IMA Fungus

IMA Fungus. 9(1): 49-73

Ustilago species causing leaf-stripe smut revisited

Julia Kruse ¹ ² , Wolfgang Dietrich ³ , Horst Zimmermann ⁴ , Friedemann Klenke ⁵ , Udo Richter ⁶ , Heidrun Richter ⁶ , Marco Thines ¹ ² ⁴

1. Goethe University Frankfurt am Main, Faculty of Biosciences, Institute of Ecology, Evolution and Diversity, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany;

2. Biodiversity and Climate Research Centre (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany

3. Barbara-Uthmann-Ring 68, 09456 Annaberg-Buchholz, Germany

4. Cluster for Integrative Fungal Research (IPF), Georg-Voigt-Str. 14-16, D-60325 Frankfurt am Main, Germany

5. Grillenburger Str. 8 c, 09627 Naundorf, Germany

6. Traubenweg 8, 06632 Freyburg / Unstrut, Germany

corresponding authors e-mail: thines@smut-fungi.net, julia.kruse@senckenberg.de

© 2018 International Mycological Association

DOI: 10.5598/imafungus.2018.09.01.05

Published online: 20 March 2018

Published in print: June 2018

Abstract

Leaf-stripe smuts on grasses are a highly polyphyletic group within Ustilaginomycotina, occurring in three genera, Tilletia, Urocystis, and Ustilago. Currently more than 12 Ustilago species inciting stripe smuts are recognised. The majority belong to the Ustilago striiformis-complex, with about 30 different taxa described from 165 different plant species. This study aims to assess whether host distinct-lineages can be observed amongst the Ustilago leaf-stripe smuts using nine different loci on a representative set. Phylogenetic reconstructions supported the monophyly of the Ustilago striiformis-complex that causes leaf-stripe and the polyphyly of other leaf-stripe smuts within Ustilago. Furthermore, smut specimens from the same host genus generally clustered together in well-supported clades that often had available species names for these lineages. In addition to already-named lineages, three new lineages were observed, and described as new species on the basis of host specificity and molecular differences: namely Ustilago jagei sp. nov. on Agrostis stolonifera, U. kummeri sp. nov. on Bromus inermis, and U. neocopinata sp. nov. on Dactylis glomerata.

INTRODUCTION

The term “stripe smut” is commonly used to refer to Ustilaginomycotina species that cause dark brown to black linear sori of varying length in the leaves of grasses (Poaceae). Black spore masses are released after the spores have matured beneath the epidermis in the mesophyll of the host leaves (Fischer 1953, Vánky 2012). The spore release process of sori is unknown, but may be facilitated either by the withering of dead epidermal cells or by enzymatic action, as in the white blister rusts (Heller & 2009). Of the smut genera that infect grasses, Ustilago is the most prevalent (Stoll et al. 2003, 2005, Vánky 2012).

The term stripe smut does not reflect phylogenetic relatedness, as at least two other genera, Tilletia and Urocystis, contain species that manifest similar symptoms. The vast majority of leaf-stripe smuts belong to Ustilago, including U. agropyri, U. bahuichivoensis, U. bethelii, U. calamagrostidis, U. calcarea, U. davisii, U. deyeuxiicola, U. echinata, U. filiformis, U. phlei, U. scrobiculata, U. serpens s. lat., U. sporoboli-indici, U. striiformis s. lat., U. trebouxii, U. trichoneurana, and U. ulei (Vánky 2012). Of these species, U. striiformis s. lat., with the type species described on Holcus lanatus, is a complex occurring on 164 species of Poaceae representing 44 different genera (Achnatherum, Agropyron, Agrostis, Alopecurus, Ammophila, Anthoxanthum, Arctagrostis, Arrhenatherum, Avena, Beckmannia, Brachypodium, Briza, Bromus, Calamagrostis, Cleistogenes, Cynosurus, Dactylis, Danthonia, Deschampsia, Deyeuxia, Elymus, Festuca, Helictotrichon, Hierochloë, Holcus, Hordeum, Hystrix, Koeleria, Leymus, Lolium, Melica, Milium, Pennisetum, Phalaris, Phleum, Piptatherum, Poa, Polypogon, Puccinellia, Sesleria, Setaria, Sitanion, Trisetaria, and Trisetum). Based on host specificity and minor differences in spore size and surface ornamentation, approximately 30 different taxa have been described in the U. striiformis species complex on various host plants (Vánky 2012, Savchenko et al. 2014a). Ustilago serpens probably represents an overlooked species complex, occuring on five host genera: Agropyron, Brachypodium, Bromus, Elymus, and Leymus. Whether other species with large warts on their spores also belong to this complex, such as U. echinata and U. scrobiculata, is currently unclear.

Ustilago striiformis s. lat. on Alopecurus pratensis has often been the sole representative of this group in phylogenetic analyses (Stoll et al. 2005, Begerow et al. 2006, McTaggart et al. 2012a). Stoll et al. (2005) supported the recognition of U. calamagrostidis, a parasite of several species of Calamagrostis, as separate from U. striiformis. The morphological difference was mainly in spore size and ornamentation. Savchenko et al. (2014a) provided a more detailed analysis of the U. striifomis species complex using several host-fungus combinations and phylogenetic reconstructions based on the nrITS and partial LSU regions. However, while two additional species were proposed as distinct in the U. striiformis-complex, the phylogenetic resolution was too low to draw further conclusions regarding host specificity and potential species boundaries. To resolve undescribed lineages within this species complex, Savchenko et al. (2014a) suggested that several additional gene loci and host-fungus combinations should be included. However, in line with Vánky (2012), Savchenko et al. (2014a) suggested that it would be difficult to distinguish between these lineages based on morphological characters. DNA-based characteristics, such as diagnostic SNPs, along with host specificity might be a solution towards characterizing and describing previously-named and new species (Denchev et al. 2009, Piątek et al. 2013). The aim of this study was to use a multigene phylogeny to infer the phylogenetic differentiation in the leaf stripe smuts in the genus Ustilago, particularly those in the U. striiformis species complex.

MATERIAL AND METHODS

Plant and fungal material

Specimens used in the study are listed in Table 1. The names of the hosts and fungi was derived from the latest version of The International Plant Names Index (www.ipni.org), Index Fungorum (www.indexfungorum.org/) and Vánky (2012), and partly following a broad generic concept for Ustilago (Thines 2016). A majority of the samples were collected in Germany (about 76) and most collections were not older than 20 years. Samples are deposited in Herbarium Senckenbergianum Görlitz (GLM). All host identifications were confirmed by ITS sequences.

DNA extraction and PCR

About 2–20 mg of infected plant tissue was taken from fungarium samples, placed in 2 mL plastic reaction tubes and homogenized in a mixer mill (MM2, Retsch) using a combination of three to five 1 mm and two 3 mm metal beads at 25 Hz for 5–10 min. Genomic DNA was extracted using the BioSprint 96 DNA Plant Kit (Qiagen, Hilden) loaded to a KingFisher Flex robot (Thermo Scientific, Dreieich).

The complete nrITS of all DNA extracts were amplified using PCR following the procedure of White et al. (1990). The primer pairs M-ITS1 (Stoll et al. 2003) / ITS4 (White et al. 1990) or M-ITS1 / smITS-R1 (Kruse et al. 2017a) were used as the reverse and forward primers, respectively. For DNA samples from historic specimens, including type specimens, the Ustilaginaceae-optimised reverse primer ITS-US3R (5’TATCAAAACCCGGCAGGGAAG3’), located at the ITS2 region, was used.

The NL1 and NL4 primer pair (O’Donnell 1993) were used to amplify the Large Subunit (LSU) of the nrDNA with an annealing temperature of 53 °C. For other loci, the following regions were amplified with their respective primer pairs and annealing temperatures in brackets: myosin R0.5/F3 (55 °C), map R6/F2 (56 °C), rpl3 R1/F1 (53 °C), tif2 R3/F3 (53 °C), ssc1 R1/F2 (53 °C), sdh1 R3/F2 (53 °C), rpl4A R1/F4 (53 °C), and atp2 R4/F6 (53 °C) (Kruse et al. 2017b).

The plant ITS was amplified using the primer pair ITS1P and ITS4 (Ridgway et al. 2003) at 53 °C annealing temperature. The cycling reaction was performed in a thermocycler (Eppendorf Mastercycler 96 vapo protect; Eppendorf, Hamburg) with an initial denaturation at 95 °C for 4 min, 36 PCR cycles of denaturation at 95 °C for 40 s, annealing between 53–56 °C (depending on the specific primer pair) for 40 s and elongation at 72 °C for 60 s, followed by a final elongation at 72 °C for 4 min. For DNA samples older than 50 years, PCR cycles were increased to 46 cycles and a larger amount of DNA (1.5 μL of extracted DNA in a reaction volume of 11 μL) was used. The resulting amplicons were sequenced at the Biodiversity and Climate Research Centre (BiK-F) laboratory using the abovementioned PCR primers. However, amplicons from M-ITS1/smITS-R1 were sequenced using the ITS4 reverse primer. The resulting sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, Table 1).

Alignments and phylogenetic tree reconstruction

We used 93 samples (including 62 of the Ustilago striiformis-complex) for the phylogenetic analysis; 93 had sequences from nrITS, atp2 (ATP synthase subunit 2) and ssc1 (member of the heat shock proteins), and 70 had additional sequences from myosin (myosin group I), map (methionine aminopeptidase), rpl3 (ribosomal protein L3), tif2 (initial translation factor of elF4A), sdh1 (succinate dehydrogenase ubiquinone flavoprotein), and rpl4A (ribosomal protein L4-A) (Table 1). About two thirds of the samples (62) belonged to the U. striiformis species complex. Sporisorium inopinatum (syn. Langdonia inopinata) was chosen as outgroup, according to the findings of McTaggart et al. (2012a).

Alignments were made on individual loci using mafft v. 7 (Katoh & Standley 2013) using the G-INS-i algorithm. Both leading and trailing gaps of the alignments were removed manually. Two different sets of concatenated alignments for the phylogenetic constructions were generated. The first multigene-alignment includes three loci (ITS, atp2, and ssc1) from 93 smut samples. The resulting total alignment was 1502 bp (ITS: 643 bp, atp2: 595 bp, ssc1: 264 bp). The second multigene-alignment included nine genes with a final alignment of 3156 bp (ITS: 643 bp, atp2: 595 bp, ssc1 264 bp, map: 251 bp, myosin: 257 bp, rpl4A: 415 bp, rpl3: 218 bp, sdh1: 269 bp, tif2: 244 bp).

The diagnostic bases for the U. striiformis species complex for all gene markers were determined using the above mentioned alignments. One further ITS alignment was created (443 bp), with the sequence of the type specimen of U. bromina (Table 1), the U. bromina sequences from GenBank (KF381006-8) and sequences from the same host-fungus-combination from this study, to check if all specimens were sequence-identical with the type collection of U. bromina on Bromus inermis (data not shown).

