Cryptogamie, Mycologie, 2013, 34 (4): 303-319 © 2013 Adac. Tous droits réservés Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces Rungtiwa PHOOKAMSAK a, b, c, Jian-Kui LIU a, b, Ekachai CHUKEATIROTE a, b, Eric H. C. McKENZIE d & Kevin D. HYDE a, b, c * a Institute of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand b School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand c International Fungal Research & Development Centre, Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming, Yunnan, 650224, China d Landcare Research, Private Bag 92170, Auckland, New Zealand Abstract – A Dothideomycete species, associated with leaf spots of sugarcane (Saccharum officinarum), was collected from Nakhonratchasima Province, Thailand. A single ascospore isolate was obtained and formed the asexual morph in culture. ITS, LSU, RPB2 and TEF1α gene regions were sequenced and analyzed with molecular data from related taxa. In a phylogenetic analysis the new isolate clustered with Leptosphaerulina americana, L. arachidicola, L. australis and L. trifolii (Didymellaceae) and the morphology was also comparable with Leptosphaerulina species. Leptosphaerulina saccharicola is introduced to accommodate this new collection which is morphologically and phylogenetically distinct from other species of Leptosphaerulina. A detailed description and illustration is provided for the new species, which is compared with similar taxa. The type specimen of Pleosphaerulina oryzae, is transferred to Leptosphaerulina. It is redescribed and is a distinct species from L. australis, with which it was formerly synonymized. Leptosphaerulina species have been linked to Pithomyces but the lack of phylogenetic support for this link is discussed. The character of the asexual morph of Leptosphaerulina, which is similar to Pithomyces, may to have evolved on separate occasions. Asexual morph / Didymellaceae / Phylogeny / Plant disease / Taxonomy INTRODUCTION Dothideomycetes are the largest and most varied class of Ascomycota comprising 22 orders, 105 families, 678 genera and more than 19,000 species (Kirk et al., 2008; Schoch et al., 2006, 2009; Lumbsch & Huhndorf, 2010; Hyde et al., 2013). Species of Dothideomycetes and their asexual morphs are found as endophytes, epiphytes or pathogens on living plants and as saprobes on decaying organic matter including dicotyledons, grasses and other monocotyledons (Schoch * Corresponding author: kdhyde3@gmail.com doi/10.7872/crym.v34.iss4.2013.303 304 R. Phookamsak et al. et al., 2006; Zhang et al., 2009). Some genera of Dothideomycetes (e.g. Botryosphaeria, Cochliobolus, Didymella, Mycosphaerella, Leptosphaeria, Leptosphaerulina, Phaeosphaeria) and their asexual morphs (e.g. Alternaria, Stemphylium) may cause serious disease of economic crops (e.g. corn, rice, banana, wheat, sugarcane) worldwide (Shoemaker & Babcock, 1989; Schoch et al., 2006, 2009; Zhang et al., 2009; Liu et al., 2012). In this study we collected a Dothideomycete from diseased sugarcane (Saccharum officinarum), and it was subsequently identified as a Leptosphaerulina species. Leptosphaerulina was introduced by McAlpine (1902) with L. australis on apricot leaves (Prunus armeniaca L.) being the type and has also been recorded as associated with Dolichos, Poa, Lolium and Vitis (McAlpine, 1902; Graham & Luttrell, 1961). The asexual state has been reported as Pithomyces (Ellis, 1971; Morgan-Jones, 1987; Zhang & Zhang, 2003; Hyde et al., 2011; Wijayawardene et al., 2012). A linkage with P. flavus Berk. & Broome (1873), the morphologically distinct type species of Pithomyces, however, has not been established with molecular data, and this relationship must be considered questionable. Leptosphaerulina species have ascomata that are ostiolate, papillate and immersed to erumpent, and the bitunicate asci are distinctly saccate. Ascospores are oblong to cylindrical, generally muriform, and mostly hyaline, but become brown at maturity (Graham & Luttrell, 1961; Crivelli, 1983; Abler, 2003; Zhang et al., 2012). The genus Leptosphaerulina was placed in the family Pseudosphaeriaceae based on its morphological characters (Höhnel, 1907; Luttrell, 1955; Graham & Luttrell, 1961; Barr 1982). Subsequently, it was accommodated in Pleosporaceae (Eriksson & Hawksworth, 1998; Kirk et al., 2001; Eriksson 