Taxonomic and Phylogenetic Placement of Nodulosphaeria

Mycol Progress (2016) 15:34 DOI 10.1007/s11557-016-1176-x

ORIGINAL ARTICLE

Taxonomic and phylogenetic placement of Nodulosphaeria

Ausana Mapook1,2,4,5 & Saranyaphat Boonmee 5 & Hiran A. Ariyawansa4,5,9 & Saowaluck Tibpromma4,5 & Erio Campesori6,7,8 & E. B. Gareth Jones3 & Ali H. Bahkali3 & K. D. Hyde1,2,3,5

Received: 27 August 2015 /Revised: 15 February 2016 /Accepted: 17 February 2016 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2016

Abstract Nodulosphaeria is a ubiquitous genus that com- the family Phaeosphaeriaceae as a distinct genus. The sexual prises saprobic, endophytic and pathogenic species associated morphs of Nodulosphaeria hirta and N. spectabilis are de- with a wide variety of substrates and has 64 species epithets scribed and illustrated using modern concepts. Two new listed in Index Fungorum. The classification of species in the Nodulosphaeria species are introduced. The phylogenetic re- genus has been a major challenge due to a lack of understand- lationships and taxonomy of the genus Nodulosphaeria are ing of the importance of characters used to distinguish taxa, as discussed, but further sampling with fresh collections, refer- well as the lack of reference strains. The present study clarifies ence or ex-type strains and molecular data are needed to obtain the phylogenetic placement of the genus and related species, a better and natural classification for the genus. using fresh collections from Italy. Four Nodulosphaeria species are characterized based on multi-loci analyses of ITS, LSU, SSU, TEF and RPB2 sequence datasets. Phylogenetic Keywords Dothideomycetes . Phaeosphaeriaceae . analyses indicate that Nodulosphaeria species group within Phylogeny . Taxonomy

Section Editor: Franz Oberwinkler

* K. D. Hyde 1 Key Laboratory for Plant Diversity and Biogeography of East Asia, [email protected] Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China Ausana Mapook 2 [email protected] World Agroforestry Centre, East and Central Asia, Kunming 650201, Yunnan, People’s Republic of China Saranyaphat Boonmee 3 Botany and Microbiology Department, College of Science, King [email protected] Saud University, Riyadh 1145, Saudi Arabia 4 School of Science, Mae Fah Luang University, Chiang Rai 57100, Hiran A. Ariyawansa Thailand [email protected] 5 Center of Excellence in Fungal Research, Mae Fah Luang University, Saowaluck Tibpromma Chiang Rai 57100, Thailand [email protected] 6 A.M.B. Gruppo Micologico Forlivese BAntonio Cicognani^,Via Roma 18, Forlì, Italy Erio Campesori 7 A.M.B. Circolo Micologico BGiovanni Carini^,C.P. [email protected] 314 Brescia, Italy E. B. Gareth Jones 8 Società per gli Studi Naturalistici della Romagna, C.P. [email protected] 144 Bagnacavallo, RA, Italy 9 Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Ali H. Bahkali Academy of Agricultural Sciences, 550006 Guiyang, Guizhou, [email protected] People’s Republic of China 34 Page 2 of 15 Mycol Progress (2016) 15:34

