5–10 ng DNA, 0.8 units Taq polymerase, 1×PCR buffer, 0.2 mM dNTP, 0.3 µm of each primer with 1.5 mM MgCl2 (Cai et al. 2009). Amplification protocols were followed according to Maharachchikumbura et al. (2013, 2014). The PCR products were visualized on 1% agarose gel stained with ethidium bromide. Purification and sequencing of PCR product were carried at Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (China). Sequences derived from this study were deposited in GenBank. 386 Phylogenetic analysis Blast searches were made to identify the closest matches in GenBank (Table 1) and recently published sequences. Combined analysis of LSU and ITS sequences of 39 taxa of Discostroma (7), Sarcostroma (2) and Seimatosporium (29) were used to confirm the phylogenetic placement of our three strains, with Pseudopestalotiopsis theae (MFLUCC 12−0055) as the outgroup taxon. Sequence data were optimized manually to allow best alignment and maximum sequence similarity as detailed in Maharachchikumbura et al. (2012). A maximum parsimony analysis (MP) was performed using PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2002). Ambiguously aligned regions were excluded and gaps were treated as missing data. The trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Maxtrees were setup to 5000, branches of zero length were collapsed and all multiple parsimonious trees were saved. Descriptive tree statistics for parsimony Tree Length [TL], Consistency Index [CI], Retention Index [RI], Relative Consistency Index [RC] and Homoplasy Index [HI] were calculated for the Maximum Parsimonious Tree (MPT). The robustness of the most parsimonious trees was evaluated by 100 bootstrap replications resulting from maximum parsimony analysis, each with ten replicates of random stepwise addition of taxa (Felsenstein 1985).The Kishino-Hasegawa tests (KHT) (Kishino & Hasegawa 1989) were performed to determine whether the trees were significantly different. Trees were viewed in TreeView v.1.6.6 (Page 1996) In addition, Bayesian Analyses (BA) were performed using MrBayes 3.2.0 (MrBayes v. 3.2.1; Ronquist et al. 2012). Suitable models for the Bayesian analysis were first selected using models of nucleotide substitution for each gene, as determined using MrModeltest v. 2.2 (Nylander 2004), and included for each gene partition. The analyses of four Markov Chain Monte Carlo (MCMC) chains were run from random trees for 1,000,000 generations and sampled every 100 generations. The temperature value was lowered to 0.15, burn-in was set to 0.10, and the run was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01.The resulting trees were printed with FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). Table 1 GenBank Accession numbers of the sequences used in phylogenetic analysis Taxon name Strain number Discostroma fuscellum (Berk. & Broome) Huhndorf NBRC 32625 NBRC 32680 10071 418-BB HKUCC 1004 NBRC 32690 NBRC 32626 GenBank Acc. No. LSU ITS AB593726 AB594794 AB593739 AB594806 − AF377284 − GU244511 AF382380 − AB593729 AB594797 AB593727 AB594795 MFLUCC 12−0055 KM116282 JQ683727 CBS 122695 CBS 118153 CBS 118154 − DQ278925 DQ278924 EU552155 DQ278923 DQ278922 CPC 13584 JN871208 JN871199 H4619 H4621 KACC 42490 KACC 42491 NBRC 32674 CPC 156 / CBS 115131 CPC 157 / CBS 110733 CPC 158 / CBS 110734 CPC 159 / CBS 114876 CPC 13580 CPC 12992 CPC 13578 NBRC 32676 AB593731 AB593732 − − AB593733 JN871209 JN871210 JN871211 JN871212 JN871214 − JN871213 AB593734 AB594799 AB594800 EF600969 EF600970 AB594801 JN871200 JN871201 − JN871202 JN871205 JN871203 JN871204 AB594802 Discostroma sp. Discostroma stoneae (H.J. Swart) Sivan. Discostroma tostum (Berk. & Broome) Brockmann Pseudopestalotiopsis theae (Sawada) Maharachch., K.D. Hyde & Crous Sarcostroma bisetulatum (Guba) Nag Raj Sarcostroma restionis S.J. Lee & Crous Seimatosporium biseptatum (H.J. Swart & M.A. Will.) P.A. Barber & Crous Seimatosporium botan Sat. Hatak. & Y. Harada Seimatosporium discosioides (Ellis & Everh.) Shoemaker Seimatosporium elegans H.J. Swart Seimatosporium eucalypti (McAlpine) H.J. Swart Seimatosporium falcatum (B. Sutton) Shoemaker Seimatosporium foliicola (Berk.) Shoemaker 387 Taxon name Seimatosporium glandigenum (Bubák & Gonz. Frag.) B. Sutton Seimatosporium grevilleae (Loos) Shoemaker Seimatosporium hakeae (B. Sutton) Shoemaker Seimatosporium hypericinum (Ces.) B. Sutton Seimatosporium kriegerianum (Bres.) Morgan-Jones & B. Sutton Seimatosporium leptospermi R.G. Bagn. & Sheridan Seimatosporium lichenicola (Corda) Shoemaker & E. Müll. Seimatosporium mariae (Clinton) Shoemaker Seimatosporium obtusum (H.J. Swart & M.A. Will.) P.A. Barber & Crous Seimatosporium parasiticum (Dearn. & House) Shoemaker Seimatosporium physocarpi C. Norphanphoun, Bulgakov & K.D. Hyde Seimatosporium pistaciae Crous & Mirab. Seimatosporium rosae Corda Seimatosporium sp. Seimatosporium vaccinii (Fuckel) B. Erikss. Seimatosporium walkeri (H.J. Swart & M.A. Will.) P.A. Barber & Crous Strain number GenBank Acc. No. LSU ITS NBRC 32677 AB593735 AB594803 ICMP 10981 AF382372 AF405304 NBRC 32678 AB593736 AB594804 NBRC 32647 AB593737 AB594805 NBRC 32679 AB593738 − ICMP 11845 MFLUCC 14−0623 NBRC 32681 AF382373 KT198725 AB593740 − KT198724 AB594807 CPC 12935 JN871215 JN871206 NBRC 32682 AB593741 AB594808 MFLUCC 14−0625 KT198723 KT198722 KP004491 KP004463 KP004492 KT198727 AF382375 AF382374 KP004464 KT198726 − − JN871216 JN871207 CPC 24455 / CBS 138865 CPC 24457 MFLUCC 14−0621 HKUCC 7986 ICMP 7003 CPC 17644 CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; H: Finnish Museum of Natural History (University of Helsinki), HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; KACC: Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology, Korea; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; NBRC: Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan; LSU: large subunit (28S) of the nrRNA gene operon; ITS: internal transcribed spacers and intervening 5.8S nrDNA. Results Phylogenetic analyses The combined LSU and ITS alignment was used to resolve the species relationships in Seimatosporium. The phylogenetic analyses were obtained from maximum parsimony (MP) and Bayesian analyses. The alignment comprised 42 taxa and 1300 total characters including gaps. Parsimony analyses indicate that 1140 characters were constant, 73 variable characters were parsimony-uninformative and 87 characters were parsimony informative. The parsimony analysis of the data matrix resulted in two equally parsimonious trees and in the first tree (TL= 288, CI=0.670, RI = 0.814, RC = 0.546, HI = 0.330) is shown here. The Bayesian analysis resulted in a tree with the same topology as the MP trees. The phylogenetic results from Fig. 1 are discussed in the descriptive notes and discussion below. Taxonomy Seimatosporium lichenicola (Corda) Shoemaker & E. Müll., Can. J. Bot. 42: 405 (1964) Fig. 2 Faces of fungi numbers: FoF00793 Asexual morph – Discostroma fuscellum (Berk. & Broome) Brockmann 1976 Saprobic and parasitic on dying twigs, leaf stalks and leaves, sometimes on living leaves of Cotinus coggygria Scop. Sexual morph: Undetermined. Asexual morph: Conidiomata 400–600 µm diam. pycnidial, solitary, immersed, unilocular, brown, with a conspicuous apex. Pycnidial wall (30–50 µm) multilayered, comprised of light brown-walled cells of textura angularis, with inner most layer thin, hyaline. Conidiophores reduced to conidiogenous cells. Conidiogenous