For phylogenetic tree constructions, Minimum Evolution (ME) analysis was done using Mega 6.06 (Tamura et al. 2013) with the Tamura-Nei substitution model and assuming complete deletion at 80 % cut-off with 1000 bootstrap replicates. All other parameters were set to default values. Maximum Likelihood (ML) analysis was done using RAxML (Stamatakis 2014) with parameters set to default values and Bayesian analysis was done using MrBayes 3.2 (Ronquist & Huelsenbeck 2003) running five times with model 6 (GTR) using four incrementally heated chains for 10 million generations, sampling every 1000th tree discarding the first 30 % of the obtained trees, all other parameters were set to default on the TrEase webserver (http://www.thines-lab.senckenberg.de/trease).

To account for potentially deviating evolutionary properties, the analysis in ME was done also on a partitioned concatenated dataset. As no supported differences within the topology of the trees were observed in comparison with the un-partitioned dataset, the other analyses were carried out without partitioning.

Morphological examination

For light microscopy, fungarium specimens (GLM-F107417, GLM-F105836, GLM-F107435, GLM-F107413, GLM-F047379, GLM-F105827) were transferred to 60 % lactic acid on a slide. Morphological examination was carried out using a Zeiss Imager M2 AX10 microscope (Carl Zeiss, Göttingen). Measurements of the spores were performed at x400. The measurements are reported as maxima and minima in parentheses, and the mean plus and minus the standard deviation of a number of measurements is given in parenthesis. The means are placed in italics.

RESULTS

Phylogenetic inference

The LSU sequence data were excluded from further analysis since sequences were identical for all members of the Ustilago striiformis species complex (data not shown). All other loci showed SNPs within the U. striiformis cluster. The diagnostic bases (SNPs) with their specific positions are given in Fig. 6.

There were no supported conflicts in the topology of the trees of the single loci and the concatenated trees. Thus, the datasets were combined and used as concatenated for further analysis. The multigene tree based on nine different loci (Fig. 1) showed strong to maximum support for a monophyly of the U. striiformis species complex. If multiple specimens from one host species were included, these grouped together with strong to maximum support, except for the clades corresponding to U. scaura s. lat. (ME 64, ML 63, BA 0.99), U. brizae (ME 63, ML 68, BA 0.99), and U. agrostidis-palustris (ME 71, ML 68, BA 0.99), which received weak to strong support (Fig. 1).

A phylogenetic reconstruction (Fig. 2) with an additional 21 specimens but based on only half of the characters per specimen (ITS, atp2, and ssc1) revealed the same groups as the double-sized alignment, but expectedly with weaker statistical support. For example, the three weak to strongly supported lineages shown in Fig. 1 still grouped together, but with no or weak support (U. brizae – ME 64, ML -, BA 0.79; U. scaura s. lat. – ME -, ML -, BA 0.79; U. agrostidis-palustris – no support), highlighting the importance of gene selection.

In the phylogenetic reconstruction based on fewer genes (Fig. 2) additional specimens were included, which further supported the high degree of genetic differentiation in conjunction with the host species infected. Specimens from Festuca and Lolium grouped together with strong support, while the monophyly of the clade containing samples from Alopecurus species was unsupported to weakly supported (ME 64, ML -, BA -). Two monophyletic groups were absent from the tree with more loci (Fig. 1): one on Calamagrostis villosa and another on Arrhenatherum elatius. Both of these groups were highly supported (Calamagrostis: ME 76, ML 94, BA 0.99; Arrhenatherum: ME 92, ML 99, BA 1) in the tree based on fewer loci (Fig. 2).

In both phylogenetic trees (Figs 1,2), U. cynodontis was inferred as the sister species to the whole U. striiformis species complex. To illustrate the relationships within this species complex further, two additional phylogenetic trees with a reduced sampling and U. cynodontis as outgroup are shown in Figs 3 (9 loci) and 4 (3 loci). The support values and the topology were comparable to the phylogenetic reconstructions in Figs 1–2. In both phylogenetic trees, U. serpens on Elymus repens and on Bromus inermis grouped together with high to maximum support. This group clustered with two further lineages with larger echinulate spores compared to the U. striiformis species complex, which is considered a synapomorphy of this lineage.

The resolution on the backbone was rather low, as highlighted also by the ambiguous placement of U. maydis, which was resolved as a sister group to the pathogens on the majority of panicoid hosts in the tree based on 9 loci (Fig. 1) with moderate to maximum support, while being inferred as a sister to the clade containing the U. species complex as well as the U. nuda species group with lacking to maximum support in the tree based on three loci (Fig. 2).

Morphology

The degree of overlap in morphological characteristics was too high in both species complexes to provide easily accessible characteristics for species delimitation (Fig. 5). The individual measurements are included in the species descriptions below and summarized in Table 3.

TAXONOMY

Based on our phylogenetic analyses, the following nomenclature and taxonomic changes are proposed for leaf stripe smuts caused by species of Ustilago. The positions given for the diagnostic bases refer to specific positions in the alignments as highlighted in the alignment consensus sequences in Fig. 4. Only selected synonyms are given here. For a complete synonymy reference should be made to Vánky (2012) and references therein.

Ustilago agrostidis-palustris W. H. Davis ex Ciferri, Ann. Mycol. 29: 54 (1931).

Type: USA: Wisconsin: Madison, on cultivated ‘redtop’ (i.e. Agrostis “palustris Huds.”, now Agrostis gigantea), 8 July 1921, W. H. & J. J. Davis (BPI 166994 – lectotype designated here, MBT 380628).

Confirmed host: Agrostis gigantea.

Confirmed distribution: Germany and USA.

Notes: Ustilago agrostidis-palustris can be distinguished from other leaf stripe smuts of the U. striiformis species complex based on its host specific occurrence on Agrostis gigantea s. lat. Furthermore, it differs in one diagnostic base from all other species of the U. striiformis-complex included in this study – in the sdh1 gene there is a C instead of a T at position 138 (Table 2, Fig. 6).

Ustilago airae-caespitosae (Lindr.) Liro, Ann. Acad. Sci. Fenn., ser. A 17 (1): 71 (1924).

Basionym: Tilletia airae-caespitosae Lindr., Acta Soc. Fauna Flora Fenn. 26:15 (1904).

Type: Finland: Nyland: Helsingfors, Hagasund, on Aira caespitosa (i.e. Deschampsia caespitosa), 10 Aug. 1902, J. I. Lindroth [Vestergren, Micr. Rar. Sel. no. 806; Sydow, Ustil. no. 316] (M-0236198 – lectotype designated here, MBT 380628; from one of the several duplicate collections treated as “lectotype” by Lindeberg, Symb. Bot. Upsal. 16 (2): 135, 1959).

Confirmed host: Deschampsia caespitosa.

Confirmed distribution: Austria and Finland.

Notes: Within the Ustilago striiformis species complex, U. airae-caespitosae can be distinguished from other species based on the host-specific occurrence on Deschampsia caespitosa. Furthermore, it differs in six diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene it has an A instead of a G at position 22 and 94, in the map gene there is a T instead of a C at position 227, in the myosin gene there is an A instead of a G at position 133, in the rpl3 gene a T instead of a C at position 199, and an A instead of a G at position 576 in the ITS region (Table 2, Fig. 6).

Ustilago alopecurivora (Ule) Liro, Ann. Acad. Sci. fenn., ser. A 17 (1): 72 (1924).

Basionym: Tilletia alopecurivora Ule, Hedwigia 25: 113 (1886).

Synonyms: Uredo longissima var. megalospora Riess, in Rabenhorst, Herb. Viv. Myc. no. 1897 (1854).

Ustilago megalospora (Riess) Cif., Nuovo Giorn. Bot. Ital. 40: 261 (1933).

Type: Germany: Bavaria: Coburg, Hofgarten, on Alopecurus pratensis, June 1879, E. Ule (B – holotype lost); Berlin: Charlottenburg-Nord, Kolonie Königsdamm, slope of ditch, 9 Aug. 1988, H. Scholz (B 70 0014985 – neotype designated here, MBT 380629).

Confirmed host: Alopecurus pratensis.

Confirmed distribution: Germany.

Notes: Within the U. striiformis species complex, U. alopecurivora can be distinguished from other species based on the host-specific occurrence on Alopecurus pratensis. Furthermore, U. alopecurivora differs in three diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene, there is an A instead of a G at position 358, in the map gene there is a G instead of a T at position 192, and in the myosin gene there is a T instead of a C at position 83 (Table 2, Fig. 6).

Ustilago brizae (Ule) Liro, Ann. Acad. Sci. Fenn., Ser. A 17 (1): 74 (1924).

Basionym: Tilletia brizae Ule, Verh. Bot. Ver. Prov. Brandenb. 25: 214 (1884).

Type: Germany: Bavaria: Coburg, Rögener Berg, on Briza media, July 1879, E. Ule [Rabenhorst, Fungi Eur. no. 3604] (M-0147750 – lectotype designated here, MBT 380630; from one of the several duplicate collections treated as “lectotype” by Lindeberg, Symb. Bot. Upsal. 16(2): 135, 1959).

Confirmed hosts: Briza media.

Confirmed distribution: Austria and Germany.

Notes: Within the U. striiformis species complex, U. brizae can be distinguished from other species based on the host-specific occurrence on Briza media. Furthermore, U. brizae differs in one diagnostic base from all other species within the U. striiformis species complex included in this study, except U. bromina on Bromus inermis, in having a C instead of a T at position 621 in the ITS region, and differs from U. bromina by having an A instead of a G at position 223 in the ITS region (Table 2, Fig. 6).

Ustilago corcontica (Bubák) Liro, Ann. Acad. Sci. Fenn., Ser. A 17 (1): 383 (1924).

Basionym: Tilletia corcontica Bubák, Houby Ceské, Hemibasidii 2: 47 (1912).

Type: Czech Republic: on the crest of Riesengebirge Mts, on Calamagrostis halleriana (i.e. C. villosa), 20 July 1872, J. Gerhardt (BPI 172761 – lectotype designated here, MBT 380631; one of the “isolectotypes” of Lindeberg, Symb. Bot. Upsal. 16(2): 114, 1959).

Confirmed host: Calamagrostis villosa.

Confirmed distribution: Czech Republic and Germany.

Notes: Within the U. striiformis species complex, U. corcontica can be distinguished from other species based on the host-specific occurrence on Calamagrostis villosa. Furthermore, U. corcontica differs in one diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene there is an T instead of a C at position 535 (Table 2, Fig. 6).

Ustilago denotarisii A. A. Fischer v. Waldheim, Aperҁu Syst. Ustil.: 22 (1877); as “de Notarisii”.

Type: Italy: on Arrhenatherum spp. (not located but could also not be confirmed as lost; a neotype may need to be designated for this species in the future).

Confirmed hosts: Arrhenatherum species.

Confirmed distribution: Germany and Italy.

