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 spe- cies 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