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Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy mycological research 112 (2008) 1297–1306 journal homepage: www.elsevier.com/locate/mycres 5 Anther smut fungi on monocots Robert BAUERa,*, Matthias LUTZa, Dominik BEGEROWb, Marcin PIA˛TEKc, Ka´lma´nVA´ NKYd, Kamila BACIGA´ LOVA´ e, Franz OBERWINKLERa aUniversita¨tTu¨bingen, Botanisches Institut, Lehrstuhl Spezielle Botanik und Mykologie, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany bRuhr-Universita¨t Bochum, AG Geobotanik, Geba¨ude ND 03/174, Universita¨tsstraße 150, 44780 Bochum, Germany cDepartment of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krako´w, Poland dHerbarium Ustilaginales Va´nky (HUV), Gabriel-Biel-Str. 5, D-72076 Tu¨bingen, Germany eInstitute of Botany, Slovak Academy of Sciences, Du´bravska´ cesta 14, SK-845-23, Bratislava, Slovakia article info abstract Article history: Teliospores, hyphal septa, cellular interactions, and nucleotide sequences from the ITS and Received 16 January 2008 LSU region of the rRNA gene of specimens of Ustilago vaillantii s. lat. on Muscari and Scilla Received in revised form species were examined and compared with findings in other Ustilaginomycotina. The data 13 May 2008 show that U. vaillantii s. lat. specimens belong to the Urocystales and represent the sister Accepted 11 June 2008 group of the Urocystaceae, standing well apart from Vankya heufleri and V. ornithogali. Within Corresponding Editor: the Urocystales, U. vaillantii s. lat. is unique in sporulating in the anthers of the host plants. Roland W. S. Weber Accordingly, the new genus Antherospora is proposed for the anther smuts on Hyacinthaceae. In addition, our data show that there is a stringent phylogenetic correlation between the Keywords: specimens of Antherospora and their respective hosts. Thus, the specimens on Scilla spp. Anther smuts as well as those on Muscari spp. form highly supported monophyla. Furthermore, on Scilla Hyacinthaceae a phylogenetic dichotomy exists between the specimens infecting Scilla bifolia and those Molecular phylogeny infecting S. vindobonensis, with the specimens of the two host species showing a difference Ultrastructure of 17 bp in the ITS nucleotide sequences. Therefore, A. vindobonensis is described as a new Urocystales species, and A. scillae and A. vaillantii are proposed as new combinations. Consequently, Ustilaginomycotina because of their sporulation in anthers and their parasitism on species of other genera Ustilago of the Hyacinthaceae, Ustilago albucae, U. peglerae, U. tourneuxii, and U. urgineae are also ascribed to Antherospora as new combinations. Descriptions are given for all species. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction 2004), phylogenetics (e.g. Kemler et al. 2006), coevolution (e.g. Begerow et al. 2004), and ecology (e.g. Thrall et al. 1993). Anther smuts on eudicots belong to the pucciniomycotinous By contrast little is known of the ustilagomycotinous genus Microbotryum (Bauer et al. 1997, 2001, 2006; Begerow anther smuts that parasitize monocots. Thus, from the et al. 1997). Va´nky (1998) discussed the history of Microbotryum described species Ustilago albucae, U. muscari-botryoidis, in detail. The core Microbotryum spp., anther smuts parasitiz- U. peglerae, U. scillae, U. tourneuxii, U. urgineae, and U. vaillantii ing members of Caryophyllaceae, are important model organ- (see Zundel 1953, Va´nky 1994, and the references therein) no isms for many biological disciplines, for example genetics ultrastructural and only scant molecular phylogenetic data (e.g. Hood & Antonovics 2004), population analysis (e.g. Giraud are available. Merely from the ITS analysis of Roux et al. 5 Part 233 in the series Studies in Heterobasidiomycetes from Botanical Institute, University of Tu¨ bingen. * Corresponding author. Tel.: þ49 7071 2973212; fax: þ49 7071 295344. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.06.002 Author's personal copy 1298 R. Bauer et al. (1998) it can be concluded that U. vaillantii belongs to the aqueous uranyl acetate for 1 h in the dark. After five washes Ustilaginomycetes and not to the Microbotryales. All these anther in distilled water, samples were dehydrated in acetone, using smut species occur on plants of the monocotyledonous family 10 min changes at 25, 50, 70, 95 %, and three times in 100 % Hyacinthaceae. Because the new system of Ustilaginomycetes, acetone. Samples were embedded in Spurr’s plastic and now considered as the subphylum Ustilaginomycotina (Bauer sectioned with a diamond knife. Serial sections were mounted et al. 2006), revealed that parallel evolution with their hosts on formvar-coated, single-slot copper grids, stained with lead was apparently a widespread phenomenon in the ustilagino- citrate at room temperature for 5 min, and washed with mycotinous phylogeny (Bauer et al. 1997, 2001), the uniform distilled water. host spectrum of the anther smuts on monocots suggests that this group is monophyletic. Molecular analyses Because of morphological similarities, Va´nky (1994) treated Ustilago muscari-botryoidis, U. scillae, U. tourneuxii, U. urgineae, Genomic DNA was isolated from herbarium specimens (for and U. vaillantii as belonging to one species, U. vaillantii. With details see Table 1). Methods of isolation and crushing of fun- the new system of Ustilagomycotina it becomes clear that gal material, DNA extraction, amplification, purification of Ustilago species are restricted to poacean hosts (Bauer et al. PCR products, sequencing, and processing of the raw data 1997, Begerow et al. 1997). Thus, based mainly on the liliaceous have been described by Lutz et al. (2004). We determined hosts, Ershad (2000) transferred U. vaillantii together with base sequences of the 5´-end of the nuLSU rDNA, including U. heufleri and U. ornithogali into the new genus Vankya typified the domains D1/D2 (LSU), and the ITS1/2 region of the rDNA, by V. ornithogali. However, anther smuts on monocots differ including the 5.8S rDNA (ITS). The LSU was amplified using in some features from foliicolous Vankya ornithogali the primer pair NL1 and NL4 (O´ Donnell 1992, 1993) or LR6 and V. heufleri. Therefore, we carried out morphological, (Vilgalys & Hester 1990), respectively. The ITS was amplified ultrastructural, and molecular studies in order to propose using the primer pair ITS1f and ITS4 (Gardes & Bruns 1993). a phylogenetic hypothesis for this group. For amplification of both regions we adjusted the annealing temperature to 45 C. DNA sequences prepared in the course of this study were deposited in GenBank, and their accession Materials and methods numbers are listed in Table 1. To ascertain the phylogenetic positions of the analysed an- The specimens of anther smuts on members of Hyacinthaceae ther smut specimens, we followed two strategies: (1) we ana- examined in this study are listed in Table 1. Additionally, DNA lysed the LSU sequences of selected anther smut specimens of the following specimens was sequenced [the first number (compare Fig 2) together with representatives of all genera of refers to the GenBank accession number of the ITS sequence the Urocystales and most of the Ustilaginales (GenBank acces- (if present) and the second number (if present) refers to the sion numbers are given in Fig 2) to support their position in GenBank accession number of the LSU sequence]: Melanustilo- Urocystales, and (2) we analysed the concatenated ITS and spora ari (-/EF517924), Urocystis alopecuri (-/EF517945), LSU sequences of all anther smut specimens growing on Mus- U. beckmanniae (-/EF517943), U. colchici (-/EF517933), U. ficariae cari and Scilla, respectively, within a dataset reduced to repre- (-/EF517938), U. floccosa (-/EF517937), U. galanthi (-/EF517922), sentatives of the Urocystales (GenBank accession numbers are U. ornithogali (-/EF517935), U. syncocca (-/EF517929), Vankya heu- given in Fig 3) to test whether distinct phylogenetic lineages fleri (EF667965/EF517925, EF653981), and V. ornithogali within these specimens could be revealed. (EF635910/-). Nomenclatural novelties were registered in Sequences were aligned for both datasets with MAFFT 5.861 MycoBank (www.MycoBank.org, see Crous et al. 2004). (Katoh et al. 2002, 2005) using the L-INS-i option. Both align- ments [length: 591 bp (LSU), 1311 bp (ITS þ LSU); variable sites: LM 305 (LSU), 385 (ITS þ LSU)] were used throughout their length. We avoided both manipulation of the alignment by hand and Sori and spore characters were studied using dried herbarium manual exclusion of any positions as recommended by Giribet material. Morphological observations and measurements of & Wheeler (1999) and Gatesy et al. (1993), respectively. spores were