Introduction Crivelli) with 5-septate ascospores were introduced by Shoemaker and Babcocck (1987). Currently, more than 60 spe- Several recent studies have been conducted on the families of cies are listed in MycoBank for Nodulosphaeria (http://www. Pleosporales in order to provide a natural classification of this mycobank.org/, 09/2014). Recent studies by Phookamsak et al. large order (Zhang et al. 2009, 2012;Ariyawansaetal.2013a, (2014) and Ariyawansa et al. (2015a) included a reference b, c, 2014a, c). Some studies have used only on morphological strain of Nodulosphaeria modesta. These authors studied mor- characterizations, while others have used molecular analysis phological characteristics and a combined dataset of LSU, SSU (Zhang et al. 2012;Ariyawansaetal.2013a, b, c, 2014a, c). and ITS sequence data. However, sequence data are not avail- Phaeosphaeriaceae is a large family in the order able in GenBank for most Nodulosphaeria taxa. Pleosporales, introduced by Barr (1979), and comprises more The aim of this study is to determine the phylogenetic than 300 species (Zhang et al. 2009;Hydeetal.2013; placement of the genus Nodulosphaeria based on molecular Wijayawardene et al. 2014). The family shares similarities with and morphological comparison with descriptions, together Leptosphaeriaceae, but can be differentiated by the peridium with illustrations of four taxa. Two reference specimens with and host. Leptosphaeriaceae species usually occur on reference sequences are proposed to stabilize their names. dicotyledons and have a thick peridium comprising scleroplectenchymatous cells (Shearer et al. 1990; Câmara et al. 2002; Zhang et al. 2009;Hydeetal.2013; Ariyawansa Materials and methods et al. 2015b). In Phaeosphaeriaceae, species usually occur on monocotyledons and have a thin peridium comprising pseudo- Sample collection and specimen examination parenchymatous cells (Câmara et al. 2002;Kirketal.2008; Zhang et al. 2009; Phookamsak et al. 2014;Ariyawansaetal. Fresh material of Nodulosphaeria species were obtained from 2015b). Ariyawansa et al. (2015b) provided data on the Italy during 2013–2014. Fresh specimens were photographed phylogeny and characters of genera of Leptosphaeriaceae. using a Carl Zeiss stereo microscope fitted with an AxioCam The two families are clearly distinguished. Hyde et al. (2013) ERC 5 S camera. Microscopic characters were examined included 26 genera in Phaeosphaeriaceae based on morpholog- using a Motic SMZ 168 Series microscope. Freehand sections ical characters and phylogenetic analysis, while nine genera of ascomata were made and characters were observed and (Ampelomyces, Chaetosphaeronema, Neosetophoma, photographed in water mounts using a Nikon ECLIPSE 80i Paraphoma, Phaeoseptoria, Phaeosphaeriopsis, Tiarospora, compound microscope, fitted with a Canon EOS 550D digital Setophoma and Wojnowicia)werelistedinthefamilyas camera. All measurements were calculated using Tarosoft asexual morphs, while Wijayawardene et al. (2014)included Image Frame Work software and images used for figures were 32 genera in the family. Recently, Phookamsak et al. (2014) processed with Adobe Photoshop CS3 Extended v.10.0 soft- studied the phylogenetic placements of genera in the family ware (Adobe Systems, USA). with descriptions and illustrations. Single spore isolations were obtained using the methods of The genus Nodulosphaeria was introduced by Rabenhorst Chomnunti et al. (2014). Ascospores germinating within 12– (1858) and typified with N. hirta Rabenh. The genus was pre- 24 h were transferred to new malt extract agar (MEA) plates viously characterized by an ascomata apex, lined with brown and incubated at 25–30 °C in the dark; some of these cultures setae, and three- to multi-septate ascospores, with a swollen cell were used for molecular work. The specimens and living cul- and some with terminal appendages (Holm 1961; Shoemaker tures are deposited in the Herbarium Mae Fah Luang 1984). Clements and Shear (1931)consideredNodulosphaeria University (Herb. MFLU) and Culture collection Mae Fah as a synonym of Leptosphaeria. The relationship of Luang University (MFLUCC), Chiang Rai, Thailand and Nodulosphaeria, Ophiobolus and Leptosphaeria was discussed Herbarium of Cryptogams, Kunming Institute of Botany by Holm (1957) who stated that Nodulosphaeria differs from Academia Sinica (HKAS), China. Facesoffungi numbers were Leptosphaeria in having hyaline ascospores with one swollen obtained as in Jayasiri et al. (2015). cell. Several species (i.e. N. erythrospora (Riess) L. Holm, N. fruticum (Roberge ex Desm.) L. Holm, N. mathieui DNA extraction, PCR amplification and sequencing (Westend.) L. Holm, N. megalosporus (Auersw. & Niessl) L. Holm, N. pseudoaffinis (Petr.) L. Holm, N. pontica (Petr.) L. Isolates were grown on MEA or PDA at 16–25 °C for Holm, N. pseudoaffinis (Petr.) L. Holm, N. robusta (Strasser) L. 2 weeks, then the fungal mycelium was scraped off and Holm and N. pellita (Fr.) Shoemaker) were transferred to transferred to 1.5-ml. microcentrifuge tubes. The fungal Ophiobolus by Shoemaker (1976). In addition, five species genomic DNA was extracted by the Biospin Fungus (i.e. N. engadinensis (E. Müll.) Shoemaker & C.E. Babc., Genomic DNA Extraction Kit (BioFlux®, China) following N. kuemmerlei Moesz, N. morthieriana (Sacc.) Shoemaker & the manufacturer’s instructions (Hangzhou, P.R. China). C.E. Babc., N. pileata (Volkart) Crivelli and N. winteri (Niessl) DNA amplifications were performed by polymerase chain Mycol Progress (2016) 15:34 Page 3 of 15 34 reaction (PCR). The internal transcribed spacer (ITS) was Phaeosphaeriaceae were used to confirm their phylogenetic amplified by using primer pairs ITS5 (5′ - placement. Didymella exigua (CBS 183.55) was used as the GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′- outgroup taxon. The sequences used for analyses, with TCCTCCGCTTATTGATATGC-3′) (White et al. 1990). accession numbers, are given in Table 1. All sequences were The partial small subunit nuclear rDNA (SSU) was ampli- aligned using MAFFT v.7.110 online program (http://mafft. fied by using primer pairs NS1 (5′-GTAGTCATA cbrc.jp/alignment/server/) (Katoh and Standley 2013). The TGCTTGTCTC-3′)andNS4(5′-CTTCCGTCAAT alignments were checked and uninformative gaps minimized TCCTTTAAG-3′) (White et al. 1990). The partial large manually where necessary in BioEdit 7.2.3 (Hall 1999). subunit nuclear rDNA (LSU) was amplified by using prim- Maximum Likelihood (ML) and Bayesian Inference (BI) were er pairs LROR (5′-ACCCGCTGAACTTAAGC-3′)and used in analyses with individual data from each partition in LR5 (5′-TCCTGAGGGAAACTTCG-3′) (Vilgalys and addition to the combined aligned dataset. Intron or variable Hester 1990). The translation elongation factor 1-alpha sequences were excluded from all analyses. gene (TEF1) was amplified by using primers EF1-983 F Maximum likelihood (ML) analysis was run in RAxML (5′-GCYCCYGGHCAYCGTGAYTTYAT-3′) and EF1- (Stamatakis et al. 2008) implemented in raxmlGUI v.0.9b2 2218R (5′-ATGACACCRACRGCRACRGTYTG-3′) (Silvestro and Michalak 2010) with 1000 rapid bootstrap rep- (Rehner 2001). The partial RNA polymerase second larg- licates using the GTR+ GAMMA model of nucleotide substi- est subunit (RPB2) was amplified by using primers fRPB2- tution. Maximum Likelihood bootstrap values (MLBP) equal 5 F and fRPB2-7cR (Liu et al. 1999). The amplification or greater than 60 % are given above each node (Fig. 1). reaction were carried out with the following protocol: The The model of nucleotide substitution was performed by final volume of the PCR reaction was 50 μlwhich using MrModeltest 2.2 (Nylander 2004) for each gene is pro- contained 2 μl of DNA template, 1.5 μl of each forward vided in Table 2. Posterior probabilities (PP) (Rannala and and reverse primers, 25 μl of 2× Easy Taq PCR SuperMix Yang 1996; Zhaxybayeva and Gogarten 2002) were determined (mixture of EasyTaqTM DNA Polymerase, dNTPs, and by Markov Chain Monte Carlo sampling (MCMC) in MrBayes optimized buffer; Beijing TransGen Biotech, Beijing, PR v.3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous China) and 20 μl of sterilized water. The PCR thermal Markov chains were run from random trees for 5,000,000 gen- cycle program for ITS and SSU genes amplification were erations and trees were sampled every 100th generation with a provided as: initially 95 °C for 3 min, followed by 40 cy- total of 50,000 trees. The first 20 % trees were discarded as the cles of denaturation at 94 °C for 30 s, annealing at 55 °C burn-inphaseandtheremainingtrees used for calculating PP in for 50 s, elongation at 72 °C for 1 min, and final extension the majority rule consensus tree (the standard deviation of split at 72 °C for 7 min. The PCR thermal cycle program for frequencies were reached to 0.01) (Ariyawansa et al. 2013b; LSU gene amplification were provided as: initially 95 °C Udayangaa et al. 2015). Bayesian posterior probabilities with for 3 min, followed by 40 cycles of denaturation at 94 °C those equal or greater than 0.90 are given below each node for 30 s, annealing at 56 °C for 50 s, elongation at 72 °C for (Fig. 1). Phylogenetic trees were drawn using Treeview 1 min, and final extension at 72 °C for 7 min. The PCR v.1.6.6 (Page 1996). The sequences are deposited in GenBank. thermal cycle program for TEF gene amplification were: initially95°Cfor3min,followedby40cyclesofdena- turation at 94 °C for 30 s, annealing at 55 °C for 50 s, Results elongation at 72 °C for 1 min, and final extension at 72 °C for 7 min. The PCR thermal cycle program for Phylogenetic analysis of combined ITS, LSU, SSU, TEF RPB2 gene amplification were: initially 95 °C for 5 min, and RPB2 sequence data followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 2 min, elongation at 72 °C for 90 s, The combined dataset includes ITS, LSU, SSU, TEF and and final extension at 72 °C for 10 min. The quality of PCR RPB2 sequence data which were analyzed by using ML and products were checked on 1 % agarose gel electrophoresis Bayesian analyses (Fig. 1). All trees were similar in topology strained with ethidium bromide. The PCR products were and did not differ significantly (data not shown). send for sequencing at BGI Tech Solutions, Beijing, China. The combined sequence alignment comprised 80 taxa, includ- ing our strains, with Didymella exigua (CBS 183.5) as the Phylogenetic analysis outgroup taxon. Bootstrap support values of ML (≥60 %) are shown on the upper branches (Fig. 1). Values of the Bayesian The most closely related taxa were determined using nucleo- PP (≥0.95) from MCMC analyses are shown below the branches. tide BLAST searches online in GenBank (http://www.ncbi. Phylogram generated from RAxML analysis based on a nlm.nih.gov/). The combined alignments of ITS, LSU, SSU, combined dataset of ITS, LSU, SSU, TEF and RPB2 datasets TEF and RPB2 sequence data from the closest relatives in shows that seven Nodulosphaeria isolates formed a well- 34 Page 4 of 15 Mycol Progress (2016) 15:34

Table 1 Taxa used in this study and their GenBank accession numbers; new sequences are in bold