cells holoblastic, phialidic, hyaline, smooth, formed from the inner most layer of pycnidium wall. Conidia (10–)12– 388 Sarcostroma bisetulatum CBS 122695 ITS Seimatosporium hakeae NBRC 32678 Sarcostroma restionis CBS 118154 56/1 Sarcostroma restionis CBS 118153 Seimatosporium grevilleae ICMP 10981 –/0.98 62/– Seimatosporium leptospermi ICMP 11845 LSU Seimatosporium mariae NBRC 32681 86/1 Discostroma stoneae NBRC 32690 84/0.98 Seimatosporium kriegerianum NBRC 32679 LSU –/– Seimatosporium elegans NBRC 32674 Seimatosporium biseptatum CPC 13584 Seimatosporium walkeri CPC 17644 Seimatosporium sp HKUCC 7986 LSU Seimatosporium eucalypti CPC 156 86/– –/– Seimatosporium eucalypti CPC 159 Seimatosporium eucalypti CPC 157 88/1 Seimatosporium falcatum CPC 13578 79/– 83/0.99 Seimatosporium falcatum CPC 13580 Seimatosporium falcatum CPC 12992 ITS 77/1 Seimatosporium eucalypti CPC 158 LSU Seimatosporium obtusum CPC 12935 –/1 Discostroma fuscellum 10071 ITS 87/0.99 Seimatosporium glandigenum NBRC 32677 Discostroma fuscellum NBRC 32625 Discostroma fuscellum 418-BB ITS –/0.99 86/1 Discostroma fuscellum NBRC 32680 52/– Seimatosporium lichenicola MFLUCC 14 0623 58/– –/– Seimatosporium vaccinii ICMP 7003 LSU 96/1 Seimatosporium parasiticum NBRC 32682 Seimatosporium physocarpi MFLUCC 14 0625 Discostroma tostum NBRC 32626 –/0.99 Seimatosporium discosioides H4621 54/– 76/1 Seimatosporium discosioides KACC 42491 ITS 54/– Seimatosporium discosioides KACC 42490 ITS 60/0.95 Seimatosporium botan H4619 60/1 Seimatosporium rosae MFLUCC 14 0621 89/1 Seimatosporium pistaciae CPC 24457 56/– Seimatosporium pistaciae CBS 138865 Discostroma sp HKUCC 1004 LSU Seimatosporium hypericinum NBRC 32647 Seimatosporium foliicola NBRC 32676 Pseudopestalotiopsis theae MFLUCC 12 0055 62/– 100/– 1 Fig. 1 – Maximum Parsimony (MP) majority rule consensus tree for the analyzed Seimatosporium isolates based on a combined dataset of LSU and ITS sequence data. MP bootstrap support values higher than 50% and Bayesian posterior probabilities (PP) above 95% are given at the nodes (MP/PP). The tree is rooted with Pseudopestalotiopsis theae (MFLUCC 12−0055). The strain numbers are mentioned after the species names. New strains are in blue bold and ex-type strains are in black bold. 14 × 4–5(–6) µm ( x = 12 × 5 µm, n = 30), initially hyaline, pale brown to dark brown at maturity, fusiform, straight , infrequently slightly curved, with 3 transverse septa, not constricted at the septa, narrowly rounded at both ends, smooth-walled, with thick walls pale brown to dark brown at maturity, narrowly rounded at both ends, smooth-walled, basal cell conic to obconic with a truncate base, subhyaline, 2.5–4 μm long ( x = 3.5 µm), with two median cells subcylindrical to doliform, with thick verruculose walls, brown to dark brown, 7.5–8 μm long ( x =7.5 µm), with apical cell conical and rounded at apex, hyaline, cylindrical to subcylindrical, 3.5–4 μm long ( x = 3.5 µm). 389 Fig. 2 – Seimatosporium lichenicola (MFLUCC 14−0623). a Leaf spot disease on Cotinus coggygria leaf. b Habit on wood. c Fruiting bodies on substrate. The blackened areas are the conidia masses. d Cross section of the conidioma. e Pycnidial wall. f Apex of conidioma. g, h, i Conidiogenous cells. j, k Mature conidia. l Germinating spore on PDA. Bars: d = 300 µm; e = 50 µm; f = 100 µm; g, h, i, l = 20 µm; j, k = 10 µm. 390 Culture characters – Colonies on PDA slow growing, reaching 2 cm diam. after 5 days at 25 °C, circular, spreading, with flat mycelium, with fimbriate margins, pale yellow clusters forming in the centre of colonies after 2 days, clustered or effuse in centre of colony, lacking aerial mycelium. Material examined – RUSSIA, Rostov Region, Oktyabrsky