Notes: Spores globose to ovoid, standard range (9.0–)10.5– (av. 11.2)–12.0 (–13.5) × (8.0–) 9.0–(av. 9.7)–10.5(–12.0) μm, length/breadth ratio of 1.10- (av. 1.20) -1.38, olive-brown, and finely echinulate. Within the U. striiformis species complex, U. denotarisii can be distinguished from other species based on the host-specific occurrence on Arrhenatherum species. Furthermore, U. denotarisii differs in two diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene there is an A instead of a G at position 346, and in the gene ssc1 there is an A instead of a C at position 182 (Table 2, Fig. 6).

Ustilago echinata J. Schröt., Abh. Schles. Ges. Vaterl. Kult., Abth. Naturwiss.: 48: 4 (1870 [“1869”].

Type: Poland: Silesia: ‘Schwarzwasserbruch’, near Legnica, on Phalaris arundinacea, June 1869, W. G. Schneider [Rabenhorst, Fungi Eur. no. 1497] (FR – lectotype designated here, MBT 380632; one of the several duplicate collections previously treated as “lectotype” in Rabenhorst, Fungi Eur. No. 1497).

Reported hosts: Glyceria grandis, Phalaris arundinacea, and Scolochloa festucacea.

Confirmed host: Phalaris arundinacea.

Known distribution: Asia, North America, and Europe.

Notes: This species shares one sequence motif (AACCCAAC) at positions 20–27 in the ITS region with other coarsely ornamented stripe smuts (U. serpens clade in Fig. 1), and many SNPs which distinguish U. echinata from species of the U. striiformis-complex. Within the U. serpens-complex, U. echinata can be distinguished from other species based on its host-specific occurrence on Phalaris arundinacea (type host). Whether the other hosts of a similar ecotype are infected by the same species could not be clarified in the current study, but the high degree of host specificity observed in Ustilago renders it possible that specimens from other host genera will have to be described as new species. Furthermore, U. echinata differs in eight diagnostic bases from all other species within the U. serpens species complex included in this study – in the atp2 gene there is a G instead of an A at position 85, in the map gene there is an A instead of a G at position 208, in the myosin gene there is a C instead of an A at position 141 and a T instead of a C at position 156, in the rpl3 gene there is a T instead of a C at position 91 and an A instead of a G at position 146, in the sdh1 gene there is an A instead of a G and at positions 58 and 256, and in the ITS locus there is a C instead of an A at position 19, a C instead of a T at position 38, an A instead of a gap at position 186 and 596 and a G instead of an A at positions 188 and 604 (Tab. 2, Fig. 6).

Due to the generally narrow host specificity of smut fungi, it is conceivable that U. echinata will be revealed to be a species group.

Ustilago jagei J. Kruse & Thines, sp. nov.

MycoBank MB819627

(Fig. 5A–B)

Etymology: Named after mycologist Horst Jage from Kemberg (Germany), who has made significant contributions to the knowledge of phytopathogenic fungi and has enabled well-sampled phylogenetic investigations in various plant pathogens by his outstanding collections.

Diagnosis: Within the U. striiformis species complex, U. jagei can be distinguished from other species based on its host-specific occurrence on Agrostis stolonifera s. lat. Furthermore, U. jagei differs in two diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene there is an A instead of a G at position 466 and in the gene rpl3 there is an A instead of a G at position 92 (Table 2, Fig. 6).

Type: Germany: Saxony-Anhalt: Dessau, Kühnauer Sea, southern shore east-southeast of Großkuhnau, wayside, on Agrostis stolonifera, 16 Sept. 2001, H. Jage (GLM-F047379 – holotype).

Description: Sori as long narrow streaks parallel to vascular bundles, mostly in the leaves, rarely ascending into the inflorescence, initially covered by the epidermis of the plants, which soon frays. Spore mass dark brown to almost black, powdery. Infection systemic, infected plants usually sterile. Spores globose to ovoid, (9.5–) 10.0–(av. 10.9) –11.5(–13.5) x (7.5–) 8.5–(av. 9.3)–10.0(–11.5) μm, length/breadth ratio 1.04-(av. 1.24)-1.5, olive-brown, finely echinulate (Table 3, Figs 3–4).

Confirmed hosts: Agrostis rupestris and A. stolonifera.

Confirmed distribution: Germany and Switzerland.

Notes: It seems possible that U. jagei on Agrostis stolonifera s. lat. represents a species complex, and further investigations with more specimens and additional gene loci are needed to clarify this situation.

Ustilago kummeri J. Kruse & Thines, sp. nov.

MycoBank MB819628

(Fig. 5I–J)

Etymology: Named after the mycologist Volker Kummer from Potsdam (Germany), who has made significant contributions to the knowledge of phytopathogenic fungi and has enabled well-sampled phylogenetic investigations in various plant pathogens by his outstanding ability to recognise easily overlooked plant pathogens.

Diagnosis: Differs from species of the U. striiformis species complex in the larger spores and taller warts. Furthermore, U. kummeri shares one sequence motif at positions 20-27 (AACCCAAC) with other coarsely ornamented stripe smuts, and many SNPs distinguishing it from species of the U. striiformis species complex. Within the U. serpens-complex, U. kummeri can be distinguished from other species based on the host-specific occurrence on Bromus inermis. Furthermore, U. kummeri differs in two diagnostic bases from U. serpens on Elymus repens – in the ITS region there is an C instead of a G at position 260 and G instead of an A at position 629 (Table 2, Fig. 6).

Type: Germany: Brandenburg: Middlemark, Uetz: Hinterer Werder, southwest corner between Sacrow-Paretzer-Channel und Havel-Channel, on Bromus inermis, 19 June 2010, V. Kummer (GLM-F107435 – holotype; VK 2577/17 – isotype).

Description: Sori as long, narrow streaks parallel to vascular bundles, mostly in the leaves, rarely ascending to the inflorescence, initially covered by the epidermis of the plants, which soon frays. Spore mass dark brown, powdery. Infection systemic, infected plants mostly sterile. Spores ovoid to globose, (11.0-) 12.0- (av. 13.0) -14.0 (-15.5) × (9.0-) 10.5- (av. 11.5) -12.0 (-13.5), length/breadth ratio 1.04- (av. 1.15) -1.41, olive-brown, coarsely verrucose to echinulate (Table 3, Figs 5–6).

Confirmed host: Bromus inermis.

Confirmed distribution: Germany.

Notes: It seems likely that additional species will be discovered in the U. serpens clade once more stripe-smuts with coarse spore ornamentation will be scrutinised.

Ustilago loliicola Ciferri, Fl. Ital. Crypt., Par. I. Fungi, Fasc. 17: 345 (1938).

Type: Germany: Berlin: Berlin-Weissensee, on Lolium perenne, Sept. 1877, E. Ule [Rabenhorst, Fungi Eur. no. 2491] (FR – lectotype designated here, MBT 380633; from one of the several duplicate collections treated as “lectotype” by Lindeberg, Symb. Bot. Upsal. 16 (2): 136, 1959).

Confirmed hosts: Festuca arundinacea s. lat. and Lolium perenne.

Confirmed distribution: Germany.

Notes: Within the U. striiformis species complex, U. loliicola can be distinguished from other species based on the specific occurrence on the closely related hosts Festuca arundinacea s. lat. and Lolium perenne. Furthermore, U. loliicola differs in four diagnostic bases from all other species within the U. striiformis species complex included in this study – in the ssc1 locus there is an A instead of a G at positions 210, 214 and 231, and a T instead of a C at position 243 (Table 2, Fig. 6).

Ustilago milii (Fuckel) Liro, Ann. Acad. Sci. Fenn., ser. A 17 (1): 78 (1924).

Basionym: Tilletia milii Fuckel, Jb. nassau. Ver. Naturk. 23–24: 40 (1870).

Type: Germany: Hesse: Rabenkopf Mt., near Oestrich, on Milium effusum, L. Fuckel [Fungi Rhenani no. 2410] (FR – lectotype designated here, MBT 380634, from one of the several duplicate collections treated as “lectotype” in Fuckel, Fungi Rhenani no. 2410).

Confirmed host: Milium effusum.

Confirmed distribution: Germany.

Notes: Within the U. striiformis species complex, U. milii can be distinguished from other species based on the host-specific occurrence on Milium effusum. Furthermore, U. milii differs in two diagnostic bases from all other species within the U. striiformis species complex included in this study – in the atp2 gene there is an A instead of a G at position 301, and in the ITS there is a T instead of a C at position 206 (Table 2, Fig. 6).

Ustilago neocopinata J. Kruse & Thines, sp. nov.

MycoBank MB819630

(Fig. 5E–F)

Etymology: Highlights the unexpected finding that there are several distinct and host-specific species within the U. striiformis species complex.

Diagnosis: Within the U. striiformis species complex, U. neocopinata can be distinguished from other species based on the host-specific occurrence on Dactylis glomerata. Furthermore, U. neocopinata differs in five diagnostic bases from all other species within the U. striiformis species complex included in this study – in the ssc1 gene there is an A instead of a G at positions 69 and 198, in the rpl4A gene there is a T instead of a C at position 120, in the rpl3 gene there is an A instead of a G at position 40, and in the ITS region there is an A instead of a G at position 617 (Table 2, Figs 5–6).

Type: Germany: Bavaria: Upper Franconia, Kronach county, Wallenfels, in the direction of the sewage treatment plant downstream of Stumpfenschneidmühle, on Dactylis glomerata, 15 July 2012, J. Kruse (GLM-F107413 – holotype).

Description: Sori as long small streaks parallel to vascular bundles, mostly in the leaves, very rarely ascending to the inflorescence, initially covered by the epidermis of the plants, which soon frays. Spore mass dark brown to almost black, powdery. Infection systemic, infected plants mostly sterile. Spores mostly globose, rarely ovoid, (9.0–) 10.0– (av. 10.5)–11.0 (–13.0) × (7.5–) 9.0– (av. 9.8) –10.5 (–11) μm, length/breadth ratio 1.00- (av. 1.07) -1.18, olive-brown, finely echinulate (Table 3, Figs 5–6).

Notes: As the host is widespread throughout the Holarctic region, it is conceivable that the species will prove to have a much wider distribution range than currently known.

Ustilago salweyi Berk. & Broome, Ann. Mag. Nat. Hist. 5: 463 (1850).

(Fig. 5G–H)

Type: UK: Channel Islands: Guernsey, St Martin’s, on Holcus lanatus [originally misidentified as Dactylis glomerata fide Hubbard, in Stevenson, PIant Dis. Rep. 30: 57, 1946], 1847, T. Salwey (K-M – holotype; K-M00022071 – isotype).

Synonyms: Uredo striiformis Westend., Bull. Acad. R. Sci. Belg., cl. sci. 18: 406 (1852); as “striaeformis”.

Uredo salveii (Berk. & Broome) Oudem., Prodromus Florae Bataviae, 2nd edn,4: 180 (1866).

Tilletia debaryana A.A. Fisch. Waldh., in Rabenhorst, Fungi eur. no. 1097 (1867).

Tilletia striiformis (Westend.) Magnus, Malpighia 1: 8 (1875).

Ustilago striiformis (Westend.) Niessl, Hedwigia 15: 1 (1876).