Taxon Culture accession no.a GenBank accession no. TEF RPB2

ITS LSU SSU

Allophaeosphaeria clematidis MFLUCC 15–0652T KT306949 KT306953 KT306956 –– Allophaeosphaeria cytisi MFLUCC 15–0649T KT306947 KT306950 KT306954 –– Allophaeosphaeria dactylidis MFLUCC 13–0618T KP744432 KP744473 KP753946 –– Allophaeosphaeria muriformia MFLUCC 13–0349T KP765680 KP765681 KP765682. –– Allophaeosphaeria subcylindrospora MFLUCC 13–0380T KT314184 KT314183 KT314185 –– Chaetosphaeronema hispidulum CBS 216.75 KF251148 KF251652 EU754045 –– Dematiopleospora mariae MFLUCC 13–0612T KJ749654 KJ749653 KJ749652 KJ749655 – Didymella exigua CBS 183.55T GU237794 EU754155 EU754056 – GU371764 Diederichomyces caloplacae CBS 129338T KP170639 JQ238643 – KP170665 – Diederichomyces cladoniicola CBS 128026T KP170642 JQ238628 – KP170668 – Diederichomyces ficuzzae CBS 128019T KP170647 JQ238616 – KP170673 – Entodesmium artemisiae MFLUCC 14–1156T KT315508 KT315509 ––– Entodesmium rude CBS 650.86 – GU301812 AF164356 GU349012 – Galliicola pseudophaeosphaeria MFLUCC 14–0524T KT326692 KT326693 ––– Loratospora aestuarii JK 5535B – GU301838 GU296168 – GU371760 Loratospora luzulae MFLUCC 14–0826T KT328497 KT328495 KT328496 –– Muriphaeosphaeria galatellae MFLUCC 14–0614T KT438333 KT438329 KT438331 –– Neosetophoma clematidis MFLUCC13–0734T KP744450 KP684153 KP684154. –– Neosetophoma italica MFLUCC 13–0388T KP711356 KP711361 KP711366 –– Neosetophoma samarorum CBS 138.96T KF251160 KF251665 GQ387517 – KF252168 Neosetophoma samarorum CBS 139.96 KF251161 GQ387579 GQ387518 – KF252169 Neostagonospora caricis CBS 135092T KF251163 KF251667 ––KF252171 Neostagonospora elegiae CBS 135101T KF251164 KF251668 ––KF252172 Nodulosphaeria aconiti MFLUCC 13–0728T KU708848 KU708844 KU708840 KU708852 KU708856 Nodulosphaeria derasa CBS 184.57 ––GU456299 GU456275 – Nodulosphaeria hirta MFLUCC 13–0867 KU708849 KU708845 KU708841 KU708853 – Nodulosphaeria modesta MFLUCC 11–0461 KM434275 KM434285 KM434294 –– Nodulosphaeria scabiosae MFLUCC 14–1111T KU708850 KU708846 KU708842 KU708854 KU708857 Nodulosphaeria senecionis MFLUCC 15–1297T KT290257 KT290258 KT290259 –– Nodulosphaeria spectabilis MFLUCC 14–1112 KU708851 KU708847 KU708843 KU708855 – Ophiobolus cirsii MFLUCC 13–0218 KM014664 KM014662 KM014663 –– Ophiobolus erythroporus MFLU 12–2225 – KM014665 KM014666 –– Ophiosimulans tanaceti MFLUCC 14–0525T KU738890 KU738891 KU738892 –– Ophiosphaerella agrostidis MFLUCC 11–0152T KM434271 KM434281 KM434290 KM434299 – Ophiosphaerella agrostidis MFLUCC 12–0007 KM434272 KM434282 KM434291 KM434300 KM434308 Ophiosphaerella aquaticus MFLU 14–0033T Paraphoma chrysanthemicola CBS 522.66 KF251166 GQ387582 GQ387521 – KF252174 Paraphoma dioscoreae CPC 11361 KF251169 KF251673 ––KF252177 Paraphoma radicina CBS 111.79T FJ427058 KF251676 EU754092 – KF252180 Paraphoma radicina CBS 102875 KF251173 KF251677 EU754091 – KF252181 Parastagonospora caricis S615T KF251176 KF251680 ––KF252184 Parastagonospora nodorum CBS 110109 KF251177 KF251681 EU754076 – KF252185 Parastagonospora poae CBS 135089 T KF251178 KF251682 ––KF252186 Phaeosphaeria alpine CBS 456.84T KF251181 KF251684 ––KF252188 Phaeosphaeria avenaria AFTOL–ID 280 KT225528 AY544684 AY544725 DQ677885 – Phaeosphaeria chiangraina MFLUCC 13–0231T KM434270 KM434280 KM434289 KM434298 KM434307 Phaeosphaeria eustoma AFTOL–ID 1570 – DQ678063 DQ678011 DQ677906 DQ677959 Mycol Progress (2016) 15:34 Page 5 of 15 34

Table 1 (continued)