District, Persianovsky Arboretum, on live leaves and dying branches of Cotinus coggygria Scop, 4 June 2014, T. Bulgakov T−095 (MFLU 14−0773, reference specimen of Seimatosporium lichenicola designated here), living culture, MFLUCC 14−0623, CBS 139966. Notes – Seimatosporium lichenicola was introduced by Shoemaker & Müller (1964) as the sexual morph of Clethridium corticola (Fuckel) Shoemaker & E. Müll. The species was described as coelomycetous, producing clavate, mostly 3 transversely septate, non-seteferous conidia. Sutton (1980) reported S. lichenicola, with 13–15 × 5.5–6.5 µm, 3-septate, fusiform conidia, which lack appendages. Nag Raj (1993) transferred S. lichenicola to Sporocadus. The type was from Rosa canina, in Lobkowitschen gardens in Prague, Czechoslovakia. Tanaka et al. (2011) reviewed these four genera and as none had a reported sexual morph, they were synonymized under Seimatosporium. The sexual morph of S. lichenicola is reported to be Discostroma fuscellum based on phylogenetic data (Tanaka et al. 2011). Our collection is consistent with the reports of Shoemaker & Müller (1964), Sutton (1980), Nag Raj (1993) and Tanaka et al. (2011), whereas our collection differs in being found on dead branches of Cotinus coggygria in Russia. Based on these findings, we name our collection as S. lichenicola. Molecular analyses show our strain to cluster with Discostroma fuscellum which is the sexual morph of S. lichenicola (Fig. 1) (Tanaka et al. 2011, Nag Raj 1993). Sequence data for S. lichenicola is not available in GenBank and therefore, we designate our collection as a reference specimen. We do not designate an epitype as the host is different (Ariyawansa et al. 2014). Seimatosporium physocarpi C. Norphanphoun, Bulgakov & K.D. Hyde, sp. nov. Fig. 3 Index Fungorum Number: IF551287 Faces of fungi number: FoF00794 Etymology – Physocarpi, refers to the the host genus from which this species was collected. HOLOTYPUS: MFLU 14−0775. Parasitic (necrotrophic) and saprobic on leaves (leaf spot), twigs and fruits of Physocarpus opulifolius. Sexual morph: Undetermined. Asexual morph: Conidiomata stromatic, erumpent, initially subglobose, becoming cornuted with an obtuse or subcylindrical head and a broad stalk, up to 350–900 µm diam, 400–900 µm high, globose, pale brown to brown, dehiscing by a rupture in the overlying host epidermis and covered with dark brown conidial masses. Pycnidial wall basal stroma up to 200 µm thick, with cell of textura angularis, moderately thick-walled, hyaline. Conidiophores arising from the upper cells of the basal stroma, branched, hyaline, smooth, up to 20 µm long, invested in mucus. Conidiogenous cells lageniform to subcylindrical with moderate apical periclinal thickening, hyaline, smooth, formed from the inner most layer of pycnidial. Conidia (10– )15–16 × 3.5–4.8(5) µm ( x =13 × 4.1 µm, n = 30), hyaline to slightly olivaceous, fusiform, straight to slightly curved, 3–septate, basal cell conic to obconic, slightly olivaceous, thin-walled and verruculose, 2–3 µm long ( x =2.5 µm), with two median cells, doliiform to cylindrical, with thick verruculose walls, constricted at the septa, concolourous, olivaceous, with septa and periclinal walls darker than the rest of the cell, together 5.5–8 µm long ( x = 6.75 µm), second cell from base 3.5–4 µm ( x = 3.7 µm); third cell 2–3.5 µm ( x = 2.75 µm); apical cell hyaline, cylindrical to subcylindrical 2–3 µm long ( x = 2.5 µm); with 1 tubular apical appendage, arising from the apex of the apical cell, 7–12 µm long ( x = 9.5 µm), unequal in length; basal appendage present, rarely one, 12– 14 µm long ( x = 13 µm). Culture characteristics – Colonies on PDA slow growing, reaching 2.5 cm diam. after 7 days at 25 °C, later producing dense mycelium, circular, erumpent, spreading, fluffy, with moderate aerial mycelium and smooth, margins lobate, pale yellow in the centre of the colony after 4 days, clustered or effuse on center of colony surface, without aerial mycelium. 