Freshwater Ascomycetes: Minutisphaera (Dothideomycetes) Revisited, Including One New Species from Japan
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Mycologia, 105(4), 2013, pp. 959–976. DOI: 10.3852/12-313 # 2013 by The Mycological Society of America, Lawrence, KS 66044-8897 Freshwater Ascomycetes: Minutisphaera (Dothideomycetes) revisited, including one new species from Japan Huzefa A. Raja1 likelihood and Bayesian analyses of combined 18S Nicholas H. Oberlies and 28S, and separate ITS sequences, as well as Mario Figueroa examination of morphology, we describe and illus- Department of Chemistry and Biochemistry, University trate a new species, M. japonica. One collection from of North Carolina at Greensboro, Greensboro, North North Carolina is confirmed as M. fimbriatispora, Carolina 27412 while two other collections are Minutisphaera-like Kazuaki Tanaka fungi that had a number of similar diagnostic Kazuyuki Hirayama morphological characters but differed only slightly Akira Hashimoto in ascospore sizes. The phylogeny inferred from the Faculty of Agriculture and Life Sciences, Hirosaki internal transcribed spacer region suggested that two University, Bunkyo-cho, Hiroskaki, Aomori 036-8561, out of the three North Carolina collections may be Japan novel and perhaps cryptic species within Minuti- Andrew N. Miller sphaera. Organic extracts of Minutisphaera from USA, Illinois Natural History Survey, University of Illinois, M. fimbriatispora (G155-1) and Minutisphaera-like Champaign, Illinois 61820 taxon (G156-1), revealed the presence of palmitic acid and (E)-hexadec-9-en-1-ol as major chemical Steven E. Zelski constituents. We discuss the placement of the Minuti- Carol A. Shearer sphaera clade within the Dothideomycetes. The Department of Plant Biology, University of Illinois, description of the genus Minutisphaera is emended Urbana, Illinois 61801 to accommodate M. japonica within Minutisphaera. Key words: Aquatic, minute fungi, submerged Abstract: During investigations of freshwater asco- wood, systematics mycetes we found one interesting taxon from Aomori (Japan), as well as three additional taxa from North INTRODUCTION Carolina (USA), which were morphologically similar to Minutisphaera, a recently described freshwater The freshwater Dothideomycetes is an ecological fungus in the Dothideomycetes. The ascomata of all group of fungi (Shearer et al. 2009) that currently the collections bore dark hair-like structures around comprise approximately 195 species, which constitute the ostiolar region, obovoid to obclavate bitunicate about 32% of the currently known freshwater asci, and one to three septate hyaline to brown ascomycetes (Shearer and Raja 2012). Most species ascospores with a sheath (in material from Japan), of freshwater Dothideomycetes belong in the Pleos- and with both sheath and appendages (in material porales (Zhang et al. 2012) or Jahnulales (Pang et al. from the USA). The apothecial ascomata of these 2002, Campbell et al. 2007, Suetrong et al. 2011), taxa, however, differ from those of the type species of although few taxa have affinities to the Capnodiales the genus, which are perithecial. Two collections of and Tubeufiaceae (Shearer et al. 2009). Minutisphaera-like fungi from the USA were morpho- Based on evaluation of morphological characters logically quite similar but differed in ascospore size. and multigene molecular phylogenetic studies, several To assess the phylogenetic affinities of Minutisphaera- new families have been assigned recently to the like taxa with the type species, M. fimbriatispora,we freshwater Dothideomycetes. These families include sequenced 18S and 28S nrDNA of five newly collected Aliquandostipitaceae (Inderbitzin et al. 2001), Amni- strains of Minutisphaera. We also sequenced the culicolaceae (Zhang et al. 2008, 2009a, b), Lentithe- nrDNA for the entire internal transcribed spacer ciaceae (Zhang et al. 2009b, c), Lindgomycetaceae region of 10 strains to assess interspecific and (Hirayama et al. 2010), Morosphaeriaceae (Suetrong intraspecific variation with M. fimbriatispora. Addi- et al. 2009, Boonmee et al. 2012) and Natipusillaceae tionally we examined the secondary metabolite (Raja et al. 2012). Although molecular sequence data profiles of two strains from USA. Based on maximum has provided phylogenetic placements for numerous freshwater Dothideomycetes, a number of Dothideo- Submitted 31 Aug 2012; accepted for publication 17 Dec 2012. mycetes from freshwater habitats occur as singletons 1 Corresponding author. E-mail: [email protected] and remain incertae sedis (Shearer et al. 2009). 959 960 MYCOLOGIA Minutisphaera Shearer, A.N. Mill. & Ferrer, typified MATERIALS AND METHODS by M. fimbriatispora Shearer, A.N. Mill. & Ferrer, is a recently described species from submerged wood in Morphological studies and fungal isolates.