Taxon Culture accession no.a GenBank accession no. TEF RPB2

ITS LSU SSU

Phaeosphaeria juncicola CBS 110108 KF251183 KF251686 ––KF252190 Phaeosphaeria musae MFLUCC 11–0133 KM434267 KM434277 KM434287 KM434296 KM434304 Phaeosphaeria musae MFLUCC 11–0151 KM434268 KM434278 KM434288 KM434297 KM434305 Phaeosphaeria nigrans CBS 307.79 KF251184. KF251687 ––KF252191 Phaeosphaeria oryzae CBS 110110T KF251186 KF251689 GQ387530 – KF252193 Phaeosphaeria oryzae MFLUCC 11–0170 KM434269 KM434279 ––KM434306 Phaeosphaeria papayae S528T KF251187 KF251690 ––KF252194 Phaeosphaeria phragmiticola CBS 459.84T KF251188 KF251691 ––KF252195 Phaeosphaeria thysanolaenicola MFLUCC 10–0563T KM434266 KM434276 KM434286 KM434295 KM434303 Phaeosphaeria typharum CBS 296.54 KF251192 GU456334 – GU456287 KF252199 Phaeosphaeriopsis dracaenicola MFLUCC 11–0157T KM434273 KM434283 KM434292 KM434301 KM434309 Phaeosphaeriopsis dracaenicola MFLUCC 11–0193 KM434274 KM434284 KM434293 KM434302 KM434310 Phaeosphaeriopsis glaucopunctata CBS 653.86 KF251199 GQ387592 GQ387531 – KF252206 Phaeosphaeriopsis glaucopunctata MFLUCC 13 0220T KJ522474 KJ522482 KJ522478 –– Phaeosphaeriopsis glaucopunctata MFLUCC 13–0265 KJ522473 KJ522477 KJ522481 –– Phaeosphaeriopsis musae CBS 120026T DQ885894 GU301862 GU296186 GU349037 – Phaeosphaeriopsis triseptata MFLUCC 13–0271T KJ522475 KJ522484 KJ522479 – KJ522485 Phaeosphaeriopsis triseptata MFLUCC 13–0347 KJ522476 KJ522480 KJ522483 – KJ522486 Premilcurensis senecionis MFLUCC 13–0575T KT728365 KT728366 ––– Setophoma chromolaena CBS 135105T KF251244 KF251747 ––KF252249 Setophoma sacchari CBS 333.39T KF251245 GQ387586 GQ387525 – KF252250 Setophoma sacchari MFLUCC 11–0154 KJ476144 KJ476146 KJ476148 KJ461319 – Setophoma sacchari MFLUCC 12–0241 KJ476145 KJ476147 KJ476149 KJ461318 – Setophoma terrestris CBS 335.87T KF251247 KF251750 GQ387528 – KF252251 Sulcispora pleurospora MFLUCC 14–0995T KP271443 KP271444 KP271445 –– Vagicola vagans CBS 604.86 KF251193 KF251696 ––KF252200 Vrystaatia aloeicola CBS 135107 KF251278 KF251781 ––– Wojnowicia dactylidicola MFLUCC 13–0738T KP744469 KP684147 KP684148 –– Wojnowicia dactylidis MFLUCC 13–0735T KP744470 KP684149 KP684150 –– Wojnowicia lonicerae MFLUCC 13–0737T KP744471 KP684151 KP684152 –– Wojnowicia viburni MFLUCC 120733T KC594286 KC594287 KC594288 –– Xenoseptoria neosaccardoi CBS 120.43 KF251280 KF251783 ––KF252285 Xenoseptoria neosaccardoi CBS 128665T KF251281 KF251784 ––KF252286 a AFTOL-ID Assembling the Fungal Tree of Life BCC Belgian Coordinated Collections of Microorganisms CBS CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC Culture collection of Pedro Crous, housed at CBS; DAOM Canadian Collection of Fungal Cultures, Ottawa, Canada; ICMP International Collection of Microorganisms from Plants; JK J. Kohlmeyer; MFLU Mae Fah Luang University Herbarium Collection; MFLUCC Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; T ex-type/ex-epitype isolates; S working collection of William Quaedvlieg resolved monophyletic clade in Phaeosphaeriaceae with rel- = Pocosphaeria (Sacc.) Berl., Icon. fung. (Abellini) 1(2): atively high bootstrap support (99 %; Fig. 1). 89 (1892). Saprobic or hemibiotrophic on herbaceous hosts. Sexual Taxonomy morph: Ascomata immersed to semi-immersed, or erumpent through host tissues at maturity, solitary or scattered, Nodulosphaeria Rabenh., Klotzschii Herb. Viv. Mycol., Edn subglobose to obpyriform, brown to dark brown, coriaceous, 2: no. 725 (in sched.) (1858) with or without subiculum covering host. Ostioles slightly =Leptosphaeriasubgen. Pocosphaeria Sacc., Syll. fung. protruding from substratum, with numerous brown to dark (Abellini) 2: 32 (1883). brown, external or internal periphyses-like setae. Peridium 34 Page 6 of 15 Mycol Progress (2016) 15:34

Fig. 1 Phylogenetic tree generated from maximum parsimony analysis and Bayesian posterior probabilities (PP, blue) equal to or greater than of a combined dataset of LSU, SSU, ITS, TEF and RPB2 sequence data. 0.95 are indicated in bold branches. The ex-type strains and reference Bootstrap support values for maximum likelihood (ML, black), equal to strains are indicated in bold. New isolates are in blue. The tree is rooted or greater than 60 %, are indicated above or below the nodes and branches with Didymella exigua CBS 183.55 Mycol Progress (2016) 15:34 Page 7 of 15 34

Table 2 Comparison of the nucleotide substitution models Gene/loci LSU ITS SSU TEF RPB2 Combined used in the analysis gene

Nucleotide substitution models for Bayesian GTR+ GTR+ GTR+ GTR+ GTR+ GTR+I+G analysis (performed by MrModeltest 2.2) I+ I+ I+ I+ I+ G G G G G

(10–)20–40 μm, comprising 2–6 layers of brown to dark constricted at septum, straight or slightly curved, smooth- brown cells of textura angularis to globulosa, darkest at the walled, guttulate. Asexual morph: Undetermined. outside. Hamathecium comprising 2–5 μm wide, cylindrical Type species: Nodulosphaeria hirta Rabenh., Klotzschii to filiform, septate, branching pseudoparaphyses. Asci 8- Herb. Viv. Mycol., Edn 2: no. 725 (in sched.) (1858) spored, bitunicate, fissitunicate, clavate to cylindric-clavate, Nodulosphaeria aconiti Mapook, Camporesi & K.D. slightly curved, sessile or with short bulbous pedicel, apically Hyde, sp. nov., rounded with a ocular chamber. Ascospores biseriate to multi- Index Fungorum number: IF551942, Facesoffunginumber: seriate, often overlapping, hyaline to pale yellowish brown or FoF00446; Fig. 2 brown, cylindrical to fusiform, multi-septate, one of the upper Etymology: Named after the host genus Aconitum, from cells swollen, enlarged near apex, with or without terminal which this species was collected. appendages and protuberance or subterminal ridges, Holotype:MFLU14–0019

Fig. 2 Nodulosphaeria aconiti (holotype). a, b Appearance of ascomata on host substrate. c Section through ascoma. d Ostiole with dark brown setae. e Peridium. f Pseudoparaphyses. g, h Immature and mature asci. i–l Ascospores. m Germinating ascospore. Scale bars (a)200μm, (b)100μm, (c, d, g, h)20μm, (e) 10 μm, (f, i–m)5μm 34 Page 8 of 15 Mycol Progress (2016) 15:34

Saprobic on dead herbaceous stems of Aconitum vulparia the rounded ends, 4-septate, broader and constricted at second Rchb., noticeable as black dots on the host surface. Sexual cell from the apex, straight to slightly curved, smooth-walled, morph: Ascomata (198–)208–210 × 113– 120(–142) μm without terminal appendages. Ascospores germinating on (̅x = 205 × 125 μm, n = 5), immersed, erumpent at maturity, MEA and PDAwithin 36 h at 18 °C and germ tubes produced solitary, scattered, subglobose to obpyriform, coriaceous, dark from both ends. Asexual morph: Undetermined. brown, Ostiole short papillate, with numerous internal dark Material examined: ITALY, Trento Province, Marilleva, brown setae. Peridium 11–16 μmwide,comprising2–3- on stems of Aconitum vulparia (Ranunculaceae), 27 June layers of thick-walled, dark brown cells of textura angularis. 2012, E. Camporesi (MFLU 14–0019, holotype), ex-type liv- Hamathecium comprising 2–2.5 μm wide, cylindrical, sep- ing culture MFLUCC 13–0728, MUCL; (isotype in HKAS, tate, branching pseudoparaphyses, anastomosing above the under the code of HKAS 90975); on dead stem of Aconitum asci. Asci (58–)62–79(–83) × 9–10 μm(̅x =69×9 μm, vulparia (Ranunculaceae), 19 August 2013, E. Camporesi n = 10), 8-spored, bitunicate, fissitunicate, cylindrical to cylin- (MFLU 15–1880, paratype) dric-subclavate, slightly curved, with a short, bulbous pedicel Notes: Nodulosphaeria aconiti was collected from dead apically rounded, with an ocular chamber. Ascospores 29– stems of Aconitum vulparia. The new species is similar to 33 × 4–5 μm(̅x =31×5μm, n = 20), overlapping 2–3-seriate, Nodulosphaeria modesta in having 4-septate ascospores, en- hyaline or pale brown, cylindric-fusiform, tapering towards larged at the second cell from the apex and a similar host

Table 3 Synopsis of Nodulosphaeria species with some similar morphological features discussed in this study

Species Ascomata (μm) Peridium Asci size (μm) Ascospores Septation Host association Source (μm) size (μm) family references