391 Fig. 3 – Seimatosporium physocarpi (holotype) a Leaf spot disease on Physocarpus opulifolius leaf. b Stromatal habit in wood. c Fruiting bodies on host substrate. d Cross section of the stroma showing perithecia. e, f Peridium. g, h Conidiogenous cells. i Immature conidia. j, k, l Mature conidia. m Germinating spore on PDA. – Bars: c = 600 µm; d = 300 µm; e, m = 50 µm; f= 20 µm; g, h = 15 µm; i, j, k, l = 10 µm. Material examined – RUSSIA, Rostov Region, Rostov-on-Don city, Botanical Garden of Southern Federal University, Systematic Arboretum, on dead branches of Physocarpus opulifolius (L.) Maxim (host family), 8 May 2014, T. Bulgakov T-126 (MFLU 14−0775, holotype); Ibid. (PDD, isotype); ex-type living culture, MFLUCC 14−0625, CBS 139968. 392 Notes – In the phylogenetic tree (Fig. 1), S. physocarpi clustered in the same clade with S. parasiticum, Discostroma tostum (S. passerinii), S. lichenicola (D. fuscellum), S. glandigenum and S. vacinii. However, the conidia of S. passerinii, S. lichenicola, S. glandigenum and S. vacinii lack appendages, while only S. parasiticum has apical and basal appendages (Nag Raj 1993). Therefore our species morphologically closely resembles S. parasiticum. Seimatosporium parasiticum was described from leaves of Physocarpus opulifolius (Rosaceae) from Germany (Sutton 1980). However, S. physocarpi can be distinguished by its smaller conidial dimensions (10–16 × 3.5–4.8 µm in S. physocarpi, 22–35 × 5–7 µm in S. parasiticum) and S. physocarpi has longer apical (7–12 µm versus 2–5 µm) and longer basal appendages (12–14 µm versus 2–8 µm) than S. parasiticum (Sutton 1980). Our new species has 3-septate conidia and occurs in European Russia (and Eastern Europe). It is distinct from S. parasiticum which occurs in Central Europe, and has 3–5-septate (mostly 5–septate) and often irregularly curved conidia (Sutton 1980, Tomoshevich 2012). A detailed morphology comparison of the related species with the new species is provided in Table 2. This species is parasitic usually causes leaf spots of Physocarpus species, and also grows on dead twigs as with many necrotrophic fungi. Seimatosporium species found on Physocarpus spp. in some regions of European Russia (Moscow and Moscow region, St.-Petersburg and Leningrad region,, Rostov region, Krasnodar region) and Asian Russia (Western Siberia, Novosibirsk region) (Bulgakov et al. 2014, Tomoshevich 2012). In some reports, the Seimatosporium species on Physocarpus were reported as Seimatosporium lonicerae (Cooke) Shoemaker, but this species is distinct from S. physocarpi by occasionally having 2-septate conidia, with mostly a single basal appendage, and sometimes with a shorter apical appendage (Melnik 1997, Nag Raj 1993). Seimatosporium rosae Corda, in Sturm, Deutschl. Fl., 3 Abt. (Pilze Deutschl.) 3(13): 79 (1833). Fig. 4 Index Fungorum Number: IF190497 Epitypification identifier: IF551303 Faces of fungi numbers: FoF00795 Type: CZECHOSLOVAKIA, Prague, Pobaba, on stems of Rosa canina L. (PRM); (ex-type collection IMI 108309). Saprobic and weak parasitic on dead and dying branches of Rosa kalmiussica Chrshan. & Lasebna (this species often includes in Rosa canina sensu lato) Sexual morph: Undetermined. Asexual morph: Conidiomata acervular, intra-epidermal, 250–400 µm diam., 150–170 µm high, unilocular, glabrous, brown, dehiscing by irregular split in the overlying host tissue; basal stroma thin, with cells