Mycol Progress (2015) 14: 88 DOI 10.1007/s11557-015-1114-3 ORIGINAL ARTICLE A molecular phylogenetic framework for Anthracocystis (Ustilaginales), including five new combinations (inter alia for the asexual Pseudozyma flocculosa), and description of Anthracocystis grodzinskae sp. nov. Marcin Piątek1 & Matthias Lutz2 & Nourou S. Yorou3 Received: 4 May 2015 /Revised: 16 July 2015 /Accepted: 2 September 2015 /Published online: 15 September 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract The genus Anthracocystis (Ustilaginales, specimens, related yeast species, and unnamed yeast strains Ustilaginaceae) was recently reinstated for grass-infecting or environmental sequences. The phylogenetic hypothesis de- species of smut fungi that have sori with a peridium composed rived from the dataset is intended to serve as a backbone tree of mostly fungal cells, filiform or slender columellae, persis- for Anthracocystis. 19 ITS and 13 LSU sequences were tent spore balls usually composed of dimorphic spores, and tracked to represent sequences generated from type specimens lacking sterile cells between spore balls. In this study, (holotypes, isotypes or paratypes). These type sequences are Anthracocystis grodzinskae sp. nov. on Euclasta recommended to be deposited in the RefSeq Targeted Loci condylotricha is described and illustrated from the Sudanian database. This study provides the first explicit evidence that savanna biome in Benin (West Africa). The new species is several asexual species are nested within the Anthracocystis compared with two other smut fungi known on Euclasta lineage. The yeast sequences were scattered in different condylotricha, namely Sporisorium euclastae and subclades of Anthracocystis and none of them could be direct- Anthracocystis ischaemoides, in Zambia. It differs from these ly assigned to a teleomorphic species. Only one of these yeast species in a number of morphological characters that are anamorphs was assigned to a species, namely Pseudozyma discussed in detail. The systematic position of flocculosa. In line with the current code of nomenclature, A. grodzinskae was investigated in a phylogenetic analysis and following recent practice of merging yeast species with with a concatenated supermatrix of the internal transcribed sexual species under the older generic name, this yeast is spacer (ITS) and large subunit (LSU) regions of ribosomal recombined into Anthracocystis as A. flocculosa. DNA. The dataset included all representatives of Additionally, new combinations are proposed for four Anthracocystis for which sequences were available in the teliosporic species (Anthracocystis andrewmitchellii, A. National Center for Biotechnology Information's (NCBI’s) christineae, A. kenyana, A. warambiensis). GenBank and that were linked to reliably identified source Keywords Anthracocystis . Phylogeny . Plant pathogens . Pseudozyma flocculosa . Smut fungi . Ustilaginomycotina . Section Editor: Dominik Begerow Yeasts * Marcin Piątek [email protected] Introduction The smut genus Anthracocystis Bref. has been described for a 1 Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Kraków, Poland causative agent of head smut of millet (Panicum miliaceum L.), Anthracocystis destruens (Schltdl.) Bref. (Brefeld 1912; 2 Plant Evolutionary Ecology, Institute of Evolution and Ecology, University of Tübingen, Auf der Morgenstelle 1, McTaggart et al. 2012b), but has not been accepted by most D-72076 Tübingen, Germany smut researchers. Until very recently, it was forgotten or con- 3 Faculty of Agronomy, University of Parakou, BP 123, sidered synonymous with Sporisorium Ehrenb. ex Link Parakou, Benin (Vánky 2002). Molecular phylogenetic studies by Stoll et al. 88 Page 2 of 15 Mycol Progress (2015) 14: 88 (2003, 2005)revealedthatSporisorium species are split into unnamed strains or environmental sequences were included in two main lineages. This finding was later confirmed by other the analyses. Therefore, a second aim of this study was to molecular analyses (Cunnington et al. 2005; Vánky et al. provide a phylogenetic framework for Anthracocystis that 2006; Vánky and Lutz 2011; McTaggart et al. 2012a; Shivas could serve as a backbone tree for future studies on the genus. et al. 2013; Zhang et al. 2013). These two main lineages in- Additionally, teleomorphic and anamorphic species are uni- cluded the type species of Sporisorium (S. sorghi Ehrenb. ex fied under one generic name, fulfilling the requirements of the Link) and Anthracocystis (A. destruens), respectively. In con- current International Code of Nomenclature for algae, fungi, sequence, McTaggart et al. (2012c)reinstatedAnthracocystis and plants (ICN). for the lineage containing A. destruens, and provided diagnos- tic