N. aconiti Mapook, (198–)208– 11–16 (58–)62–79(– 29–33 × 4– 4-septate, without Ranunculaceae This study Camporesi & K.D. 210 × 113– 83) × 9–10 5 terminal appendages. Hyde 120(–142) (MFLUCC 13-0728) N. derasa 250–350 × 250– 35–40 90–110 × 11– 28–55 × 5– 8-septate, with small Asteraceae Shoemaker (Berk. & Broome) 300 14 6 curved cylindric 1984. L. Holm 1957 terminal appendages. N. derasa 200–250(– 20–40 90–120 × 11– (35–)40– 8-septate, with small Asteraceae Holm 1957 (Berk. & Broome) 300) × 250–300 14 52 × 4– curved cylindric L. Holm 1957 4.5 terminal appendages. N. dolioloides Auersw. 175–250 × 35–50 70–85 × 12– 36– 8-septate, without Asteraceae Shoemaker 1863 200–300 16 42 × 5.5– terminal appendages. 1984. 6.5 N. hirta Rabenh. 1858 230–380 × 21–44 (83–)90–110( 48–55 × 5– 8-septate, terminal Undetermined Phookamsak (S-F70410) 225–395 −115) × (1- 7(−7.5) appendages not et al. 2014. 3.5–)15–20 reported. N. hirta Rabenh. 1858 260–330 × 15–30 100– 48–63 × 5– 8-septate, without Undetermined Zhang et al. (BR 101945–95) 260–330 123 × 12.5– 6.5 terminal appendages. 2012. 15 (−17.5) N. hirta Rabenh. 1858 273–277 (16–)19– 95–97 × 12– (40–)56– 8-septate, without Asteraceae This study (MFLUCC 13-0867) (–303) × 23(– 15 62 × (4– terminal appendages. 220– 250(–285) 25) )5–6 N. modesta (Rabenh.) 130–200 × 7–10 60–70 × 13– 29–40 × 5– 4-septate, with terminal Asteraceae, Shoemaker Munk ex L. Holm 150–250 17 7 appendages. Ranunculaceae, 1984. 1957 Rosaceae, Apiaceae N. pellita (Fr.) 200–400 × 40–55 100– 50– 8-septate, with terminal Asteraceae Shoemaker Shoemaker 1968 200–500 120 × 17– 60 × 4.5– appendages. 1984. 20 5.5 N. scabiosae Mapook, (195–)200–210(– 25–30 105– 35–40 × 3– 4-septate, without Caprifoliaceae This study Camporesi & K.D. 236) × (192–) 125 × 11– 6 terminal appendages. Hyde (MFLUCC 215–232(–265) 13 14-1111) N. spectabilis (Niessl) 200–250 × 25–30 110– 34– 4-septate, with terminal Apiaceae Shoemaker L. Holm 1961 210–240 135 × 13– 44 × 5.5– appendages. 1984. 15 6.5 N. spectabilis (Niessl) 210–290 × 250– 20–25(– (95–)100– 30–42 × 5– 4-septate, terminal Apiaceae This study L. Holm 1961 300 43) 125 × 13– 7 appendages could not (MFLUCC 14-1112) 16 see. Mycol Progress (2016) 15:34 Page 9 of 15 34 family association (Holm 1961; Shoemaker 1984; Ostiole papillate, protruding from substratum, with numer- Phookamsak et al. 2014). Nodulosphaeria aconiti differs from ous, internal brown to dark brown setae, protruding exter- N. modesta in having distinct setae in the ostiole and larger nally. Peridium (16–)19–23(–25) μm wide, comprising 4– ascomata [(198–)208–210 × 113–120(–142) μmvs.130– 5 layers of dark brown cells of textura angularis, flattened 200 × 150–250 μm] (Table 3). Nodulosphaeria aconiti is also at the inside. Hamathecium comprising 2.5–3.5 μmwide, similar to N. scabiosae which we introduce as a new species, filiform, septate, branching pseudoparaphyses. Asci 95– but differs in having distinct setae around the ostiole, larger 97 × 12–15 μm(̅x =96×14 μm, n = 5), 8-spored, ascomata and smaller ascospores (29–33 × 4–5 μm vs. 35– bitunicate, cylindric-clavate, slightly curved, short pedicel- 40 × (3–)5–6 μm). The phylogeny also supports this as a dis- late, apically rounded, with an ocular chamber. Ascospores tinct species with high bootstrap support (Fig. 1). (40–)56–62 × (4–)5–6 μm(̅x =55×5μm, n = 10), overlapping Nodulosphaeria hirta Rabenh., Klotzschii Herb. Viv. 3–4-seriate, hyaline or pale yellowish brown, long fusiform, Mycol., Edn 2: no. 725 (in sched.) (1858) 8–9-septate, enlarged at the fourth cell from the apex, con- Facesoffunginumber: FoF01892; Fig. 3 stricted at the septum, straight or slightly curved, thick and Reference specimen:MFLU15–1128 smooth walled, without terminal appendages. Ascospores ger- Saprobic on a dead stems of Achillea sp. Sexual morph: minating on MEA within 36 hr. at 18 °C and germ tubes Ascomata 273–277(–303) × 220–250(–285) μm produced from both ends. Asexual morph: Undetermined. (̅x = 285 × 252 μm, n = 5), immersed or semi-immersed, Material examined: ITALY, Trento Province, Stavel – erumpent, solitary or scattered, subglobose to obpyriform, Vermiglio, on dead stems of Achillea sp. (Asteraceae), 28 coriaceous, dark brown, with subiculum covering the host. June 2012, E. Camporesi (MFLU 15–1128/HKAS 90972,

Fig. 3 Nodulosphaeria hirta (reference specimen). a, b Appearance of ascomata on substrate. c Section through ascoma. d Ostiole with external dark brown setae. e Peridium. f Pseudoparaphyses. g, h Immature and mature asci. i–l Ascospores. m Germinating ascospore. Scale bars (a)200μm, (b)100μm, (c) 50 μm, (d, g, h)20μm, (e, i–m) 10 μm, (f)5μm 34 Page 10 of 15 Mycol Progress (2016) 15:34 reference specimendesignatedhere), living culture Nodulosphaeria modesta (Rabenh.) Munk ex L. Holm, in MFLUCC 13–0867, MUCL. Holm, Symb. bot. upsal. 14 (no. 3): 80 (1957). Notes: Nodulosphaeria hirta was introduced by Facesoffunginumber: FoF00253 Rabenhorst (1858) as the generic type based on Sphaeria See Phookamsak et al. 2014 for description; Fig. 4 hirta. The type material was examined and described by Nodulosphaeria scabiosae Mapook, Camporesi & K.D. Phookamsak et al. (2014), but molecular data is lacking Hyde, sp. nov. for this species. Nodulosphaeria hirta was isolated from Index Fungorum number: IF551943, Faces of fungi Achillea sp. from Italy and is similar to N. hirta number: FoF01893; Fig. 5 (Rabenhorst 1858)andN. dolioloides (Auerswald 1863) Etymology: Name reflects the host genus Scabiosa,from in having 8-septate ascospores, enlarged at the fourth cell which this species was collected. from the apex. It differs from N. dolioloides in having Holotype:MFLU15–1129 larger ascospores [(40–)56–62 × (4–)5–6 μmvs.36– Saprobic on dead stems of Scabiosa sp., noticeable as 42 × 5.5–6.5 μm] with distinct setae at the apex of the black dots on host surface. Sexual morph: Ascomata ostiole. Our collection is identical to the holotype, al- (195–)200–210(–236) × (192–)215–232(–265) μm though the host genus differs. As the type specimen at S (x̅= 210 × 225 μm, n = 5), immersed, erumpent at maturity, is in good condition we designate isolate MFLUCC 13- solitary, scattered, subglobose to obpyriform, coriaceous, dark 0867 as a reference specimen (sensu Ariyawansa et al. brown, without a subiculum covering the host. Ostiole papil- 2014b)forNodulosphaeria hirta. late, protruding from the substrate, with numerous, external