Tilletia salveii (Berk. & Broome) P. Karst., Bidrag. Kännedom. Finlands Naurt. Folk. 6: 102 (1884).

Confirmed hosts: Holcus lanatus and H. mollis.

Confirmed distribution: Belgium, Germany, and UK.

Notes: Spores globose to ovoid, standard range (9.5–)10.0–(av. 10.6) –11.0 (–12.5) × (7.5–) 9.0–(av. 9.4)–10.0(–10.5) μm, finely echinulate, length/breadth ratio 1.00–(av. 1.15)–1.39. Within the U. striiformis species complex, U. salweyi can be distinguished from other species based on the host-specific occurrence on Holcus lanatus and H. mollis. Furthermore, U. salweyi differs in three diagnostic bases from all other species within the striiformis species complex included in this study – in the rpl4A gene there is a T instead of a C at position 85, in rpl3 there is a T instead of a C at position 133, and in the ITS region there is an A instead of a G at positions 103 (Table 2, Fig. 6).

The original host was misidentified as Dactylis glomerata, but this was found to be incorrect and actually Holcus lanatus by the leading grass specialist C.E. Hubbard (in Stevenson 1946). David Hawksworth also studied the type materials in K-M and concurs. Hosts in their vegetative stage can be misidentified, as some characteristics, such as leaf shape, ligula, and general habit can be modified as a consequence of infection.

Ustilago scaura Liro s. lat. , Ann. Acad. Sci. Fenn., ser. A, 17(1): 73 (1924).

Replaced name: Tilletia avenae Ule, Verh. Bot. Vereins Prov. Brandenburg 25: 214 (1884).

Type: Germany: Bavaria: Coburg, Fortress, on Avena pratensis (i.e. Helictotrichon pratense), June 1879, E. Ule (s. n. – lost); Hesse: county Tann/Rhön, at Galgenmount, on Avena pubescens [now, Helictotrichon pubescens], 16 Sept. 1990, H. Scholz (B 70 0014830 – neotype designated here, MBT 380637).

Non Ustilago avenae (Pers.) Rostrup, Overs. K. danske Vidensk. Selsk. Forh. Medlemmers Arbeider: 13 (1890).

Confirmed host: Helictotrichon pubescens, H. pratense?

Confirmed distribution: Germany.

Notes: Within the U. striiformis species complex, U. scaura s. lat. can be distinguished from other species based on the host-specific occurrence on Helictotrichon pratense and H. pubescens. Furthermore, U. scaura s. lat. differs in one diagnostic base from all other species within the U. salweyi species complex included in this study, except U. denotarisii on Arrhenatherum spp., in having a T instead of a C at position 628 in the ITS region, and from U. denotarisii on Arrhenatherum elatius in having a 13 nucleotide deletion at positions 222-241 in the ITS alignment (Table 2, Fig. 6).

Since the type has been lost, we designate a neotype for Ustilago scaura with material on the closely related H. pubescens.

Ustilago scrobiculata Liro, Ann. Acad. Sci. Fenn., ser. A 17(1): 68 (1924).

Type: Finland: Nyland: Pornainen, Kirveskoski, on Calamagrostis arundinacea, 9 Aug. 1916, T. Putkonen & J. I. Liro (H – lectotype, designated by Lindeberg, Symb. Bot. Upsal. 16 (2): 130 (1959).

Synonym: ? Ustilago deyeuxiae L. Guo, Mycosystema 6: 51 (1993).

Reported hosts: Calamagrostis spp. (see Vánky 2012: 1265).

Reported distribution: Asia and Europe.

Notes: This species shares one sequence motif with other coarsely ornamented stripe smuts (AACCCAAC at positions 20–27), which distinguishes it from species of the Ustilago striiformis species complex, and many additional single SNPs. Within the U. serpens species complex, U. scrobiculata differs in 21 diagnostic bases from other species (Table 2, Fig. 6). It seems possible that U. deyeuxiae has not been sampled on Calamagrostis arundinacea, as the host of U. deyeuxiae is given as “Deyeuxia arundinacea” by Guo (1993), which is often seen as a synonym of D. pyramidalis in Asian literature (e.g. Shenglian et al. 2006). Thus, it seems possible that the species needs to be reconsidered as independent from U. scrobiculata once sequence data from the type specimen become available.

Ustilago serpens (P. Karst.) B. Lindeb., Symb. Bot. Upsal. 16(2): 133 (1959).

Basionym: Tilletia serpens P. Karst., Fungi Fenn. Exs., fasc. 6 : no. 599 (1866).

Type: Finland: Merimasku, on “Dactylis glomerata” [re-determined as Elymus repens by Lindeberg, Symb. Bot. Upsal. 16(2): 133, 1959], July 1862, P. Karsten [Fungi Fenn. Exs no. 599] (HUV 10432 – lectotype designated here; MBT 380638 from one of the several duplicate collections treated as “lectotype” by Lindeberg, Symb. Bot. Upsal. 16(2): 133, 1959).

Confirmed host: Elymus repens.

Confirmed distribution: Finland and Germany.

Notes: The spores are small to medium sized, (11.5–) 13.0– (av. 13.5) –14.5 (–15.5) × (10.5–) 11.5 (av. 12.5) –13.0 (–14.0) μm, with a length/breadth ratio of 1.00–(av. 1.09)–1.23 and with coarsely verrucose ornamentation. This species shares one sequence motif with other coarsely ornamented stripe smuts (AACCCAAC at position 20–27), which distinguishes it from species of the U. striiformis species complex and many additional SNPs. Within the U. serpens-complex, U. serpens can be distinguished from other species based on four diagnostic bases: in the myosin gene there is an A instead of G at position 93, in rpl4A gene there is a T instead of a C at position 232 and in the ITS locus there is a C instead of a T at position 260, and a G instead of an A at position 629 (Table 2, Fig. 6).

Vánky (2012) lists several additional hosts for U. serpens. Due to the narrow specialization of stripe-smut revealed in this study, however, it seems likely that these harbour several distinct species. Until sequence data become available for these host-pathogen combinations, Ustilago on these other hosts is probably best referred to as U. serpens s. lat.

DISCUSSION

In this study, the closely related species of the Ustilago striiformis-complex and some other leaf stripe Ustilago smuts were investigated using multigene phylogenetic reconstructions to clarify their relationships. In total, 62 specimens of the U. striiformis species complex (incl. U. calamagrostidis) and four other leaf stripe smuts (U. echinata, U. filiformis, U. scrobiculata, and U. serpens s. lat.) were studied.

Phylogenetic analyses provided strong support for the polyphyly of the leaf-stripe smuts within Ustilago. However, the multilocus-based phylogenetic trees support the monophyly of the U. striiformis species complex, in contrast to the analysis by Savchenko et al. (2014a), where it was concluded that the U. striiformis group was polyphyletic and the segregation of two species was necessary to render it monophyletic. That interpretation was mainly based on a combined LSU-ITS tree of U. striiformis species, where U. bromina and U. nunavutica were located outside the U. striiformis s. lat. clade. Because of this conflicting result, the ITS region of the type specimen of U. bromina was sequenced (Table 1) and compared with the deposited GenBank sequences of Savchenko et al. (2014a). The type specimen of U. bromina on Bromus inermis had an ITS sequence nearly identical (except for a base exchange in a poly A/T region) with the other specimens identified as this species in the current study. It differed in nine bases compared to the three sequences labelled as U. bromina in Savchenko et al. (2014a). It is conceivable that these specimens belong to another undescribed smut species (the three sequences were obtained from material from Israel and USA, while the type collection was from Germany), or the quality of the sequences was not optimal; almost all differences in the sequences from Savchenko et al. (2014a) in comparison to the sequences from this study were located behind a poly A/T site, which necessitated re-sequencing for several of the specimens used in this study. Furthermore, misidentification of the host plant seems also possible, as no records were found for the occurrence of Bromus inermis in the floras of Israel (http://flora.org.il/en/plants/) or Palestine (Feinbrun-Dothan 1986).

Ustilago nunavutica was the second species that led Savchenko et al. (2014a) to assume that the U. striiformis species complex was polyphyletic. Comparing the ITS and LSU sequences of U. nunavutica with sequences from the current study, the LSU sequence used by Savchenko et al. (2014a) showed several SNPs (data not shown), while all other U. striiformis samples investigated in this study were identical in the LSU region. In contrast, the ITS sequence of U. nunavutica has only few SNPs in comparison to other members of the U. striiformis species complex and is identical with U. neocopinata. It seems possible that the LSU sequence of U. nunavutica either was of bad quality or shows the amplification of a contaminant smut fungus. However, as the genera Puccinellia and Dactylis are not closely related (Schneider et al. 2009) and very high host specificity has been revealed for the closely related species of the U. striiformis species complex in this study, it is unlikely that U. neocopinata and U. nunavutica are conspecific.

In agreement with Stoll et al. (2005) and Spooner & Legon (2006), we found that U. calamagrostidis and U. corcontica belonged to the U. striiformis species complex. However, further resolution within the U. striiformis species complex was only achieved when the protein-coding loci introduced by Kruse et al. (2017b) were employed. The trees revealed a host genus or host species specific occurrence for almost all lineages within the U. striiformis species complex, thus they should be treated as distinct species, supported by the observations of Liro (1924). All specimens from a single host species formed a clade according to the host species (or the host genus, in case of Holcus), with the exception of the rather closely related species Lolium perenne and Festuca arundinacea (Malik & Thomas 1966, Catalán et al. 2004, Hand et al. 2010). As most of these clades received high to maximum support, they should be considered to represent distinct species, which can be distinguished based on the host and diagnostic SNPs (Fig. 6). For most of the 14 lineages of the U. striiformis species complex validly published names are available, necessitating the description of only two new species in this complex, U. neocopinata on Dactylis glomerata and U. jagei on Agrostis stolonifera s. lat. Vánky (2012) and Savchenko et al. (2014a) mentioned that different species on different hosts within this complex vary remarkably in spore shape, size, and ornamentation. However, morphological variation was observed to be high even within the same host species in the current study and also by Vánky (2012). Thus it is difficult to distinguish these closely related species based on morphology, necessitating the consideration of hosts and SNPs for diagnosis. The host range of at least two species of Ustilago parasitic to Agrostis could not be inferred with certainty, as both ITS and chloroplast loci did not resolve closely related species in the A. stolonifera and A. gigantea clusters (Amundsen & Warnke 2012).

While investigating synonymies of the U. striiformis species complex, it was found that the name U. salweyi is the correct name for the stripe smut on Holcus lanatus. Stevenson (1946) flagged U. salweyi as a “nomen ambiguum”, although no action was taken to formally reject the name. Following the ICN (McNeill et al. 2012), the name U. salweyi has priority over Uredo striiformis as it was published two years earlier (Berkeley & Broome 1850: 463). Although the group generally referred to as the U. striiformis-group does not contain a species with that as the correct name, as it is still included as a synonym we feel that it is best to continue to use “U. striiformis-group” or “species complex” for these fungi as it is so well established and recalls the symptoms all species of the complex exhibit, although this feature is shared by some leaf-stripe smuts not belonging to this complex.