moderately thick-walled, almost colourless at the base, pale brown in the lateral tissue and cells of textura angularis. Conidiophores arising from the upper cells of the basal and lateral tissue, sparsely septate and branched, colourless, thin-walled, smooth, up to 10 µm long. Conidiogenous cells lageniform to cylindrical, colourless, thin-walled,smooth, formed from the inner most layer of pycnidium wall. Conidia (11–)12–15 × 3.5–4.5(–5) µm ( x = 13.5 × 4.5 µm, n = 30), hyaline or slightly olivaceous, fusiform, straight to slightly curved, 3-septate, basal cell conic to obconic, thin-walled and verruculose, 2–3.5 µm long ( x = 3µm), with two median cells, doliiform to cylindrical, with thick verruculose walls, constricted at septa, concolourous, olivaceous, with septa and periclinal walls darker than the rest of the cell, together 5–7 µm long ( x = 17 µm) second cell from base 3–4.5 µm ( x = 4 µm); third cell 3–4 µm ( x = 3.5 µm); apical cell hyaline, cylindrical to subcylindrical 2.5–4 µm long ( x = 3.25 µm); with 1 tubular apical appendage, arising from the apex of the apical cell, 5–10(–10.4) µm long ( x = 8 µm), unequal in length; basal appendage present, 5–7(–8) µm long ( x = 5.5 µm). Culture characteristics – Colonies on PDA slow growing, reaching 3 cm diam. after 13 days at 25 °C, later producing dense mycelium, circular, rough margin white at first with dark green clusters in the center of the colony after 5 days, clustered or effuse on center of colony surface, without aerial mycelium. Hyphae septate branched, hyaline, thin. 393 Fig. 4 – Seimatosporium rosae (epitype). a Habit in wood. b, c Fruiting bodies on host substrate. d Cross section of fruiting body. e, f Peridium. g, h, i Conidiogenous cells. j, k, l Mature conidia. m Germinating spore on PDA. n from above. o from below. Bars: c = 200 µm; d = 300 µm; e, g, h, i, m = 20 µm; f= 50 µm; j, k, l = 10 µm. Material examined – RUSSIA, Rostov Region, Krasnosulinsky District, Donskoye Forestry, edge of ravine forest, on dying and dead branches of Rosa kalmiussica Chrshan. & Lasebna, 21 May 2014, T. Bulgakov T−056 (MFLU 14–0771, epitype of Seimatosporium rosae designated here); living cultures MFLUCC 14–0621, CBS 139823. Notes – Seimatosporium rosae is the generic type and was found on Rosa canina (L.) from Podbaba in Prague (Corda 1833). Our isolate was collected on Rosa kalmiussica Chrshan. & 394 Lasebna from the Rostov Region of Russia and has the same conidial size range (11–15 × 3.5–5 µm) and same host as S. rosae, which is consistent with reports of Sutton (1980), and Nag Raj (1993). In molecular analyses (Fig. 1), our isolate clusters together with S. pistaciae, a species introduced by Crous & Mirab (2014) from buds of Pistacia vera from Iran. Although, S. pistaciae morphologically closely matches S. rosae, Crous & Mirab distinguished these taxa, the former having larger conidia (15–22 × 4–5 µm, Crous et al. 2014). We designate our collection of S. rosae as an epitype specimen (sensu Ariyawansa et al. 2014) as it was collected on same host in Europe. Discussion The genus Seimatosporium previously belonged in the family Amphisphaeriaceae (Shoemaker & Müller 1964, Brockmann 1976, Paulus et al. 2006, Maharachchikumbura et al. 2015) and later transferred to newly introduced Discosiaceae (Senanayake et al. 2015). Amphisphaeriaceae was established by Winter (1887) as a large heterogeneous family which mainly contains Pestalotiopsis-like asexual morphs (Jeewon et al. 2002). These taxa are characterized by ascomata immersed in the host, with dark peridial walls and asci with amyloid apical apparatii (Barr 1975). Their asexual morphs are generally characterized by coelomycetous conidiomata producing