Fig. 4 Nodulosphaeria modesta (MFLU12–2214). a, b Appearance of ascomata on host substrate. c, d Section through ascoma. e Close up of the peridium. f Pseudoparaphyses. g– j Immature and mature asci. k–n Ascospores. Scale bars (c, k–n) 5 μm, (d)50μm, (e–j)20μm Mycol Progress (2016) 15:34 Page 11 of 15 34

Fig. 5 Nodulosphaeria scabiosae (holotype). a Appearance of immersed ascomata on host substrate. b Section through ascoma. c Ostiole with external dark brown setae. d Peridium. e Pseudoparaphyses. f– i Immature and mature asci. j–o Ascospores. Scale bars (b, f–i) 50 μm, (j–o)20μm, (c)10μm, (d, e)5μm

brown to dark brown setae. Peridium 25–30 μm wide, com- 4-septate, tapering towards the rounded ends, enlarged at the prising 4–5 layers of dark brown cells of textura angularis. second cell from the apex, constricted at septum, straight to Hamathecium comprising 2.5–3 μm wide, filiform, septate, slightly curved, guttulate, lacking terminal appendages. branching pseudoparaphyses. Asci 105–125 × 11–13 μm Ascospores germinating on MEA within 36 hr. at 18 °C and (x̅= 115 × 12.5 μm, n = 10), 8-spored, bitunicate, fissitunicate, germ tubes produced from both ends. Asexual morph: cylindrical to cylindric-subclavate, slightly curved, short ped- Undetermined. icellate, apically rounded, with an ocular chamber. Ascospores Material examined: ITALY, Forlì-Cesena Province, 35–40 × (3–)5–6 μm(x̅=37×5μm, n = 20), overlapping 2–3- Fiumicello – Premilcuore, on dead stems of Scabiosa sp. seriate, hyaline or pale yellowish brown, cylindric-fusiform, (Caprifoliaceae), 3 June 2014, E. Camporesi (MFLU 15– 34 Page 12 of 15 Mycol Progress (2016) 15:34

1129, holotype), ex-type living culture, MFLUCC 14–1111, different from other Nodulosphaeria species and designate MUCL; (isotype in HKAS, under the code of HKAS 90973); this as a new species based on morphology. The molecular on dead stem of Scabiosa sp. (Caprifoliaceae), 16 June 2014, phylogeny also indicates this as a new species with high boot- E. Camporesi (MFLU 15–1878, paratype) strap support (Fig. 1). Notes: Nodulosphaeria scabiosae is similar to Nodulosphaeria spectabilis (Niessl) L. Holm, Svensk bot. Nodulosphaeria modesta in having 4-septate ascospores, en- Tidskr., 55: 70. 1961. larged at the second cell from the apex (Holm 1957; ≡ Leptosphaeria spectabilis Niessl, Verh. nat. Ver. Brünn Shoemaker 1984; Phookamsak et al. 2014), but N. scabiosae 10: 179 (1872) differs from N. modesta in having longer, narrower and larger Faces of fungi number: FoF01894; Fig. 6 asci (105–125 × 11–13 μm vs. 60–70 × 13–17 μm), distinct Reference specimen:MFLU15–1130 ostiolar setae and a different host in the family Saprobic on dead stem of Peucedanum cervaria (L.) Caprifoliaceae (Table 3). We consider that our strain is Lapeyr., noticeable as black dots on host surface. Sexual