The species within the U. striiformis species complex have sometimes been recognised as special forms based on infection trials (Liro 1924, Davis 1930, 1935, Fischer 1940). However, it has been shown for various biotrophic pathogens that the special form concept, in which there is a population continuum with somewhat specialised forms, cannot be upheld (Göker et al. 2004, Lutz et al. 2005, Kemler et al. 2009, Thines et al. 2009, Ploch et al. 2011, Savchenko et al. 2014b, Choi & Thines 2015).

Similar to the situation in the U. striiformis species complex, Ustilago serpens s. lat. on different hosts clustered in phylogenetically distinct subgroups. As the type host for U. serpens is Elymus repens, the collections from Bromus inermis warrants recognition as a new species. Ustilago serpens is another example illustrating the narrow host specialization among smut fungi. As for both the coarsely ornamented stripe-smuts (U. serpens clade) and the finely ornamented stripe smuts (U. striiformis clade) only a subset of the known hosts could be included in the current study. It is therefore conceivable that some older names published for specific host-pathogen combinations in these groups warrant recognition and several new species await discovery.

With respect to the global phylogeny of Ustilago it is noteworthy that even based on nine loci the backbone of the phylogenetic tree was only poorly resolved. Conflicting supported topologies were inferred with respect to the phylogenetic position of U. maydis in the reconstructions based on three (sister to a clade comprising, among others, the U. nuda and the U. salweyi clade) and nine loci (sister to a clade comprising the majority of smuts on panicoid grasses).

This highlights the high degree of uncertainty that there still is with respect to the global phylogeny of Ustilago s. lat. (Thines 2016). Considering the diversity of anatomical characteristics and disease syndromes caused, many of which have arisen several times independently (such as the stripe-smut habit; McTaggart et al. 2012a, b, c), any splitting of Ustilago s. lat. into smaller genera as suggested by McTaggart et al. (2012a, 2016) is probably premature and might become obsolete due to the high degree of parallel evolution and associated homoplasy.

Fig. 1.View larger versionFig. 1. Phylogenetic tree based on Minimum Evolution analysis of nine loci (ITS, myosin, map, rpl3, tif2, ssc1, sdh1, rpl4A, atp2). Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

Fig. 2.View larger versionFig. 2. Phylogenetic tree based on Minimum Evolution analysis of three loci (ITS, ssc1, atp2). Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘.. The bar indicates the number of substitutions per site.

Fig. 3.View larger versionFig. 3. Phylogenetic tree based on Minimum Evolution analysis of nine loci (ITS, myosin, map, rpl3, tif2, ssc1, sdh1, rpl4A, atp2) detailed showing the Ustilago striiformis-complex with the outgroup U. cynodontis. Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

Fig. 4.View larger versionFig. 4. Phylogenetic tree based on Minimum Evolution analysis of three loci (ITS, ssc1, atp2) detailed showing the Ustilago striiformis-complex with the outgroup U. cynodontis. Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

Fig. 5.View larger versionFig. 5. Sori and spores of Ustilago jagei (A–B), U. denotarisii (C–D), U. neocopinata (E–F), U. salweyi (G–H), U. kummeri (I–J), and U. serpens s. str. (K–L). A. Sori of U. jagei on Agrostis stolonifera (GLM-F047379); B. Teliospores seen by LM; C. Sori of U. denotarisii on Arrhenatherum elatius (GLM-F105836); D. Teliospores seen by LM; E. Sori of U. neocopinata on Dactylis glomerata (GLM-F107413); F. Teliospores seen by LM; G. Sori of U. salweyi on Holcus lanatus (GLM-F107417); H. Teliospores seen by LM; I. Sori of U. kummeri on Bromus inermis (GLM-F107435); J. Teliospores seen by LM; K. Sori of U. serpens s. str. on Elymus repens (GLM-F105827); and L. Teliospores seen by LM.

Fig. 6.View larger versionFig. 6. Alignment consensus sequences for the alignments used in this study with positions of diagnostic bases highlighted in bold face.

See full tableTable 1. Smut specimens used for phylogenetic analysis.

See full tableTable 2. Diagnostic bases within the Ustilago striiformis and the Ustilago serpens complexes.

See full tableTable 3. Measurements from 100 teliospores for four different species of the Ustilago striiformis-complex on Agrostis stolonifera, Dactylis glomerata, Arrhenatherum elatius, and Holcus lanatus, as well as two species of the Ustilago serpens-complex on Elymus repens and Bromus inermis.

Acknowledgments

This study was funded by the LOEWE research funding programme in the framework of the Cluster for Integrative Fungal Research (IPF). We are grateful to the curators of B (Berlin), GLM (Görlitz), M (Munich), and TUB (Tübingen) for allowing the investigation of specimens in their keeping, and David Hawksworth for important information on Ustilago salweyi types in K-M (Kew fungarium) and nomenclatural corrections. We furthermore want to thank the curators of B (Berlin), HBG (Hamburg) and L (Leiden) for searching for the type collection of Ustilago scaura. We thank the private collectors Horst Jage and Volker Kummer for allowing us to include some of their collections in this study. We want to thank Bagdevi Mishra for the opportunity to use TrEase (thines-lab@senckenberg.de/trease) for phylogenetic analysis. Furthermore, we are grateful to Y.-J. Choi for help with the primer design for an internal ITS primer and to Reuel Bennett for proof-reading an initial draft of the manuscript. MT and JK conceived the study; JK made collection trips; FK, HJ, HR, HZ, UR, WD, and VK provided material; JK performed laboratory experiments and microscopy, and also analysed the data; JK and MT interpreted the data and prepared the manuscript with contributions from the other authors.

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KG SavchenkoLM CarrisLA CastleburyVP HelutaSP WasserE Nevo (2014a) Stripe smuts of grasses: one lineage or high levels of polyphyly? Persoonia 33: 169–181.25737599

KG SavchenkoLM CarrisLA CastleburyVP HelutaSP WasserE Nevo (2014b) Revision of Entyloma (Entylomatales, Exobasidiomycetes) on Eryngium. Mycologia 106: 797–810.24987132

J SchneiderE DöringKW HiluM Röser (2009) Phylogenetic structure of the grass subfamily Pooideae based on comparison of plastid matK gene–3’trnK exon and nuclear ITS sequences. Taxon 58: 405–424.

L ShenglianC WenliSM Phillips (2006) 87. Deyeuxia Clarion ex P. Beauvois, Ess. Agrostogr. 43. 1812. In: Flora of China (W ZhengyiPH RavenH Deyuan, eds) 22: 348–359. Beijing: Science Press.

BM SpoonerNW Legon (2006) Additions and amendments to the list of British smut fungi. Mycologist 20: 90–96.

A Stamatakis (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312–1313.24451623

JA Stevenson (1946) A nomenclatorial discussion of Ustilago striiformis. PIant Disease Reporter 30: 53–59.

M StollD BegerowF Oberwinkler (2005) Molecular phylogeny of Ustilago, Sporisorium, and related taxa based on combined analyses of rDNA sequences. Mycological Research 109: 342–356.15912952

M StollM PiepenbringD BegerowF Oberwinkler (2003) Molecular phylogeny of Ustilago and Sporisorium species (Basidiomycota, Ustilaginales) based on internal transcribed spacer (ITS) sequences. Canadian Journal of Botany 81: 976–984.

K TamuraG StecherD PetersonA FilipskiS Kumar (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 2725–2729.24132122

M Thines (2016) Proposal to conserve the name Ustilago (Basidiomycota) with a conserved type. Taxon 65: 1170–1171.

M ThinesY-J ChoiE KemenS PlochEB HolubH-D ShinJDG Jones (2009) A new species of Albugo parasitic to Arabidopsis thaliana reveals new evolutionary patterns in white blister rusts (Albuginaceae). Persoonia 22: 123–128.20198144

K Vánky (2012) Smut Fungi of the World. St Paul, MN: American Phytopathological Society Press.

K Vánky (2007) Taxonomic studies on Ustilaginomycetes – 27. Mycotaxon 99: 1–70.

TJ WhiteT BrunsS LeeJ Taylor (1990) Amplification and direct sequencing of fungal ribosomal RNA sequences for phylogenetics. In: PCR Protocols: a guide to methods and applications (N InnisD GelfandJ SninskyT White, eds): 315–322. San Diego: Academic Press.

[Back]

Fig. 1.

Phylogenetic tree based on Minimum Evolution analysis of nine loci (ITS, myosin, map, rpl3, tif2, ssc1, sdh1, rpl4A, atp2). Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

[Back]

[Back]

Fig. 2.

Phylogenetic tree based on Minimum Evolution analysis of three loci (ITS, ssc1, atp2). Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘.. The bar indicates the number of substitutions per site.

[Back]

[Back]

Fig. 3.

Phylogenetic tree based on Minimum Evolution analysis of nine loci (ITS, myosin, map, rpl3, tif2, ssc1, sdh1, rpl4A, atp2) detailed showing the Ustilago striiformis-complex with the outgroup U. cynodontis. Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

[Back]

[Back]

Fig. 4.

Phylogenetic tree based on Minimum Evolution analysis of three loci (ITS, ssc1, atp2) detailed showing the Ustilago striiformis-complex with the outgroup U. cynodontis. Numbers on branches denote support in Minimum Evolution, Maximum Likelihood and Bayesian Analyses, in the respective order. Values below 55 % are denoted by ‘-‘. The bar indicates the number of substitutions per site.

[Back]

[Back]

Fig. 5.

Sori and spores of Ustilago jagei (A–B), U. denotarisii (C–D), U. neocopinata (E–F), U. salweyi (G–H), U. kummeri (I–J), and U. serpens s. str. (K–L). A. Sori of U. jagei on Agrostis stolonifera (GLM-F047379); B. Teliospores seen by LM; C. Sori of U. denotarisii on Arrhenatherum elatius (GLM-F105836); D. Teliospores seen by LM; E. Sori of U. neocopinata on Dactylis glomerata (GLM-F107413); F. Teliospores seen by LM; G. Sori of U. salweyi on Holcus lanatus (GLM-F107417); H. Teliospores seen by LM; I. Sori of U. kummeri on Bromus inermis (GLM-F107435); J. Teliospores seen by LM; K. Sori of U. serpens s. str. on Elymus repens (GLM-F105827); and L. Teliospores seen by LM.

[Back]

[Back]

Fig. 6.

Alignment consensus sequences for the alignments used in this study with positions of diagnostic bases highlighted in bold face.

[Back]

[Back]

Table 1.

Smut specimens used for phylogenetic analysis.