2005). However Kodsueb et al. (2006) studied this genus based on phylogenetic analysis and were unable to resolve the placement of Leptosphaerulina in either of these families. Recent molecular data have confirmed the placement of Leptosphaerulina in Didymellaceae (Aveskamp et al., 2010; Zhang et al., 2012; Hyde et al., 2013). Species of Leptosphaerulina and their asexual morphs are reported as saprobic or parasitic on leaves or stems of various plants including important crop plants. For example, L. trifolii (Rostr.) Petr. causes pepper spot on Trifolium, Medicago and various other hosts, L. arachidicola W.Y. Yen, M.J. Chen & K.T. Huang is pathogenic on leaves of Arachis and L. brassicae Karan causes leaf spots on Brassica (Yen et al., 1956; Karan, 1964; Roux, 1986; Abler, 2003; Ahonsi et al., 2010). Leptosphaerulina americana (Ellis & Everh.) J.H. Graham & Luttr., and L. australis McAlpine have been reported as saprobes on dead plant materials (Graham & Luttrell, 1961; Irwin & Davis, 1985; Zhang et al., 2012). The genus has morphologically been relatively well-studied. Graham & Luttrell (1961) recognized six species (Leptosphaerulina australis, L. americana, L. arachidicola, L. argentinensis (Speg.) J.H. Graham & Luttr., L. Briosiana (Pollacci) Graham & Luttr. and L. trifolii from forage plants. These species were differentiated based on the size and septation of ascospores, the host and characteristics in culture. Irwin & Davis (1985) accepted only four species (L. americana, L. arachidicola, L. argentinensis and L. trifolii), and placed L. australis and L. briosiana in synonymy with L. trifolii, following Booth & Pirozynski (1967). Olanya & Campbell (1990) found that ascospores were not a good character to distinguish species of Leptosphaerulina. It appears that morphological characters in Leptosphaerulina overlap and may comprise species complexes as has been shown in several other recently evolving plant pathogenic genera (e.g. Bipolaris, Manamgoda et al., 2012; Diaporthe, Udayanga et al., 2012; Phyllosticta, Wikee et al., 2011; Wulandari et al., 2009). We are carrying out research of Dothideomycetes on monocotyledons in Thailand, and the present study details one pathogenic and one saprobic taxa on Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces 305 leaves of Saccharum officinarum and Oryza sativa, respectively. One is a new species, Leptosphaerulina saccharicola which is introduced in this paper based on both of morphology and molecular study. A Pithomyces-like asexual state of this species was obtained in culture and thus these genera are discussed and compared using molecular and morphological data. The second taxon is a new combination for Leptosphaerulina oryzae (basionym: Pleosphaerulina oryzae I. Miyake), which we carry out based on the morphology of the re-examined type species. MATERIAL AND METHODS Isolation and identification The specimen was collected from Nakhonratchasima Province in Thailand and returned to the laboratory for examination following the methods described by Taylor & Hyde (2003), Chomnunti et al. (2011) and Liu et al. (2011). The specimen was observed and examined under a Motic SMZ 168 Series stereomicroscope. Micromorphological images were captured using a Nikon ECLIPSE 80i compound microscope with a Canon EOS 550D digital camera. Indian ink was used to reveal any mucilaginous sheath surrounding the ascospores. Measurements were made with Tarosoft (R) Image Frame Work version 0.9.7 (Liu et al., 2010; Chomnunti et al., 2011). A culture was derived via single spore isolation following the methods described in Chomnunti et al. (2011). Ascomata were cut with a razor blade, the centrum tissue containing ascospores were removed using a sterile needle and placed in sterile water. The spore drop which contained the ascospores suspension were placed on quarter strength Sabouraud Dextrose Agar (SDA 3.9 g / 0.6 g yeast extract / 9.0 g bacteriological agar in 600 ml sterile distilled water) and incubated overnight at room temperature. The germinated spores were removed to Malt Extract Agar (MEA; 33.6 g/L sterile distilled water, Difco malt extract) and Potato Dextrose Agar (PDA; 39 g/L distilled water, Difco potato dextrose). The culture is deposited at Mae Fah Luang University Culture Collection (MFLUCC). The specimens