Fig. 6 Nodulosphaeria spectabilis (reference specimen). a, b Appearance of immersed ascomata on substrate. c Section through of ascoma. d Ostiole with dark brown setae. e Peridium. f Pseudoparaphyses. g–j Immature and mature asci. k–p Ascospores. Scale bars (c, g–j)50μm, (d, e, k–p )20μm, (f)5μm Mycol Progress (2016) 15:34 Page 13 of 15 34 morph: Ascomata 210– 290 × 250– 300 μ m Senanayake et al. 2015; Tibpromma et al. 2015,andin (x̅=275×250 μm, n = 5), immersed or erumpent at maturity, preparation). In this study, we introduce two new species solitary, scattered, subglobose to obpyriform, coriaceous, dark and provide reference specimens for two species, and confirm brown, without a subiculum covering the host. Ostiole papil- the phylogenetic placement of Nodulosphaeria in the late, protruding slightly from substrate, with numerous, inter- Phaeosphaeriaceae. nal brown to dark brown setae, protrude externally. Peridium 20–25(–43) μm wide, comprising 3–4layersofdarkbrown Acknowledgments This work was supported by the International cells of textura angularis to t. globulosa. Hamathecium com- Research Group Program (IRG-14-27), Deanship of Scientific prising 2–3 μm wide, filiform, septate, branching Research, King Saud University, Saudi Arabia. Ausana Mapook is grate- pseudoparaphyses. Asci (95–)100–125 × 13–16 μm ful to Research and Researchers for Industries (RRI) PHD57I0012 under the Thailand Research Fund for providing financial support. Saranyaphat (x̅=110×15 μm, n = 20), 8-spored, bitunicate, cylindric- Boonmee thanks to Thailand Research Fund, project number subclavate, slightly curved, with short bulbous pedicel, apical- TRG5880152. K.D. Hyde thanks the Chinese Academy of Sciences, ly rounded, with an ocular chamber. Ascospores 30–42 × 5– project number 2013T2S0030, for the award of Visiting Professorship 7 μm(x̅=38×6μm, n = 20), overlapping 2–4-seriate, yellow- for Senior International Scientists at Kunming Institute of Botany. We thanks the Plant Germplasm and Genomics Center in Germplasm Bank ish brown to brown, cylindric-fusiform, 4-septate, tapering of Wild Species, Kunming Institute of Botany for the help with molecular towards the rounded ends, broader and constricted at second work. Thanks are extended to Chonticha Singtripop, Li Junfu, Dhanushka cell from the apex, with subterminal ridges, straight to slightly N. Wanasinghe for their help in obtaining sequence data. Ausana Mapook curved, guttulate, lacking terminal appendages. Asexual also thanks Alan J.L. Phillips, Shaun Pennycook, Rungtiwa Phookamsak, Mingkwan Doilom, Saranyaphat Boonmee, J.K. Lui and K.W. Thilini morph: Undetermined. Chethana for their valuable suggestions and help. Material examined: ITALY, Forlì-Cesena Province, Passo delle Forche – Galeata, on dead stems of Peucedanum cervaria (Apiaceae), 16 July 2014, E. Camporesi (MFLU References 15–1130/HKAS 90974, reference specimen designated – here), living culture MFLUCC 14 1112, MUCL. Ariyawansa HA, Jones EBG, Suetrong S, Alias SA, Kang JC, Hyde KD Notes: Our collection is identical to N. spectabilis (Niessl) (2013a) Halojulellaceae a new family of the order Pleosporales. L. Holm., which was introduced by Niessl (1872)as Phytotaxa 130(1):14–24 Leptosphaeria spectabilis Niessl. Important characteristics Ariyawansa HA, Kang JC, Alias SA, Chukeatirote E, Hyde KD (2013b) are the 4-septate ascospores, enlarged at the second cell from Towards a natural classification of Dothideomycetes: the genera Dermatodothella, Dothideopsella, Grandigallia, Hysteropeltella the apex and subterminal ridges or protuberance at both ends and Gloeodiscus (Dothideomycetes incertae sedis). Phytotaxa (Holm 1961). The type and our collection are from the same 147(2):35–47 host family Apiaceae and from the same continent, but are Ariyawansa HA, Maharachchikumbura SSN, Karunarathne SC, from different hosts (Peucedanum cervaria in our collection Chukeatirote E, Bahkali AH, Kang JC, Bhat JD, Hyde KD (2013c) Deniquelata barringtoniae from Barringtonia asiatica,as- vs. Laserpitium latifolium in the type). Therefore, we desig- sociated with leaf spots of Barringtonia asiatica. Phytotaxa 105(1): nate our collection as a reference specimen (sensu Ariyawansa 11–20 et al. 2014b)forNodulosphaeria spectabilis. Ariyawansa HA, Phookamsak R, Tibpromma S, Kang JC, Hyde KD (2014a) A molecular and morphological reassessment of Diademaceae. Sci World J 2014:1–11 Ariyawansa HA, Camporesi E, Thambugala KM, Mapook A, Kang JC, Discussion Alias SA, Chukeatirote E, Thines M, Mckenzie EHC, Hyde KD (2014b) Confusion surrounding Didymosphaeria, phylogenetic Nodulosphaeria (Phaeosphaeriaceae) was introduced by and morphological evidence suggest Didymosphaeriaceae is not a – Rabenhorst (1858) and is characterized by brown setae at distinct family. Phytotaxa 176(1):102 119 Ariyawansa HA, Hawksworth DL, Hyde KD, Jones EBG, the ascomata apex, and three- to multi-septate ascospores, Maharachchikumbura SSN, Manamgoda DS, Thambugala KM, with a swollen cell and some with terminal appendages Udayanga D, Camporesi E, Daranagama A, Jayawardena R, Liu (Holm 1961; Shoemaker 1984). The genus was re-examined JK, McKenzie EHC, Phookamsak R, Senanayake IC, Shivas RG, by Zhang et al. (2012) and Phookamsak et al. (2014)basedon Tian Q, Xu JC (2014c) Epitypification and neotypification: guide- lines with appropriate and inappropriate examples. Fungal Divers type material (N. hirta). In this study, we conclude that 69(1):57–91 Nodulosphaeria should be placed in the Phaeosphaeriaceae, Ariyawansa HA, Hyde KD, Jayasiri SC et al (2015a) Fungal diversity as the genus forms a monophyletic group, sister to the genera notes 111–252—taxonomic and phylogenetic contributions to fun- Chaetosphaeronema, Dematiopleospora, Entodesmium, gal taxa. Fungal Divers 75(1):27–274 Loratospora, Muriphaeosphaeria, Ophiobolus, Ariyawansa HA, Phukhamsakda C, Thambugala KM, Wanasinghe DN, Perera RH, Mapook A, Camporesi E, Kang JC, Jones EBG, Bahkali Ophiosimulans, Premilcurensis and Sulcispora,asinrecent AH, Bhat JD, Hyde KD (2015b) Revision and phylogeny of studies (Ariyawansa et al. 2015a; Liu et al. 2015; Leptosphaeriaceae. Fungal Divers 74(1):19–51 Phookamsak et al. 2014; Phukhamsakda et al. 2015; Auerswald (1863) Fungi Europaei Exsiccati: no. 547 34 Page 14 of 15 Mycol Progress (2016) 15:34