DNA-no.
Species
Host
Collection details

gene loci

Location
Date
Collector
Fungarium no.
ITS
atp2
ssc1
map
myosin
rpl4A
rpl3
sdh1
tif2

2354
Sporisorium aff. inopiatum (Langdonia)
Aristida adscensionis
Zambia
12 Apr. 2001
C., T. & K. Vánky
M-0215944
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929824
KY929964
KY930127

474
Ustilago agrostidis-palustris
Agrostis cf. gigantea
Germany, Bavaria
22 Jun. 2012
J. Kruse
GLM-F105832
KY929551
KY930157
KY929994
KY929709
KY929639
KY929849
KY929779
KY929919
KY930082

1374

Agrostis gigantea
Switzerland
9 Jul. 2004
V. Kummer
GLM-F107425
KY929582
KY930188
KY930025
KY929729
KY929659
KY929869
KY929799
KY929939
KY930102

2395

Agrostis sp.
Germany, Lower Saxony
12 Jul. 2014
J. Kruse & H. Jage
GLM-F107439
KY929596
KY930202
KY930039
KY929739
KY929669
KY929879
KY929809
KY929949
KY930112

2287
Ustilago airae-caespitosae
Deschampsia caespitosa
Polen
13 Jul. 1994
H. Scholz
B 70 0014901
KY929526
KY930132
KY929969
KY929688
KY929618
KY929828
KY929758
KY929898
KY930061

2401

Deschampsia caespitosa
Austria, Upper Austria
15 Aug. 2014
J. Kruse
GLM-F107444
KY929601
KY930207
KY930044
KY929744
KY929674
KY929884
KY929814
KY929954
KY930117

2402

Deschampsia caespitosa
Austria, Upper Austria
15 Aug. 2014
J. Kruse
GLM-F107445
KY929602
KY930208
KY930045
KY929745
KY929675
KY929885
KY929815
KY929955
KY930118

477
Ustilago alopecurivora
Alopecurus pratensis
Germany, Hesse
22 May 2010
J. Kruse
GLM-F105834
KY929553
KY930159
KY929996
KY929711
KY929641
KY929851
KY929781
KY929921
KY930084

1376

Alopecurus pratensis
Germany, Saxony-Anhalt
20 May 2013
H. Jage
GLM-F107426
KY929583
KY930189
KY930026
–
–
–
–
–
–

1822
Ustilago aff. andropogonis (Sporisorium)
Bothriochloa ischaemum
Germany, Saxony-Anhalt
25 Jul. 2004
H. Jage & H. John
GLM-F062665
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929764
KY929904
KY930067

432
Ustilago perennans
Arrhenatherum elatius
Germany, Schleswig-Holstein
21 Jun. 2007
J. Kruse
GLM-F105817
KY929536
KY930142
KY929979
KY929697
KY929627
KY929837
KY929767
KY929907
KY930070

2398
Ustilago brizae
Briza media
Austria, Tirol
21 Jul. 2014
J. Kruse
GLM-F107442
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929812
KY929952
KY930115

2399

Briza media
Germany, Bavaria
19 Jul. 2014
J. Kruse
GLM-F107443
KY929600
KY930206
KY930043
KY929743
KY929673
KY929883
KY929813
KY929953
KY930116

498
Ustilago bromina
Bromus inermis
Germany, Saxony-Anhalt
04 Jun. 2011
J. Kruse
GLM-F105843
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929785
KY929925
KY930088

500

Bromus inermis
Germany, Thuringia
15 Jun. 2013
J. Kruse
GLM-F105844
KY929563
KY930169
KY930006
KY929716
KY929646
KY929856
KY929786
KY929926
KY930089

1180

Bromus inermis
Germany, Berlin
May 1983
H. Scholz
HUV No 498 (TUB)
KY929613
KY930219
KY930056
–
–
–
–
–
–

2070

Bromus inermis
Germany, Berlin
Aug. 1892
P. Sydow
B 70 0014775
KY929525
–
–
–
–
–
–
–
–

2275

Bromus inermis
Germany, Brandenburg
17 Jul. 2005
H. & I. Scholz
B 70 0014755
KY929524
KY930131
KY929968
–
–
–
–
–
–

2276

Bromus inermis
Germany, Thuringia
10 Sep. 1999
I. Scholz
B 70 0021843
KY929527
KY930133
KY929970
–
–
–
–
–
–

1591
Ustilago aff. bromivora
Bromus rigidus
Greece
23 Apr. 2013
C. & F. Klenke
GLM-F107429
KY929586
KY930192
KY930029
KY929731
KY929661
KY929871
KY929801
KY929941
KY930104

3370

Bromus sterilis
Spain, Andalusia
2 May 2015
J. Kruse
GLM-F107449
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929818
KY929958
KY930121

442
Ustilago calamagrostidis
Calamagrostis epigejos
Germany, Lower Saxony
03 Aug. 2011
J. Kruse
GLM-F105818
KY929537
KY930143
KY929980
–
–
–
–
–
–

445

Calamagrostis epigejos
Germany, Baden-Württemberg
20 Jul. 2013
J. Kruse
GLM-F105819
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929768
KY929908
KY930071

1383

Calamagrostis epigejos
Germany, Saxony-Anhalt
29 Jun. 2013
H. Zimmermann, U. Richter
GLM-F107427
KY929584
KY930190
KY930027
KY929730
KY929660
KY929870
KY929800
KY929940
KY930103

1912

Calamagrostis epigejos
Germany, Saxony-Anhalt
09 Aug. 1996
H. Jage
GLM-F048100
KY929530
KY930136
KY929973
KY929691
KY929621
KY929831
KY929761
KY929901
KY930064

1182
Ustilago corcontica
Calamagrostis villosa
Germany, Saxony
22 Aug. 1987
W. Dietrich
HUV No 794 (TUB)
KY929615
KY930221
KY930058
–
–
–
–
–
–

1611

Calamagrostis villosa
Germany, Saxony-Anhalt
26 Jul. 2003
H. & U. Richter
GLM-F107434
KY929591
KY930197
KY930034
–
–
–
–
–
–

1825
Ustilago cruenta (Sporisorium)
Sorghum bicolor
Greece
11 May 2006
H-W, Otto
GLM-F078871
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929766
KY929906
KY930069

3375
Ustilago cynodontis
Cynodon dactylon
Spain, Andalusia
3 May 2015
J. Kruse
GLM-F107450
KY929607
KY930213
KY930050
KY929749
KY929679
KY929889
KY929819
KY929959
KY930122

3376

Cynodon dactylon
Spain, Andalusia
3 May 2015
J. Kruse
GLM-F107451
KY929608
KY930214
KY930051
KY929750
KY929680
KY929890
KY929820
KY929960
KY930123

1596
Ustilago aff. dactyloctaenii (Sporisorium)
Dactyloctenium australe
South-Africa
22 Feb. 2000
V. Kummer
GLM-F107430
KY929587
KY930193
KY930030
KY929732
KY929662
KY929872
KY929802
KY929942
KY930105

478
Ustilago denotarisii
Arrhenatherum elatius
Germany, Schleswig-Holstein
13 May 2007
J. Kruse
GLM-F105835
KY929554
KY930160
KY929997
–
–
–
–
–
–

481

Arrhenatherum elatius
Germany, Rhineland-Palatinate
23 May 2010
J. Kruse
GLM-F105836
KY929555
KY930161
KY929998
–
–
–
–
–
–

483

Arrhenatherum elatius
Germany, Lower Saxony
31 Jul. 2011
J. Kruse
GLM-F105837
KY929556
KY930162
KY929999
–
–
–
–
–
–

486

Arrhenatherum elatius
Germany, Thuringia
04 Jun. 2012
J. Kruse
GLM-F105838
KY929557
KY930163
KY930000
–
–
–
–
–
–

488

Arrhenatherum elatius
Germany, Bavaria
16 May 2013
J. Kruse
GLM-F105839
KY929558
KY930164
KY930001
–
–
–
–
–
–

447
Ustilago echinata
Phalaris arundinacea
Germany, Lower Saxony
01 Jul. 2010
J. Kruse
GLM-F105820
KY929539
KY930145
KY929982
KY929699
KY929629
KY929839
KY929769
KY929909
KY930072

449

Phalaris arundinacea
Germany, Lower Saxony
29 Aug. 2011
J. Kruse
GLM-F105821
KY929540
KY930146
KY929983
KY929700
KY929630
KY929840
KY929770
KY929910
KY930073

1914

Phalaris arundinacea
Switzerland, St. Gallen
26 Jul. 2000
H. Jage
GLM-F048338
KY929531
KY930137
KY929974
KY929692
KY929622
KY929832
KY929762
KY929902
KY930065

451
Ustilago aff. filiformis
Glyceria fluitans
Germany, Lower Saxony
17 May 2007
J. Kruse
GLM-F105822
KY929541
KY930147
KY929984
KY929701
KY929631
KY929841
KY929771
KY929911
KY930074

454

Glyceria fluitans
Germany, Bavaria
24 Jun. 2012
J. Kruse
GLM-F105823
KY929542
KY930148
KY929985
KY929702
KY929632
KY929842
KY929772
KY929912
KY930075

455

Glyceria fluitans
Germany, Bavaria
10 May 2013
J. Kruse
GLM-F105824
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929773
KY929913
KY930076

456
Ustilago filiformis
Glyceria maxima
Germany, Lower Saxony
01 Jul. 2010
J. Kruse
GLM-F105825
KY929544
KY930150
KY929987
KY929704
KY929634
KY929844
KY929774
KY929914
KY930077

472
Ustilago jagei sp. nov.
Agrostis rupestris
Switzerland, Grisons
02 Aug. 2009
J. Kruse
GLM-F105830
KY929549
KY930155
KY929992
–
–
–
–
–
–

473

Agrostis stolonifera
Germany, Bavaria
20 May 2012
J. Kruse
GLM-F105831
KY929550
KY930156
KY929993
–
–
–
–
–
–

476

Agrostis stolonifera
Germany, Hesse
22 May 2010
J. Kruse
GLM-F105833
KY929552
KY930158
KY929995
KY929710
KY929640
KY929850
KY929780
KY929920
KY930083

551

Agrostis sp.
Germany, Lower Saxony
11 Jun. 2010
J. Kruse
GLM-F107423
KY929580
KY930186
KY930023
KY929727
KY929657
KY929867
KY929797
KY929937
KY930100

2396

Agrostis stolonifera
Germany, Bavaria
20 Jul. 2014
J. Kruse
GLM-F107440
KY929597
KY930203
KY930040
KY929740
KY929670
KY929880
KY929810
KY929950
KY930113

2397

Agrostis stolonifera
Germany, Hesse
27 Jun. 2014
J. Kruse
GLM-F107441
KY929598
KY930204
KY930041
KY929741
KY929671
KY929881
KY929811
KY929951
KY930114

494

Agrostis sp.
Germany, Bavaria
04 Jul. 2013
J. Kruse
GLM-F105841
KY929560
KY930166
KY930003
KY929713
KY929643
KY929853
KY929783
KY929923
KY930086

1375

Agrostis stolonifera
Germany, Saxony-Anhalt
16 Sep. 2001
H. Jage
GLM-F047379
KY929528
KY930134
KY929971
KY929689
KY929619
KY929829
KY929759
KY929899
KY930062