septate conidia with filiform apical appendages (Barr 1990, Nag Raj 1993). Nag Raj (1993) has reported conidia with pigmented median cells, with or without one apical and basal appendage. The present findings that species of Vermisporium, characterized by their falcate to elongate-fusiform conidia with pigmented cells, a beak-like apical cell, and a podiform, tubular, unbranched, eccentric basal appendage, clustering in Seimatosporium was rather unexpected. Nag Raj (1993) has reported that one of the main characters used for distinguishing species was the length to width ratio, even though this had previously been found to be inadequate (Swart & Williamson 1983). The taxonomy has traditionally placed a great deal of emphasis on length and width of conidia, while distinguishing species in Pestalotiopsis (Jeewon et al. 2003), and Seimatosporium, as a genus sharing many similarities. Spore length has been used as a key character, with many new species of Pestalotiopsis being described based on subtle differences in spore size (Mordue 1985, 1986, Nag Raj 1985, 1986, Venkatasubbaiah et al. 1991). Further, this finding also questions the value of appendage type sensu Nag Raj (1993) as informative features at generic level. Sutton (1980) treated Seimatosporium as heterogeneous with a broad concept. However, Nag Raj (1993) segregated Seimatosporium in to five genera based on the conidial morphology. Therefore, the generic concept of Seimatosporium remains unresolved and this was discussed by Tanaka et al. (2011) with the support of molecular data. In their study, Tanaka et al. (2011), included isolates representing all the five genera discussed by Nag Raj (1993) and strains representing Seimatosporium sensu stricto. They observed that all Seimatosporium species and Discostroma sexual morphs clustered in a monophyletic group, but with several subgroups among the Seimatosporium sensu lato clade. These may be related to the colour of median cells and may have taxonomic value. For example, in the present study, the clade represented by Sarcostroma bisetulatum, S. restionis, Seimatosporium hakeae, S. grevilleae, S. leptospermi and S. mariae has coloured median cells. Its sister clade represented by Seimatosporium kriegerianum, S. elegans, S. biseptatum, Seimatosporium walkeri, S. eucalypti S. falcatum and S. obtusum however, have hyaline median cells. The colour of the median cells in Pestalotiopsis (Amphisphaeriales) has been shown to have taxonomic value, and based on pigmentation the genus was segregated in Pestalotiopsis, Neopestalotiopsis and Pseudopestalotiopsis (Maharachchikumbura et al. 2014). The same approach may helpful in the subdivision of Seimatosporium. However, further sampling of more genera/species, fresh collection with type strains, and establishing asexual and sexual morph connections are required for reassessment of this genus complex. In addition species of Discostroma and Seimatosporium have been linked by molecular data (Tanaka et al. 2011), however the molecular links have not been shown for the types of either genera. Both names should therefore be retained pending fresh collections of the types and further molecular study (Maharachchikumbura et al. 2015). 