are deposited at the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand with duplicates culture in International Collection of Microorganisms from Plants (ICMP), Landcare Research, New Zealand. DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from fresh fungal mycelium grown on MEA/PDA media agar at 25-27°C. The Biospin Fungus Genomic DNA Extraction Kit (BioFlux®, Hangzhou, and P.R. China) was used to extract DNA according to the manufacturer’s instructions. DNA amplification was performed by Polymerase Chain Reaction (PCR). Four partial gene portions were used in this study: the large subunits of the nuclear ribosomal RNA genes (LSU), the internal transcribed spacers (ITS) and two protein coding genes, namely the translation elongation factor 1-alpha gene (TEF1α) and the partial RNA polymerase second largest subunit (RPB2). The primers used were LROR and LR5 (Vilgalys & Hester, 1990) for LSU, ITS5 and ITS4 (White et al., 1990) for ITS, EF1-983F and EF1-2218R (Rehner, 2001) for TEF1α and fRPB2-5F and fRPB2-7cR (Liu et al., 1999) for RPB2. The PCR 306 R. Phookamsak et al. thermal cycle program for ITS, LSU and TEF1α amplification were as follows: initially 94°C for 3 mins, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 50 seconds, elongation at 72°C for 1 mins, and final extension at 72°C for 10 mins. The PCR thermal cycle program for the partial RNA polymerase second largest subunit (RPB2) was followed as initially 95°C for 5 mins, followed by 40 cycle of denaturation at 95°C for 1 mins, annealing at 52°C for 2 mins, elongation at 72°C for 90 seconds, and final extension at 72°C for 10 mins. The PCR products were purified by using the PCR Purification Kit according to manufacturer’s protocol. DNA sequencing was performed by Shanghai Sangon Biological Engineering Technology & Services Co. (Shanghai, P.R. China). Phylogenetic analysis The generated ITS, LSU and TEF1 were analyzed with other GenBank sequences (Table 1). The sequences were performed to indicate the closest matches with taxa in Didymellaceae by Blast search. In addition, the fungal members of Cucurbitariaceae, Dothidotthiaceae, Melanommaceae, Leptosphaeriaceae, Phaeosphaeriaceae, Pithomyces and Pleosporaceae were included in this analysis. Botryosphaeria dothidea was selected as outgroup. The fungal sequence strains were combined and aligned with SATé (Liu et al., 2009) using MAFFT v. 7.036 (Katoh & Standley, 2013) and improved in MEGA5 (Tamura et al., 2011). The alignments were checked and improved manually where necessary. The Phylogenetic relationships were inferred using Maximum-parsimony (MP) in PAUP* 4.0b10 (Swoord, 2002), and MrBayes v. 3.0b4 (Ronquist & Huelsenbeck, 2003) for Bayesian analysis. Maximun likelihood analysis used RAxML v.7.2.8 as part of the RAxML-HPC2 on TG” tool (Stamatakis, 2006; Stamatakis et al., 2008) and was performed at the CIPRES webportal (Miller et al., 2010). Maximum-parsimony (MP) search was performed using the tree heuristic search option with TBR branch swapping and 1,000 random sequence additions. Maxtrees were set up to 500, branches of zero length were collapsed and all multiple parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated by 1,000 bootstrap replications resulting from maximum parsimony analysis, each with 10 replicates of random stemwise addition of taxa ((Hillis & Bull, 1993). Other measures used were consistency index (CI), retention index (RI), rescaled consistency index [RC] and homoplasy index (HI). Bayesian analysis was performed by MrBayes v. 3.0b4 (Ronquist & Huelsenbeck, 2003) with the best-fit model of sequences evolution estimated with MrModeltest 2.2 (Nylander, 2004). Markov Chain Monte Carlo sampling (BMCMC) was used to determine the posterior probabilities (PP) (Rannala & Yang, 1996; Zhaxybayeva & Gogarten, 2002) in MrBayes v. 3.0b4 (Huelsenbeck & Ronquist, 2001). Six simultaneous Markov chains were run for 1000000 generations sampling one tree every 100th generations of trees (resulting 10001 total trees). The burn-in (first 2000 trees) which represented the phase of the analysis were discarded and the remaining 8000 trees were used to built a majority rule consensus tree (Cai et al., 2006; Cai et al., 2008; Liu et al., 2011) with posterior probabilities (PP). A consensus phylogram (50% majority rule) was