Barr ME (1979) A classification of Loculoascomycetes. Mycologia 71: Abdel -Aziz FA, Adamčík S, Bahkali AH, Boonyuen N, Bulgakov 935–957 T, Callac P, Chomnunti P, Greiner K, Hashimoto A, Hofstetter V, Câmara MPS, Palm ME, van Berkum P, O’Neill NR (2002) Molecular Kang JC, Lewis DA, Li XH, Liu XX, Liu ZY, Matumura M, phylogeny of Leptosphaeria and Phaeosphaeria. Mycologia 94: Mortimer PE, Rambold G, Randrianjohany E, Sato G, Indrasutdhi 630–640 VS, Tian CM, Verbeken A, von Brackel W, Wang Y, Wen TC, Xu Clements FE, Shear CL (1931) Genera of fungi, 2nd edn. H.W. Wilson, JC, Yan JY, Zhao RL, Camporesi E, Hyde KD (2015) Fungal diver- New York sity notes 1–110: taxonomic and phylogenetic contributions to fun- Chomnunti P, Hongsanan S, Hudson BA, Tian Q, Peršoh D, Dhami MK, gal species. Fungal Divers 72:1–197 Alias AS, Xu J, Liu X, Stadler M, Hyde KD (2014) The sooty Niessl (1872) Beiträge zur Kenntniss der Pilze. Beschreibung neuer und moulds. Fungal Divers 66:1–36 wenig bekannter Pilze. Verh Naturforsch Ver Brünn 10:153–217 Hall TA (1999) BioEdit: a user-friendly biologicalsequence alignment Nylander JAA (2004) MrModeltest 2.0. Program distributed by the au- editor and analysis programfor Windows 95/98/NT.Nucl Acids thor. Evolutionary Biology Centre, Uppsala University Symp Ser41:95–98 Page RDM (1996) TreeView: an application to display phylogenetic trees Holm L (1957) Etudes taxonomiques sur les pléosporacées. Symb Bot on personal computers. Comput Appl Biosci 12:357–358 Upsaliens 14:1–188 Phookamsak R, Liu JK, McKenzie EHC, Manamgoda DS, Ariyawansa Holm L (1961) Taxonomical notes on Ascomycetes. IV. Notes of H, Thambugala KM, Dai DQ, Camporesi E, Chukeatirote E, Nodulosphaeria Rbh. Sven Bot Tidskr 55:63–80 Wijayawardene NN, Bahkali AH, Mortimer PE, Xu JC, Hyde K Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of (2014) Revision of Phaeosphaeriaceae. Fungal Divers 68:159–238 phylogenetic trees. Bioinformatics 17:754–755 Phukhamsakda C, Ariyawansa HA, Phookamsak R, Chomnunti P, Hyde KD, Jones EBG, Liu JK, Ariyawansa HA, Boehm E, Boonmee S, Bulgakov TS, Yang JB, Bhat DJ, Bahkali AH, Hyde KD (2015) Braun U, Chomnunti P, Crous PW, Dai DQ, Diederich P, Muriphaeosphaeria galatellae gen. et sp. nov. in Dissanayake AJ, Doilom M, Doveri F, Hongsanan S, Jayawardena Phaeosphaeriaceae (Pleosporales). Phytotaxa 277(1):55–65 R, Lawrey JD, Li YM, Liu YX, Lücking R, Monkai J, Muggia L, Rabenhorst (1858) Herb myc, ed. 2 no. 725 (in sched.) Nelsen MP, Pang KL, Phookamsak R, Senanayake I, Shearer CA, Rannala B, Yang Z (1996) Probability distribution of molecular evolutionary Seutrong S, Tanaka K, Thambugala KM, Wijayawardene NN, trees: a new method of phylogenetic inference. J Mol Evol 43:304–311 Wikee S, Wu HX, Zhang Y, Aguirre-Hudson B, Alias SA, Aptroot Rehner S (2001) Primers for elongation factor 1-α (EF1-α). http://ocid. A, Bahkali AH, Bezerra JL, Bhat JD, Camporesi E, Chukeatirote E, NACSE.ORG/research/deephyphae/EF1primer.pdf Gueidan C, Hawksworth DL, Hirayama K, De Hoog S, Kang JC, Senanayake IC, Bahkali AH, Camporesi E, Chomnunti P, Knudsen K, Li WJ, Li X, Liu ZY, Mapook A, McKenzie EHC, Maharachchikumbura SS, Mortimer PE, Xu J, Hyde KD (2015) Miller AN, Mortimer PE, Phillips AJL, Raja HA, Scheuer C, Phylogeny and morphology of Sulcispora pleurospora Schumm F, Taylor JE, Tian Q, Tibpromma S, Wanasinghe DN, (Phaeosphaeriaceae, Pleosporales) on Anthoxanthum odoratum Wang Y, Xu JC, Yan J, Yacharoen S, Zhang M (2013) Families of from Italy. Mycotaxon (in press) Dothideomycetes. Fungal Divers 63:1–313 Shearer CA, Crane JL, Chandra Reddy KR (1990) Studies in Jayasiri SC, Hyde KD, Ariyawansa HA, Bhat J, Buyck B, Cai L, Dai YC, Leptosphaeria. Lectotypification of Sphaeria doliolum. Mycologia Abd-Elsalam KA, Ertz D, Hidayat I, Jeewon R, Jones EBG, Bahkali 82(4):496–500 AH, Karunarathna SC, Liu JK, Luangsa-ard JJ, Lumbsch HT, Shoemaker RA (1968) Type studies of Pleospora calvescens, Pleospova Maharachchikumbura SSN, McKenzie EHC, Moncalvo JM, papaveracea, and some allied species. Can J Bot 46(9):1143–1150 Ghobad-Nejhad M, Nilsson H, Pang KA, Pereira OL, Phillips Shoemaker RA (1976) Canadian and some extralimital Ophiobolus spe- AJL, Raspé O, Rollins AW, Romero AI, Etayo J, Selçuk F, cies. Can J Bot 54:2365–2404 Stephenson SL, Suetrong S, Taylor JE, Tsui CKM, Vizzini A, Shoemaker RA (1984) Canadian and some extralimital Nodulosphaeria Abdel-Wahab MA, Wen TC, Boonmee S, Dai DQ, Daranagama and Entodesmium species. Can J Bot 62:2730–2753 DA, Dissanayake AJ, Ekanayaka AH, Fryar SC, Hongsanan S, Shoemaker RA, Babcocck E (1987) Additions to Nodulosphaeria.CanJ Jayawardena RS, Li WJ, Perera RH, Phookamsak R, de Silva NI, Bot 65:1921–1926 Thambugala KM, Tian Q, Wijayawardene NN, Zhao RL, Zhao Q, Silvestro D, Michalak I (2010) raxmlGUI: a graphical front-end for Kang JC, Promputtha I (2015) The faces of fungi database: fungal RAxML Program. and documentation available from http://www. names linked with morphology, phylogeny and human impacts. sourceforge.com Fungal Divers 74(1):3–18 Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algo- Katoh K, Standley DM (2013) MAFFT multiple sequence alignment rithm for the RAxML web-servers. Syst Biol 75:758–771 software version 7: improvements in performance and usability. Tibpromma S, Phookamsak R, Boonmee S, Camporesi E, Yang JB, MolBiolEvol30:772–780 Bahkali AH, McKenzie EHC, Hyde KD (2015) Phylogeny and Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Ainsworth & morphology of Premilcurensis gen. nov. (Pleosporales) from stems Bisby’s Dictionary of the Fungi, 10th edn. CABI, Wallingford of Senecio in Italy. Phytotaxa 236(1):40–52 Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among Udayangaa D, Castleburya LA, Rossmana AY,Chukeatirote E, Hyde KD ascomycetes: evidence from an RNA polymerase II subunit. Mol (2015) The Diaporthe sojae species complex: phylogenetic re- Biol Evol 16:1799–1808 assessment of pathogens associated with soybean, cucurbits and Liu JK, Jones EBG, Ariyawansa HA, Bhat DJ, Boonmee S, other field crops. Fungal Biol 119(5):383–407 Maharachchikumbura S, McKenzie EHC, Phookamsak R, Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of Phukhamsakda C, Shenoy BD, Abdel-Wahab MA, Buyck B, enzymatically amplified ribosomal DNA from several Cryptococcus Chen J, Chethana KWT, Singtripop C, Dai DQ, Dai YC, species. J Bacteriol 172:4239–4246 Daranagama DA, Dissanayake AJ, Doliom M, D’souza MJ, Fan White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct se- XL, Goonasekara ID, Hirayama K, Hongsanan S, Jayasiri SC, quencing of fungal ribosomal RNA genes for phylogenetics. In: Innis Jayawardena RS, Karunarathna SC, Li WJ, Mapook A, MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide Norphanphoun C, Pang KL, Perera RH, Peršoh D, Pinruan U, to methods and applications, Academic, San Diego, pp 315–322 Senanayake IC, Somrithipol S, Suetrong S, Tanaka K, Wijayawardene NN, Crous PW, Kirk PM, Hawksworth DL, Boonmee S, Thambugala KM, Tian Q, Tibpromma S, Udayanga D, Braun U, Dai DQ, D’souza MJ, Diederich P, Dissanayake A, Wijayawardene NN, Wanasinghe DN, Wisitrassameewong K, Doilom M, Hongsanan S, Jones EBG, Groenewald JZ, Mycol Progress (2016) 15:34 Page 15 of 15 34

JayawardenaR,LawreyJD,LiuJK,LückingR,MadridH, Zhang Y, Schoch CL, Fournier J, Crous PW, De Gruyter J, Woudenberg Manamgoda DS, Muggia L, Nelsen MP, Phookamsak R, Suetrong JHC, Hirayama K, Tanaka K, Pointing SB, Hyde KD (2009) Multi- S, Tanaka K, Thambugala KM, Wanasinghe DN, Wikee S, Zhang Y, locus phylogeny of the Pleosporales: a taxonomic, ecological and Aptroot A, Ariyawansa HA, Bahkali AH, Bhat DJ, Gueidan C, evolutionary re-evaluation. Stud Mycol 64:85–102 Chomnunti P, Hoog GSD, Knudsen K, Li WJ, McKenzie EHC, Zhang Y, Crous PW, Schoch CL, Hyde KD (2012) Pleosporales. Fungal Miller AN, Phillips AJL, Piątek M, Raja HA, Shivas RS, Slippers Divers 53:1–221 B, Taylor JE, Tian Q, Wang Y, Woudenberg JHC, Cai L, Jaklitsch Zhaxybayeva O, Gogarten JP (2002) Bootstrap, Bayesian probability and WM, Hyde KD (2014) Naming and outline of Dothideomycetes maximum likelihood mapping: exploring new tools for comparative 2014. Fungal Divers 69(1):1–55 genome analyses. BMC Genomics 3(1):4