characters to separate Sporisorium and Anthracocystis. Thus, the most important diagnostic characters of the Materials and methods resurrected and emended genus Anthracocystis are: a peridi- um composed of mostly fungal cells, filiform or slender col- Specimen sampling and morphological examination umellae, persistent spore balls usually composed of dimorphic spores (i.e., morphologically different inner and outer spores), The specimens examined are listed in Table 1. The herbarium and a lack of sterile cells between spore balls. specimens are deposited in KRAM F. The characteristics of The members of Anthracocystis are predominantly tropical sori, spore balls, spores, and peridial cells were studied using species, with several species occurring in warm temperate dried herbarium material. The specimens were examined by regions, and few species introduced together with their hosts light microscopy (LM) and scanning electron microscopy to temperate areas. Two species are pathogenic to cultivated (SEM). For LM, small pieces of sori were mounted in 80 % crops, i.e., Anthracocystis destruens causing head smut of lactic acid, heated to boiling and cooled, and then examined millet and Anthracocystis ehrenbergii (J.G. Kühn) under a Nikon Eclipse 80i light microscope. LM micrographs McTaggart & R.G. Shivas causing long smut of sorghum. were taken with a Nikon DS-Fi1 camera. 20 spore balls, 50 Currently, 126 Anthracocystis species are known spores and 10 peridial cells were measured from each speci- (McTaggart et al. 2012c; Denchev and Denchev 2013), of men, at a magnification of×1000, using NIS-Elements BR 3.0 which 60 species were reported from Africa (Vánky imaging software. Except for the walls of spores and peridial et al. 2011). The number of Anthracocystis species in cells, the measurements were adjusted to the nearest 0.5 μm. Africa may, however, be significantly larger, as this con- Spore size range, and the mean and standard deviation were tinent has not been sufficiently explored for smut fungi. calculatedforeachinvestigatedspecimen(Table1). The spe- There are, potentially, many interesting species within cies descriptions include the combined values from all mea- continental or regional scales, as was demonstrated by sured specimens. For SEM, spore balls with spores were recent findings of smut fungi from herbarium materials mounted on carbon tabs and fixed to an aluminium stub with and field studies (Piątek 2006, 2009, 2015;Piątek and double-sided transparent tape. The stubs were sputter-coated Vánky 2007;Piątek et al. 2008, 2012, 2014, 2015). with carbon using a Cressington sputter-coater and viewed In recent surveys to West Africa, many grasses were under a Hitachi S-4700 scanning electron microscope, with screened for smut infections. Euclasta condylotricha a working distance of ca. 12–13 mm. SEM micrographs were (Hochst. ex Steud.) Stapf was continuously smut-disease- taken in the Laboratory of Field Emission Scanning Electron free in most of the surveyed locations. In 2012, however, at Microscopy and Microanalysis at the Institute of Geological two places near the southern border of the Pendjari National Sciences of the Jagiellonian University, Kraków (Poland). Park in northern Benin, moderate infections on inflorescences of Euclasta condylotricha were found that were caused by an DNA extraction, polymerase chain reaction (PCR), ovaricolous smut of the genus Anthracocystis.Thisstudy and sequencing aimed to resolve the specific identity of this smut using light and scanning electron microscopy and DNA sequence analy- Genomic DNA was isolated directly from herbarium speci- ses, and to provide some ecological information obtained from mens. For methods of isolation and crushing of fungal mate- its natural environment. rial, DNA extraction, amplification of the ITS 1 and ITS 2 The phylogenetic placement of this smut fungus was inves- regions of the rDNA including the 5.8S rDNA (ITS, about tigated with the internal transcribed spacer (ITS) and large 740 bp) and the 5′-end of the nuclear large subunit ribosomal subunit (LSU) regions of ribosomal DNA (rDNA). DNA (LSU, about 640 bp), purification of PCR prod- Sequences from all representatives of Anthracocystis that ucts, sequencing, and processing of the raw data see were available in the National Center for Biotechnology Lutz et al. (2004, 2012). DNA sequences determined for this Information's (NCBI’s) GenBank and that were linked to re- study were deposited in GenBank (accession numbers are liably identified source specimens, related yeast species, and given in Fig. 1, Tables 1 and 2). Mycol Progress (2015) 14: 88 Page 3 of 15 88 Table 1 Spore size ranges, mean spore sizes with standard deviations and GenBank