presented in Fig. 2. Maximum likelihood analysis was performed by RAxML v.7.2.8 as part of the RAxML-HPC2 on TG” tool (Stamatakis, 2006; Stamatakis et al., 2008) at the CIPRES webportal (Miller et al., 2010). A discrete gamma and the four rate classes were relevant with a general time revisable model (GTR) and fifty through Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces 307 Table 1. Isolates used in this study and their GenBank accession numbers. The newly generated sequences are indicated in bold Taxon Boeremia exigua var. exigua Botryosphaeria dothidae Cochliobolus sativus Cucurbitaria berberidis Cucurbitaria berberidis Didymella cucurbitacearum Didymella exigua Didymella fabae Dothidotthia aspera Dothidotthia symphoricarpi Entodesmium rude Herpotrichia juniperi Leptosphaeria doliolum Leptosphaeria maculans Leptosphaeria maculans Leptosphaerulina americana Leptosphaerulina arachidicola Leptosphaerulina australis Leptosphaerulina australis Leptosphaerulina saccharicola Leptosphaerulina trifolii Melanomma pulvis-pyrius Neosetophoma samarorum Ophiosphaerella herpotricha Paraphoma radicina Phaeosphaeria oryzae Phaeosphaeriopsis musae Phoma herbarum Pithomyces chartarum Pithomyces valparadisiacus Plenodomus biglobosus Plenodomus chrysanthemi Pleospora herbarum Pyrenochaeta nobilis Pyrenochaeta nobilis Pyrenophora phaeocomes Pyrenophora tritici-repentis Setomelanomma holmii Setosphaeria monoceras Culture Accession No1 CBS 431.74 CMW 8000 DAOM 226212 CBS 394.84 CBS 363.93 IMI 373225 CBS 183.55T CBS 524.77 CPC 12933 CPC 12929 CBS 650.86 CBS 468.64 CBS 505.75T DAOM 229267 CBS 275.63 CBS 213.55 CBS 275.59 CBS 311.51T CBS 317.83 MFLUCC 11-0169 CBS 235.58 CBS 371.75 CBS 138.96T CBS 240.31 CBS 111.79T CBS 110110 CBS 120026T CBS 276.37T DS1bioJ1b CBS 113339 CBS 298.36 CBS 539.63T CBS 191.86T CBS 407.76T CBS 566.75 DAOM 222769 AFTOL-ID 173 CBS 110217 CBS 154.26 GenBank Accession No2 LSU ITS TEF1α EU754183 AY928047 DQ678045 GQ387605 GQ387606 AY293792 EU754155 EU754133 EU673276 EU673273 GU301812 DQ384093 GU301827 DQ470946 JF740306 GU237981 GU237983 FJ795500 EU754166 KF670716 GU237982 GU301845 GQ387578 DQ767656 EU754191 GQ387591 GU301862 DQ678066 HM216194 EU552152 GU237980 GU238151 DQ247804 EU754206 GQ387616 DQ499596 AY544672 GU301871 AY016368 FJ427001 AY236949 – – JF740191 AY293804 GU237794 GU237880 – – – GQ203759 JF740205 – JF740234 GU237799 GU237820 – GU237829 KF670717 GU237806 – FJ427061 – FJ427058 – – JF810524 HM216213 EU552152 – JF740253 – EU930011 – DQ491507 – – – GU349080 AY236898 – – – – – – – – GU349012 – GU349069 DQ471062 – – – GU456272 GU349070 KF670715 – GU349019 – DQ767639 GU349076 – GU349037 DQ677909 – – – – DQ471090 – – DQ497607 DQ677882 GU349028 – Abbreviations: AFTOL: Assembling the Fungal Tree of Life; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CMW: Tree Pathology Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CPC: Collection of Pedro Crous housed at CBS; DAOM: Plant Research Institute, Department of Agriculture (Mycology), Ottawa, Canada; ICMP: International Collection of Micro-organisms from Plants, Landcare Research, New Zealand; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bake-ham Lane, U.K.; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; T ex-type/ex-epitype isolates. 308 R. Phookamsak et al. maximum likelihood (ML) tree were performed at the same model in RAxML v.7.2.7 with each one starting from the separate randomized trees. One thousand non parametric bootstrap iterations were run with the GTR model and a discrete gamma distribution; the resulting replicates were plotted on to the best scoring tree obtained previously. The phylogram was visualized in Treeview (Page, 1996). The sequences in this study are deposited in GenBank (KF670716, KF670717, KF670715). RESULTS AND DISCUSSION Phylogenetic analysis The combined LSU, ITS and TEF1α gene data set consists of 39 taxa, with Botryosphaeria dothidea as the outgroup taxon. The dataset consists of 2512 characters after alignment, of which 1854 characters are constant and 418 sites (18%) are parsimony informative. A heuristic search with random addition sequences (1000 replicates) and treating gaps as missing characters generated six equally parsimonious trees. All trees were similar in topology and not significantly different (data not shown). The best