—Methods of morphological observation are described by Tanaka et al. freshwater habitats from USA (Ferrer et al. 2011). It is (2009). For ascospore septum position, the decimal system characterized by small globose to subglobose asco- (Shoemaker 1984) was used. Single ascospore cultures were mata with dark brown to black hairs around the obtained according to Shearer et al. (2004). For the ostiole, fissitunicate, oblong to obclavate, eight- Japanese collections, ascomata formation was induced by spored asci, and one-septate, multiguttulate, hyaline placing a small piece of mycelial culture on rice straw agar to pale brown ascospores equipped with a gelatinous (RSA; Tanaka and Harada 2003). Colony colors on potato- sheath and numerous filamentous appendages radi- dextrose agar (PDA; Difco), cornmeal agar (CMA; Difco) ating around the spore at the mid-septum. Minuti- and weak oatmeal agar (wOA; 15 g Difco oatmeal agar, 6 g sphaera currently is placed in the Dothideomycetes agar, 1 L water; Zhao and Shamoun 2006) were character- based on morphological as well as nuclear ribosomal ized using Rayner (1970). Fungal cultures obtained from sequence data, but its relationship with other taxa Japan were deposited at the Japan Collection of Microor- ganisms (JCM) and the National Institute of Agrobiological within the Dothideomycetes remains unresolved. Sciences (MAFF). Cultures from USA were deposited in the During ongoing investigations of freshwater asco- Department of Plant Biology Culture Collection at the mycetes in the USA (Raja et al. 2011b) and Japan University of Illinois and Department of Chemistry and (Hirayama et al. 2010), we found interesting taxa Biochemistry Culture Collection at the University of North from both Aomori (Japan) and North Carolina Carolina at Greensboro (UNCG). (USA), which were morphologically similar to Minuti- DNA extraction and amplification.—Detailed protocols for sphaera. These taxa have dark hair-like structures DNA extraction and PCR amplification were described by around the ostiolar region, broadly shaped bitunicate Hirayama et al. (2010). DNA from mycelia was extracted asci, and 1–3-septate, hyaline to brown ascospores with the ISOPLANT Kit (Nippon Gene Co., Tokyo, Japan) with a sheath in material from Japan, and with both a according to the manufacturer’s instructions. Partial SSU sheath and appendages in material from USA. The and LSU and the complete ITS region of nrDNA were ascomata of these taxa, however, appeared more amplified with three primer sets, NS1–NS4 (White et al. apothecioid than those of the type species of the 1990), LROR–LR7 (Rehner and Samuels 1994) and ITS1/ genus. In addition, collections of Minutisphaera-like 1F–ITS4 (White et al. 1990, Gardes and Bruns 1993). fungi from USA showed variation in ascospore size. Sequences were assembled with Sequencher 4.9 (Gene For example, the ascospores of G155-1 were more Codes Corp.), optimized by eye and manually corrected similar to those reported for M. fimbriatispora but the when necessary. ascospores of G156-1 and G156-2 were comparatively Taxon sampling and phylogenetic analyses.—Four datasets smaller than those of M. fimbriatispora (Ferrer et al. were assembled for phylogenetic analyses: (i) a SSU dataset 2011). Whether these size variations are intraspecific that consisted of 80 taxa; (ii) an LSU dataset consisting of or result from cryptic speciation and therefore mostly the same taxa as in the SSU dataset; (iii) a combined interspecific within Minutisphaera is unknown at this 83 taxa SSU and LSU dataset; and (iv) an ITS dataset with time. 10 strains of Minutisphaera spp. to assess interspecific and The goal of the present study, therefore, was to intraspecific relationships among members of the genus. For the SSU and LSU datasets, we sampled taxa from the understand the phylogenetic relationships of Minuti- major orders of the Pleosporomycetidae and Dothideomy- sphaera-like taxa collected from Japan and North cetidae currently included in the Dothideomycetes (Schoch Carolina (USA) with the original collections of M. et al. 2009). Taxa included in the present study were fimbriatispora and within the Dothideomycetes. To obtained from a study on the molecular phylogeny of address this goal, we undertook a molecular phyloge- freshwater Dothideomycetes (Shearer et al. 2009), as well as netic study using partial 18S small subunit nrDNA other studies on the phylogenetic relationships among (SSU) and 28S large subunit nrDNA (LSU). To better dothideomyceteous fungi (Wu et al. 2011, Zhang et al. understand species boundaries of our collections 2012). We also included members of the Patellariales and within Minutisphaera, we conducted phylogenetic other apothecial dothideomycete members such as Catinella analyses based on ITS sequences and compared taxa olivacea (Batsch) Boudier to test whether Minutisphaera-like based