395 Table 2 Synopsis of morphological features of species of Seimatosporium (Related to this research) Conidia Conidioma ta (μm) Septate Conidia size (μm) Two median cell (µm) Apical appendage (µm) Basal appendage (µm) – 3 septate 16–20 × 4–5 8–11 4–8 4–8 Duan et al., (2011) 100–200 3 septate 11.5–18 × 3.5–5 7.5–10 2–7 2–6 Sutton, 1980 Quercus ballota, >250 3 septate 15–18 × 5–6.5 7.5–10 None None Sutton, 1980 Rosa sp. >350 3 septate 13–15 × 5.5–6.5 – None None Sutton, 1980 150 2-3 septate 11-19 x 3-5 6.5-10 4-10 2-9 Sutton, 1980 400–600 3 septate (10–)12–14 × 4– 5(–6) 7.5–8 None None In this study >100 3–5 septate (mostly 5 septate) 24–31 × 4.5–6.5 17–24 1–8.5 1.5–9 Sutton, 1980 400–800 3 septate 14–20 × (3–2)–4 – 10–18 None Syndowia, 28: 320 350–900 3 septate 5.5–8 7–12 12– 14 In this study >150 4 septate – 10–14 12–20 12.5–16.5 × 3.5–4 7-10 11-20 9-19 250–400 3 septate 10–15 × 3–4 5–9 1–6.5 1–8 Sutton, 1980 Species Host Seimatosporium botan Paeonia suffruticosa (stems) S. discosioides Rosa heliophila (leaves) S. glandigenum S. lichenicola S. lonicerae S. lichenicola (MFLUCC 14–0623) Physocarpus opulifolius (twigs) Cotinus coggygria (dying branches, live leaves) S. parasiticum Physocarpus opulifolius (leaves) S. passerinii Epilobium fleischeri S. physocarpi (MFLUCC 14–0625) Physocarpus opulifolius (leaves, twigs and fruits) Physocarpus opulifolius (bud) S. pistaciae S. rosae Rosa kalmiussica Chrshan. & Lasebna (10–)15–16 × 3.5–4.8 (5) (15–)17–20(–22) × (4–)4.5(–5) Reference Persoonia 33, 2014 Nag Raj, 1993 S. rosae (MFLUCC 14–0621) Rosa sp. (branches) 250–400 × 150–170 3 septate (11–)12–15 × 3.5–4.5(–5) 5–7 5–10(–10.4) 5–7(–8) In this study S. vaccinii Vaccinium myrtillus >100 3 septate 13–18 × 4.5–5.5 – None None Sutton, 1980 ―–― no result 396 In the present study, combined LSU and ITS alignment is used to resolve species relationships in Seimatosporium (Fig 1), following (Tanaka et al. 2011). The phylogenetic tree shows that our collection clearly groups with Seimatosporium. The application of molecular data can provide reliable genetic evidence to define species boundaries in taxonomic studies, as shown in this study. Combined multi-gene data phylogenies can better resolve the taxonomy of genera such as Seimatosporium (Tanaka et al. 2011). Seimatosporium species are common as saprobes and pathogens on plants, but are not considered to be destructive foliar pathogens. For example, Vermisporium acutum (≡ Seimatosporium acutum), was found on a single necrotic lesion on Eucalyptus nicholii leaves (Barber et al. 2003). Species of Seimatosporium have been reported from necrotic spots and associated with disease on branches, twigs, leaves and fruits (Bulgakov et al. 2014). Seimatosporium physocarpi causes leaf spots and grows on living and dying branches. Thus, the Seimatosporium species in this study cannot be considered as saprobic in nature. In temperate arid climate zones, many necrotrophic fungi are specific on host-plants and grow during the cold and wet seasons. Endophytes, may mostly be pathogens, but they often may be treated as saprobes by mycologists, because during the summer they can only be found on dead branches (Bulgakov et al. 2014). Leaf spots of eucalyptus caused by Vermisporium have been reported from the Southern Hemisphere (Barber et al. 2011), while Vermisporium quercinum was found on the bark of Quercus suber (Fagaceae) in Sardinia, Italy (Franceschini et al. 1995). Vermisporium tenzingii was reported from leaves of Osbeckia crinita from Darjeeling, India and Osbeckia stellata in Kaltani, Nepal (Wu & Sutton 1996). Some species such as S. brevicentrum and S. eucalypti were associated with insect damaged leaves, presumably as secondary invaders of leaf tissue damaged previously by insects or as endophytes sporulating in necrotic tissues (Barber et al. 2011). These results suggest that species of Seimatosporium are not likely to be a serious commercial problem. Acknowledgements Chada Norphanphoun thanks for the Mushroom Research Foundation, Chiang Mai, Thailand, for supporting this research. Kevin D. Hyde thanks the Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. 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