scoring RAxML tree is shown in Fig. 1. Bootstrap support (BS) values of ML and MP (equal to or greater 50% based on 1,000 replicates) are shown above the nodes and values of the Bayesian posterior probabilities (PP) (equal to or above 95% based on 1,000 replicates) from MCMC analyses are indicated below nodes. The phylogenetic trees obtained from maximum likelihood, maximum parsimony and Bayesian analyses gave similar results relating to family. The families Cucurbitariaceae, Didymellaceae, Dothidotthiaceae, Leptosphaeriaceae, Phaeosphaeriaceae and Pleosporaceae clustered into the suborder Pleosporineae. The strains in Leptosphaeriaceae did not form a good clade none of the phylogenetic models (ML, MP and Bayesian), however once more genes (RPB2) were added this could be improved. In this study, because of the limitation of multi-loci combination, we chose three genes (LSU, ITS and TEF1α) for our analysis, the strain of Leptosphaeria doliolum (CBS 505.75) did not cluster with the other four selected strains of Leptosphaeriaceae. Leptosphaerulina saccharicola (MFLUCC11-0169) clustered together with four other Leptosphaerulina species and formed a well supported clade (99% ML, 98% MP and 1.00 PP) with L. arachidicola (CBS 275.59) in Didymellaceae. The phylogenetic tree resulting from the combined genes showed that L. saccharicola is distinct from L. americana, L. arachidicola, L. australis and L. trifolii, with the latter three species clades probably being species complexes. Two strains of Pithomyces did not cluster in the suborder Pleosporineae and were included in Didymellaceae with Leptosphaerulina. Based on the phylogenetic analysis and morphology, therefore we introduce a new species, Leptosphaerulina saccharicola R. Phookamsak, J.K. Liu & K.D. Hyde, sp. nov. The description and illustrations of this new species are provided as below, as well as the culture characters and details of asexual morphs. Taxonomy Leptosphaerulina saccharicola Phookamsak, J.K. Liu & K.D. Hyde., sp. nov. Fig. 2 MycoBank: MB 805600 Etymology: Referring to the host on which the fungus was found. Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces 309 Fig. 1. RAxML tree based on a combined dataset of LSU, ITS and TEF1 sequences. Bootstrap support values for maximum likelihood (ML, green) and maximum parsimony (MP, red) greater than 50% are given above the nodes. Bayesian posterior probabilities (BYPP, blue) above 0.95 are given below the nodes. The tree was rooted to Botryosphaeria dothidea. HOLOTYPUS: MFLU11-0205 Pathogenic, associated with leaf spots, about 8-40 mm diam., visible as pale brown to brown regions, separated from healthy part of leaf by dark brown margins. Sexual state: Ascomata 70-110 µm high, 100-140 µm diam, immersed under epidermis, uniloculate, globose to subglobose, membranous, brown, gregarious, scattered to clustered. Ostioles circular, central, papillate. Peridium 8-16 µm wide, comprising a few layers, the outer layers comprised of brown walled cells of textura angularis, the inner layer comprised of pale brown to hyaline, 310 R. Phookamsak et al. Fig. 2. Leptosphaerulina saccharicola (Holotype: MFLU11-0205). a. Fruiting bodies on host tissue. b. Section through ascomata. c. Section of peridium. d-f. Asci. g. Germinating ascospore. h-j. Ascospores. k. Ascospore stained in Indian ink showing sheath. l-m. Living culture on PDA. n. Ascomata developing in living culture. o. Section through ascomata forming on culture. p-r. Conidiogenous cells. s-w. Conidia. Scale bars: b, o = 100 µm, g = 50 µm, c, d, e, f, s = 20 µm. h, i, j, k, p, q, r, t, uv, w = 10 µm. Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces 311 elongate thick-walled cells, of textura angularis. Hamathecium lacking pseudoparaphyses. Asci (49-)60-80(-86) × (32-)35-45(-49) µm ( x = 67.9 × 39.4 µm, n = 25), 8-spored, bitunicate, fissitunicate, saccate, obpyriform, ovoid or amygdaliform, apedicellate, apically rounded with well-developed ocular chamber, thick-walled at the apex, arising from the base of the ascoma. Ascospores (25-)27-32(-35.5) × (9-)10-11.5(-12) µm ( x = 29.6 × 11 µm, n = 30), overlapping or irregularly triseriate, oblong to cylindrical or ellipsoidal, muriform or phragmosporous, hyaline, with 4 transverse septa, and 0-2 longitudinal septa, usually widest in the second