1612
Ustilago kummeri sp. nov.
Bromus inermis
Germany, Brandenburg
19 Jun. 2010
V. Kummer
GLM-F107435
KY929592
KY930198
KY930035
KY929736
KY929666
KY929876
KY929806
KY929946
KY930109

1948

Bromus inermis
Germany, Saxony-Anhalt
17 Jul. 2001
H. Jage, W. Lehman
GLM-F047380
KY929529
KY930135
KY929972
KY929690
KY929620
KY929830
KY929760
KY929900
KY930063

501
Ustilago loliicola
Lolium perenne
Germany, Bavaria
14 May 2013
J. Kruse
GLM-F105845
KY929564
KY930170
KY930007
–
–
–
–
–
–

2288A

Festuca pratensis
Germany, Hesse
25 May 2014
J. Kruse
GLM-F107437
KY929594
KY930200
KY930037
–
–
–
–
–
–

3386

Festuca arundinacea
Germany, Hesse
02 Nov. 2014
J. Kruse
GLM-F107454
KY929611
KY930217
KY930054
KY929753
KY929683
KY929893
KY929823
KY929963
KY930126

2815A
Ustilago maydis
Zea mays
Germany, Saxony-Anhalt
10 Jul. 2007
H. Jage
GLM-F107446
KY929603
KY930209
KY930046
KY929746
KY929676
KY929886
KY929816
KY929956
KY930119

1404
Ustilago milii
Milium effusum
Germany, Saxony-Anhalt
02 Jun. 2002
H. Jage
GLM-F107428
KY929585
KY930191
KY930028
–
–
–
–
–
–

2303

Milium effusum
Germany, Saxony
03 Jun. 2012
W. Dietrich
GLM-F107438
KY929595
KY930201
KY930038
KY929738
KY929668
KY929878
KY929808
KY929948
KY930111

3385

Milium effusum
Germany, Hesse
11 Jun. 2015
J. Kruse
GLM-F107453
KY929610
KY930216
KY930053
KY929752
KY929682
KY929892
KY929822
KY929962
KY930125

503
Ustilago neocopinata sp. nov.
Dactylis glomerata
Germany, Lower Saxony
01 Jul. 2010
J. Kruse
GLM-F105846
KY929565
KY930171
KY930008
–
–
–
–
–
–

505

Dactylis glomerata
Germany, Bavaria
20 Jun. 2010
J. Kruse
GLM-F105847
KY929566
KY930172
KY930009
–
–
–
–
–
–

506

Dactylis glomerata
Germany, Lower Saxony
19 May 2011
J. Kruse
GLM-F105848
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929787
KY929927
KY930090

508

Dactylis glomerata
Germany, Bavaria
19 Jul. 2011
J. Kruse
GLM-F105849
KY929568
KY930174
KY930011
KY929718
KY929648
KY929858
KY929788
KY929928
KY930091

510

Dactylis glomerata
Germany, Bavaria
24 May 2012
J. Kruse
GLM-F105850
KY929569
KY930175
KY930012
KY929719
KY929649
KY929859
KY929789
KY929929
KY930092

512

Dactylis glomerata
Germany, Bavaria
15 Jun. 2012
J. Kruse
GLM-F107413
KY929570
KY930176
KY930013
–
–
–
–
–
–

521

Dactylis glomerata
Germany, Thuringia
15 Jun. 2013
J. Kruse
GLM-F107414
KY929571
KY930177
KY930014
–
–
–
–
–
–

463
Ustilago nuda
Hordeum vulgare
Germany, Bavaria
12 May 2012
J. Kruse
GLM-F105826
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929775
KY929915
KY930078

884
Sporisorium aff. occidentale
Andropogon gerardii
USA
30 Jul. 1989
not known
HUV No 758 (TUB)
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929825
KY929965
KY930128

471
Ustilago salweyi
Holcus mollis
Germany, Bavaria
11 Jun. 2012
J. Kruse
GLM-F105829
KY929548
KY930154
KY929991
KY929708
KY929638
KY929848
KY929778
KY929918
KY930081

489

Holcus mollis
Germany, Bavaria
16 May 2013
J. Kruse
GLM-F105840
KY929559
KY930165
KY930002
KY929712
KY929642
KY929852
KY929782
KY929922
KY930085

523

Holcus lanatus
Germany, Lower Saxony
24 May 2009
J. Kruse
GLM-F107415
KY929572
KY930178
KY930015
KY929720
KY929650
KY929860
KY929790
KY929930
KY930093

524

Holcus lanatus
Germany, Lower Saxony
22 May 2010
J. Kruse
GLM-F107416
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929791
KY929931
KY930094

525

Holcus lanatus
Germany, Lower Saxony
27 May 2010
J. Kruse
GLM-F107417
KY929574
KY930180
KY930017
KY929722
KY929652
KY929862
KY929792
KY929932
KY930095

531

Holcus lanatus
Germany, Bavaria
17 May 2012
J. Kruse
GLM-F107418
KY929575
KY930181
KY930018
–
–
–
–
–
–

541

Holcus mollis
Germany, Saxony
03 Jun. 2011
J. Kruse
GLM-F107419
KY929576
KY930182
KY930019
KY929723
KY929653
KY929863
KY929793
KY929933
KY930096

543

Holcus mollis
Germany, Saxony-Anhalt
05 Jun. 2011
J. Kruse
GLM-F107420
KY929577
KY930183
KY930020
KY929724
KY929654
KY929864
KY929794
KY929934
KY930097

544

Holcus mollis
Germany, Saxony-Anhalt
05 Jun. 2011
J. Kruse
GLM-F107421
KY929578
KY930184
KY930021
KY929725
KY929655
KY929865
KY929795
KY929935
KY930098

545

Holcus mollis
Germany, Lower Saxony
17 Aug. 2011
J. Kruse
GLM-F107422
KY929579
KY930185
KY930022
KY929726
KY929656
KY929866
KY929796
KY929936
KY930099

497
Ustilago scaura
Helictotrichon pubescens
Germany, Rhineland-Palatinate
23 May 2010
J. Kruse
GLM-F105842
KY929561
KY930167
KY930004
KY929714
KY929644
KY929854
KY929784
KY929924
KY930087

3384

Helictotrichon pubescens
Germany, Hesse
10 Jun. 2015
J. Kruse
GLM-F107452
KY929609
KY930215
KY930052
KY929751
KY929681
KY929891
KY929821
KY929961
KY930124

1359
Ustilago aff. schroeteriana (Sporisorium)
Paspalum virgatum
Costa Rica
15 Mar. 1991
T. & K. Vánky
HUV No 888 (TUB)
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929826
KY929966
KY930129

1608
Ustilago scrobiculata
Calamagrostis epigejos
Germany, Brandenburg
17 Aug. 2011
V. Kummer & C. Buhr
GLM-F107431
KY929588
KY930194
KY930031
KY929733
KY929663
KY929873
KY929803
KY929943
KY930106

1609

Calamagrostis epigejos
Germany, Thuringia
27 May 2010
V. Kummer
GLM-F107432
KY929589
KY930195
KY930032
KY929734
KY929664
KY929874
KY929804
KY929944
KY930107

1610

Calamagrostis epigejos
Germany, Brandenburg
24 Jun. 2007
V. Kummer
GLM-F107433
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929805
KY929945
KY930108

467
Ustilago serpens
Elymus repens
Germany, Schleswig-Holstein
31 Jul. 2012
J. Kruse
GLM-F105827
KY929546
KY930152
KY929989
KY929706
KY929636
KY929846
KY929776
KY929916
KY930079

469

Elymus repens
Germany, Thuringia
15 Jun. 2013
J. Kruse
GLM-F105828
KY929547
KY930153
KY929990
KY929707
KY929637
KY929847
KY929777
KY929917
KY930080

3110

Elymus repens
Germany, Brandenburg
29 Jun. 2014
V. Kummer
GLM-F107447
KY929604
KY930210
KY930047
–
–
–
–
–
–

1305
Ustilago aff. sorghi (Sporisorium)
Sorghum plumosum
Australia
20 Feb. 1996
A. A. Mitchell, C. & K. Vánky
HUV No 970 (TUB)
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929827
KY929967
KY930130

1951
Ustilago aff. syntherismae
Digitaria sanguinalis
Germany, Saxony-Anhalt
01 Oct . 2004
H. Jage
GLM-F064759
KY929534
KY930140
KY929977
KY929695
KY929625
KY929835
KY929765
KY929905
KY930068

1617

Digitaria sanguinalis
Germany, Brandenburg
11 Aug. 2001
V. Kummer
GLM-F107436
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929807
KY929947
KY930110

553
Ustilago trichophora
Echinochloa crus-galli
Germany, North Rhine-Westphalia
04 Oct . 2010
J. Kruse
GLM-F107424
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
Kruse et al. 2017b
KY929798
KY929938
KY930101

1957

Echinochloa crus-galli
Germany, Saxony-Anhalt
01 Oct . 2003
H. Jage
GLM-F062638
KY929532
KY930138
KY929975
KY929693
KY929623
KY929833
KY929763
KY929903
KY930066

3347
Ustilago aff. vanderystii (Sporisorium)
Hyparrhenia hirta
Spain, Andalusia
22 Apr. 2015
J. Kruse
GLM-F107448
KY929605
KY930211
KY930048
KY929747
KY929677
KY929887
KY929817
KY929957
KY930120

Type specimens are printed in bold face.

[Back]

[Back]

Table 2.

Diagnostic bases within the Ustilago striiformis and the Ustilago serpens complexes.

Gen Loci

atp2
map
ssc1
myosin
rpl4A
rpl3
sdh1
tif2
ITS

U. striiformis-complex
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base

on Agrostis gigantea
x
x
x
x
x
x
x
x
x
x
x
x
138
C / T
x
x
x
x

on Agrostis stolonifera and A. rupestris
466
A / G
x
x
x
x
x
x
x
x
92
A / G
x
x
x
x
x
x

on Alopecurus pratensis
358
A / G
192
G / T
x
x
83
T / C
x
x
x
x
x
x
x
x
x
x

on Arrhenatherum elatius
346
A / G
x
x
182
A / C
x
x
x
x
x
x
x
x
x
x
x
x

on Bromus inermis
191, 244
G / A
x
x
232
C / T
x
x
228, 311
292
A / G

x
x
x
x
23
A / G
621
C / T

on Calamagrostis epigejos
91
A / G
x
x
x
x
x
x
x
x
x
x
x
x
65
T / C
102
T / C

on Calamagrostis villosa
535
T / C
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

on Dactylis glomerata
x
x
x
x
69, 198
A / G
x
x
120
T / C
40
A / G
x
x
x
x
617
A / G

on Deschampsia caespitosa
22, 94
A / G
227
T / C
x
x
133
A / G
x
x
199
T / C
x
x
x
x
576
A / G

on Festuca spp. and Lolium spp.
x
x
x
x
210, 214, 231
243
A / G

x
x
x
x
x
x
x
x
x
x
x
x

on Holcus spp.
x
x
x
x
x
x
x
x
85
T / C
133
T / C
x
x
x
x
103
A / G

on Milium effusum
301
A / G
x
x
x
x
x
x
x
x
x
x
x
x
x
x
206
T / C

U. serpens-complex
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base
Pos.
Base

on Bromus inermis
x
x
X
x
x
x
x
x
x
x
x
x
x
x
x
x
260
629
C / T
G / A

on Calamagrostis epigejos
175
G / A
92
A / C
88, 99
C / G
70
A / G
7
C / G
28
G / T
13
G / A
x
x
215
C / T

181, 429, 496
T / C
203
C / T
93
G / A
225
T / C
91
T / C
25
G / T
522
T / -

352
A / G

255
G / T

187
G / T
100
A / G
597
A / T

606
A / G

on Elymus repens
x
x
x
x
x
x
93
A / G
232
T / C
x
x
x
x
x
x
157
170
A / G
C / T

on Phalaris arundinacea
85
G / A
208
A / G
x
x
141
C / A
x
x
91
T / C
58, 256
A / G
x
x
19
C / A

156
T / C
146
A / G
38
C / T

186, 596
A / -

188, 604
G / A

Slash (/) = instead of, x = no diagnostic bases.