cell, smooth-walled, with small guttules, surrounded by distinctive structured mucilaginous sheath. Spore germinating initially at both ends and later in all cells, germinating within 12 hours on WA, newly emerging hyphae broadly branching. Asexual state produced on PDA and CMA, hyphae usually hyaline when young, becoming brown to dark brown when mature. Conidiophores 6-11.5 × 2.5-5 µm, mononematous, solitary, narrower than vegetative hyphae, with one apical pore, mostly unbranched, septate, initially hyaline, becoming light brown, walls smooth. Conidiogenous cells 3-8 × 2-5 µm, holoblastic, integrated, terminal, cylindrical, hyaline to brown. Conidia (23-)(24-)25-30(-32)(-33) × (9-)10-12(-13) µm ( x = 29.2 × 11.5 µm, n = 30), solitary thalloconidia, oblong to cylindrical or ellipsoidal, muriform or phragmosporous, initially hyaline, becoming brown to dark brown, mostly with 3-4 transverse septa and 0-1 longitudinal septa. Culture characters: Colonies on PDA 35-45 mm diameter after 1 week at 25-30°C; circular, brown to dark brown, white to pale yellow at the edge, sometimes dark grey, covered by white fluffy hyphae, flattened; reverse white to pale yellow at the edge, brown to dark brown in the middle with fimbriate edge, opaque, floccose to fluffy, no pigments produced, after 4 weeks black ascomata produced on colony and submerged in agar. Material examined: THAILAND, Nakhonratchasima, Huaithalaeng District, Loongpradoo Village, on living leaves of Saccharum officinarum L., 1 November 2010, R. Phookamsak, RP0085 (MFLU11-0205, holotype); extype living culture = MFLUCC 11-0169 = ICMP 19875. Gene sequence data: ITS (KF670717), LSU (KF670716), TEF1α (KF670715) and RPB2 (KF670714). Commentary: Leptosphaerulina saccharicola shares similar morphological characters with L. arachidicola, L. australis and L. trifolii in having ascospores of similar size and septation and in culture characteristic and differs from L. americana by size and septation of ascospores and host preferences (Graham & Luttrell, 1961). Leptosphaerulina saccharicola is most similar to L. Arachidicola, although the latter was reported as causing leaf disease of only Arachis spp. while L. saccharicola is associated with leaf disease of Saccharum officinarum. Our phylogenetic analysis also showed that L. saccharicola is most similar to L. arachidicola, but was in a different clade. Leptosphaerulina saccharicola differs from L. australis in having ascospores with transverse septa and in the pigment production on media. In L. australis ascospores have 5 transverse septa and produce a pink pigment when grown in V-8 juice agar, while L. saccharicola has ascospores with 4 transverse septa and does not produce pigment in V-8 juice agar. Leptosphaerulina arachidicola, L. australis and L. saccharicola are morphologically similar to L. trifolii, and Booth & Pirozynski (1967) synonymised L. australis under L. trifolii. However, these species differ in the size of ascospores, growth on media and host preferences. Leptosphaerulina trifolii has larger ascospores and asci; it is associated with leaf disease of Medicago and Trifolium, grows slowly on V-8 juice agar, while other species are fast growing (Graham & Luttrell, 1961; Miles, 1925; Miller, 1925; Abler, 2003). We have compared the 312 R. Phookamsak et al. morphological characters of species of Leptosphaerulina on other monocotyledonous plants with Leptosphaerulina saccharicola; our species differs in ascospore septation and host (Table 2). The asexual morph of Leptosphaerulina saccharicola formed in culture, and has similar characters with some putative Pithomyces species. The type species of Pithomyces is P. flavus which was collected from a monocotyledonous plant in Sri Lanka (Peradeniya) by Berkeley and Broome in December 1868 (Berkeley & Broome, 1873). P. flavus produces yellow or olive-yellow, soft, spreading colonies on the host and has 15 × 10 µm, 4-5-septate, straw-colored to dark brown, echinulate, doliiform conidia, with darkened septal bands, which are widest in the centre (Berkeley & Broome, 1873; Ellis, 1971). The asexual state of L. saccharicola differs from P. flavus in colony appearance, conidia shape (generally being wider at one end), a part of the conidiophore not remaining attached to the base of the conidia, lack of longitudinal septa and septal bands. Thus, we do not consider L. saccharicola to be related to the type species of Pithomyces. Several species with somewhat similar characters to Pithomyces such as P. chartarum (Berk. & M.A. Curtis) M.B. Ellis have also been placed in Pithomyces. The link between P. chartarum and L. chartarum was reported by Roux (1986), based on cultural studies. The conidia of P. chartarum are morphologically similar to the ascospores of L. chartarum, although the latter are smooth and hyaline to light brown. P. saccharicola was reported from China by Zhang & Zhang (2003), also from Saccharum officinarum; however the asexual morph of L. saccharicola differs from P. saccharicola in septation of conidia. The asexual morph of L. saccharicola has 3-4 transverse septa and 0-1 longitudinal septum, while P. saccharicola has 1-2 transverse septa with longitudinal septa being absent or rare (Zhang & Zhang, 2003). These Pithomyces species have not been linked to the type species, P. flavus. Thus, there is considerable confusion surrounded the genus Pithomyces which requires further work at the molecular level using fresh collections. Pithomyces chartarum (strain DS1bioJ1b) and P. valparadisiacus (strain CBS113339) were clearly unrelated to Leptosphaerulina in our phylogenetic analysis. We therefore treat Pithomyces in a strict sense based on P. flavus as having yellow or olive-yellow, soft, spreading colonies on the host and straw-colored to dark brown, echinulate, doliiform, trans-septate conidia, with darkened septal bands, and widest in the centre, while Pithomyces-like species related to Leptosphaerulina have conidia that are brown to dark brown, smooth-walled, cylindrical, with 3-4 transverse septa and 0-1 longitudinal septa and without a part of the conidiophore remaining attached to the base of the conidia. A third group which may include P. chartarum, does not cluster with Leptosphaerulina and may need to be accommodated in a separate genus. However, further studies are needed. It is likely that the genus Leptosphaerulina comprises several species complexes with each complex having several morphologically similar taxa, but which are phylogenetically distinct and infect different hosts. Detailed studies of species in this genus should be carried out using multigene phylogeny to establish if this is the case. Recent studies have shown that pathogenic genera on economic crops usually comprise species complexes with each complex comprising morphologically similar, but phylogenetically well differentiated species. For example, Colletotrichum contains nine species complexes, with C. Gloeosporioides sensu lato comprising 22 cryptic species plus one subspecies (Cannon et al., 2012; Weir et al., 2012). Similar scenarios have been found in the pathogenic genera Alternaria, Diaporthe, Fusarium, Pestalotiopsis and Phyllosticta (Summerell et al., Table 2. Synopsis of Leptosphaerulina species discussed in this study Septation Hosts Source references Ascospores Asci Ascomata (diam) Transverse septa Longitudinal septa L. americana 34-49 × 13-18 101-106 × 45-48 126-140 5-6 2-5 Trifolium pretense, Graham & Luttrell (1961) Phleum pratense L. arachidicola 23-40 × 11-17 53-87 × 28-42 64-140 3.5 0-2 Arachis spp. Graham & Luttrell (1961) 30-32 × 11 75-80 × 28-50 150 5 2 Various hosts McAlpine (1902), http://nt.ars- grin.gov/ fungaldatabases/fungushost/ new_frameFungusHost Report.cfm 19-23.1 × 6.3-7.3 63-81.9 × 14.7-16.8 126-175 3-5 1-3 Calamagrostis epigeios Pisareva (1964) 23(25-)27 × 7(-8)-12 100-150 × 60-100 3 1 14-20 × 5-9(12) 45-65 × 15-20(-29) 60-170 3-6 1-2 Carex firma, Dryas Nograsek (1990) octopetala 28-30 × 10-11 60-78 × 38-51 120-170 4-5 2-3 Oryza sativa This study 27-32 × 10-11.5 60-80 × 35-45 100-140 4 0-2 Saccharum officinarum This study 25-49 × 11-21 62-95 × 42-59 124-207 3-4 0-2 Various hosts Graham & Luttrell (1961), http://nt.ars- grin.gov/ fungaldatabases/fungushost/ new_frameFungusHost Report.cfm L. australis L. calamagrostidis L. chartarum L. olivaceogrisea L. oryzae L. saccharicola L. trifolii Galenia procumbens Roux (1986) Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces Size (µm) Species 313 314 R. Phookamsak et al. 2010; Glienke et al., 2011; Summerell & Leslie, 2011; Maharachchikumbura et al., 2011, 2012; Udayanga et al., 2011, 2012; Wang et al., 2011; Wikee et al., 2011; Weir et al., 2012; Woudenberg et al., 2013). The molecular data presented here, indicates that Leptosphaerulina comprises species complexes. Analysis of LSU gene sequence data alone cannot separate the species, although they have different morphological characters, and different asexual morphs. Combined gene analysis of five genes, which included two protein genes (TEF1α and RPB2), indicated that the Leptosphaerulina species used in the analysis are distinct species. The species appear to be host or family specific, but further taxa sampling and gene data is required to address the diversity in this genus. Leptosphaerulina oryzae (I. Miyake) Phookamsak, J.K. Liu & K.D. Hyde, comb. nov. Fig. 3 MycoBank: MB 805608 ⬅ Pleosphaerulina oryzae I. Miyake, J. Coll. Agric. imp. Univ. Tokyo2: 250 (1910) ⬅ Pringsheimia oryzae (T. Miyake) Hara, A Monograph of Rice Diseases: 78 (1959) Saprobic on Oryza sativa. Sexual state: Ascomata 100-120 µm high, 120-170 µm diam, immersed below epidermis, solitary, scattered, uniloculate, globose to subglobose, membranous, pale brown to brown. Ostioles circular, central, papillate. Peridium 2.5-8 µm wide, comprising 1-2 layers of brown to dark brown, thin-walled cells, of textura angularis. Hamathecium lacking pseudoparaphyses. Asci (58-)60-78(-85) × (36-)38-51(-54) µm ( x = 70.8 × 45.4 µm, n = 25), 8-spored, bitunicate, fissitunicate, saccate, obpyriform or ovoid, sessile, apically rounded with well-developed ocular chamber, thick-walled, arising from the base of ascoma. Ascospores (26.5-)28-30(-33) × (9.5-)10-11(-12) µm ( x = 30.1 × 10.9 µm, n = 30), irregularly tri-seriate, oblong to cylindrical or ellipsoidal, hyaline to brown, muriform, with 4-5 transverse septa, and 2-3 longitudinal septa, usually widest at the second cell, smooth-walled, surrounded by distinct mucilaginous sheath. Material examined: Japan, on dead leaves of Oryza sativa, September 1907 (Hara, F8478-type, as Pleosphaerulina oryzae). Commentary: Pleosphaerulina oryzae was introduced by Miyake (1910) as saprobic on Oryza sativa in Japan. This species was placed as a synonym of Leptosphaerulina australis (Graham & Luttrell 1961). We observed the type specimens of Pleosphaerulina oryzae and compared the morphological characters with the original description of L. australis (McAlpine 1902). Pleosphaerulina oryzae is similar to L. australis, but they differ in the septation of ascospores, with P. oryzae mostly having 4 (rarely 5) transverse septa, while L. australis has 5 transverse septa (McAlpine 1902). The hosts also differ — rice (Oryza sativa, Poaceae), for P. oryzae and apricots (Prunus armeniaca L., Rosaceae) for L. australis. The new combination is supported by morphology but, unfortunately, type specimens of Leptosphaerulina australis could not be located. Epitypification and molecular data is required to resolve this species. Leptosphaerulina saccharicola has similar characters to those of L. oryzae in the size and shape of ascomata, asci and ascospores. However, L. oryzae has more longitudinal septa (2-3) than L. saccharicola (0-2). Phylogeny and morphology of Leptosphaerulina saccharicola sp. nov. and Pleosphaerulina oryzae and relationships with Pithomyces 315 Fig. 3. Leptosphaerulina oryzae (S, F8478_type of Pleosphaerulina oryzae). a. Herbarium specimens and label of Pleosphaerulina oryzae. b. Ascoma on substrate. c. Section through ascoma. d. Section through peridium. e. Asci. f-i. Ascus. j-l. Immature ascospore. m-n. Mature ascospore. Scale bars: c, d, e, f, g, h, i = 20 µm, j, k, l, m, n = 10 µm. Acknowledgements. This study is supported by Royal Golden Jubilee Ph. D. Program (PHD/0090/2551) under Thailand Research Fund and Mae Fah Luang University (56 1 01 02 00 32), both of which are gratefully acknowledged for scholarship and laboratory support. Wen Jing Li and the International Fungal Research & Development Centre, Research Institute of Resource Insects, Chinese Academy of Forestry are grateful for use of molecular data. The curator of the herbarium S is thanked for lending the herbarium specimen. 316 R. Phookamsak et al. REFERENCES ABLER S.W., 2003 — Ecology and taxonomy of Leptosphaerulina spp. associated with turfgrasses in the United States. 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