[Back]

[Back]

Table 3.

Measurements from 100 teliospores for four different species of the Ustilago striiformis-complex on Agrostis stolonifera, Dactylis glomerata, Arrhenatherum elatius, and Holcus lanatus, as well as two species of the Ustilago serpens-complex on Elymus repens and Bromus inermis.

| Ustilago striiformis-complex | Ustilago serpens-complex
| U. jagei sp. nov. | U. denotarisii | U. neocopinata sp. nov. | U. salveii | U. serpens | U. kummeri sp. nov.
| on Agrostis stolonifera | on Arrhenatherum elatius | on Dactylis glomerata | on Holcus lanatus | on Elymus repens | on Bromus inermis
| spores | spores | spores | spores | spores | spores
No. | length | width | l/b | length | width | l/b | length | width | l/b | length | width | l/b | length | width | l/b | length | width | l/b
1 | 10.5 | 9.5 | 1.11 | 10 | 9 | 1.11 | 11 | 10.5 | 1.05 | 11.5 | 10.5 | 1.1 | 11 | 10.5 | 1.05 | 14 | 13 | 1.08
2 | 10 | 7.5 | 1.33 | 11.5 | 9 | 1.28 | 9.5 | 9.5 | 1 | 11.5 | 10 | 1.15 | 12 | 10 | 1.2 | 13.5 | 11 | 1.23
3 | 10.5 | 8.5 | 1.24 | 9 | 8 | 1.13 | 11 | 10 | 1.1 | 10 | 9.5 | 1.05 | 12.5 | 10 | 1.25 | 14.5 | 12 | 1.21
4 | 13.5 | 9.5 | 1.42 | 10 | 8 | 1.25 | 10.5 | 9.5 | 1.11 | 11 | 9.5 | 1.16 | 13 | 12 | 1.08 | 14 | 12.5 | 1.12
5 | 11 | 9 | 1.22 | 10.5 | 8.5 | 1.24 | 10.5 | 10 | 1.05 | 12 | 9.5 | 1.26 | 12.5 | 10.5 | 1.19 | 14 | 12 | 1.17
6 | 11 | 10 | 1.1 | 11.5 | 9 | 1.28 | 11 | 9.5 | 1.16 | 12 | 9 | 1.33 | 13 | 12.5 | 1.04 | 11.5 | 11.5 | 1
7 | 9.5 | 8 | 1.19 | 10.5 | 9.5 | 1.11 | 10 | 8.5 | 1.18 | 11 | 9 | 1.22 | 12.5 | 11.5 | 1.09 | 14 | 12 | 1.17
8 | 11 | 8 | 1.38 | 10.5 | 9.5 | 1.11 | 10.5 | 10 | 1.05 | 10.5 | 9 | 1.17 | 12.5 | 9.5 | 1.32 | 14 | 13.5 | 1.04
9 | 10.5 | 10 | 1.05 | 11.5 | 10 | 1.15 | 10.5 | 10 | 1.05 | 10 | 10 | 1 | 13.5 | 11 | 1.23 | 13 | 12.5 | 1.04
10 | 11.5 | 9 | 1.28 | 11.5 | 8.5 | 1.35 | 10.5 | 10.5 | 1 | 10.5 | 9.5 | 1.11 | 13 | 11 | 1.18 | 13.5 | 13.5 | 1
11 | 11.5 | 10 | 1.15 | 11 | 8 | 1.38 | 11 | 10 | 1.1 | 10.5 | 9.5 | 1.11 | 14.5 | 13.5 | 1.07 | 13.5 | 11.5 | 1.17
12 | 11.5 | 8 | 1.44 | 11 | 10 | 1.1 | 11 | 11 | 1 | 10.5 | 10 | 1.05 | 14.5 | 12 | 1.21 | 12.5 | 11 | 1.14
13 | 12 | 8 | 1.5 | 10.5 | 9 | 1.17 | 12 | 10.5 | 1.14 | 12.5 | 9 | 1.39 | 15.5 | 11 | 1.41 | 13.5 | 12.5 | 1.08
14 | 12 | 10.5 | 1.14 | 12 | 9 | 1.33 | 10 | 10 | 1 | 10 | 8 | 1.25 | 13 | 12.5 | 1.04 | 12 | 12 | 1
15 | 10 | 8.5 | 1.18 | 10.5 | 9 | 1.17 | 10.5 | 10.5 | 1 | 11 | 10 | 1.1 | 12.5 | 12 | 1.04 | 13.5 | 12.5 | 1.08
16 | 12 | 11.5 | 1.04 | 10.5 | 9.5 | 1.11 | 10.5 | 9.5 | 1.11 | 10.5 | 9.5 | 1.11 | 13 | 12.5 | 1.04 | 12 | 11.5 | 1.04
17 | 11 | 8 | 1.38 | 12 | 9 | 1.33 | 10 | 9 | 1.11 | 10 | 9 | 1.11 | 12 | 11.5 | 1.04 | 13.5 | 13 | 1.04
18 | 11 | 9.5 | 1.16 | 12.5 | 10.5 | 1.19 | 10 | 9.5 | 1.05 | 11.5 | 9.5 | 1.21 | 13 | 10.5 | 1.24 | 14.5 | 13 | 1.12
19 | 11 | 9 | 1.22 | 10 | 9 | 1.11 | 10.5 | 9.5 | 1.11 | 10 | 9 | 1.11 | 13 | 11.5 | 1.13 | 13 | 11.5 | 1.13
20 | 12 | 9.5 | 1.26 | 12.5 | 11 | 1.14 | 11 | 10.5 | 1.05 | 10.5 | 9.5 | 1.11 | 13 | 12 | 1.08 | 13.5 | 13 | 1.04
21 | 11 | 9.5 | 1.16 | 12.5 | 11.5 | 1.09 | 10.5 | 10 | 1.05 | 10.5 | 9 | 1.17 | 12.5 | 11 | 1.14 | 14.5 | 12 | 1.21
22 | 13 | 9.5 | 1.37 | 13.5 | 12 | 1.13 | 11.5 | 10.5 | 1.1 | 10 | 8.5 | 1.18 | 12 | 11.5 | 1.04 | 13.5 | 12.5 | 1.08
23 | 12.5 | 10 | 1.25 | 13.5 | 10 | 1.35 | 11 | 11 | 1 | 11.5 | 9 | 1.28 | 13 | 11.5 | 1.13 | 13 | 12.5 | 1.04
24 | 11.5 | 10 | 1.15 | 11.5 | 10.5 | 1.1 | 10 | 9.5 | 1.05 | 10 | 9.5 | 1.05 | 13.5 | 12 | 1.13 | 12.5 | 12 | 1.04
25 | 10.5 | 8.5 | 1.24 | 11.5 | 9.5 | 1.21 | 11 | 10.5 | 1.05 | 11 | 9.5 | 1.16 | 13 | 10.5 | 1.24 | 15 | 13.5 | 1.11
26 | 10.5 | 10 | 1.05 | 12.5 | 11 | 1.14 | 11 | 10.5 | 1.05 | 9.5 | 9 | 1.06 | 12 | 10.5 | 1.14 | 13 | 11.5 | 1.13
27 | 11 | 9 | 1.22 | 12.5 | 11.5 | 1.09 | 11 | 10 | 1.1 | 10.5 | 9 | 1.17 | 12.5 | 12 | 1.04 | 13.5 | 13.5 | 1
28 | 10.5 | 10.5 | 1 | 11 | 10.5 | 1.05 | 10.5 | 9 | 1.17 | 10 | 9.5 | 1.05 | 13 | 12 | 1.08 | 13.5 | 11.5 | 1.17
29 | 11 | 9.5 | 1.16 | 11 | 11 | 1 | 11 | 11 | 1 | 10 | 9 | 1.11 | 14 | 12.5 | 1.12 | 13.5 | 11.5 | 1.17
30 | 10.5 | 7.5 | 1.4 | 11 | 9.5 | 1.16 | 10 | 10 | 1 | 10 | 9 | 1.11 | 12 | 11 | 1.09 | 13 | 12.5 | 1.04
31 | 10.5 | 9 | 1.17 | 11 | 9 | 1.22 | 10 | 9.5 | 1.05 | 10 | 9 | 1.11 | 12.5 | 11.5 | 1.09 | 13 | 11 | 1.18
32 | 10 | 8.5 | 1.18 | 11.5 | 10.5 | 1.1 | 10 | 9.5 | 1.05 | 11 | 10 | 1.1 | 14.5 | 12.5 | 1.16 | 14 | 13 | 1.08
33 | 10.5 | 9.5 | 1.11 | 11 | 8.5 | 1.29 | 10.5 | 10.5 | 1 | 10 | 9 | 1.11 | 13 | 11.5 | 1.13 | 14 | 13 | 1.08
34 | 10.5 | 9.5 | 1.11 | 11.5 | 9 | 1.28 | 10.5 | 10.5 | 1 | 11 | 8.5 | 1.29 | 14 | 12.5 | 1.12 | 14 | 13 | 1.08
35 | 11.5 | 10 | 1.15 | 12.5 | 9.5 | 1.32 | 11 | 10 | 1.1 | 11 | 10 | 1.1 | 12 | 11.5 | 1.04 | 12.5 | 12 | 1.04
36 | 12 | 9 | 1.33 | 10.5 | 8.5 | 1.24 | 10.5 | 9.5 | 1.11 | 10 | 9 | 1.11 | 14.5 | 11.5 | 1.26 | 13 | 11 | 1.18
37 | 11 | 9.5 | 1.16 | 12.5 | 10.5 | 1.19 | 11 | 10.5 | 1.05 | 10 | 8.5 | 1.18 | 12.5 | 11.5 | 1.09 | 15 | 13.5 | 1.11
38 | 10.5 | 9 | 1.17 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 10.5 | 9.5 | 1.11 | 14 | 12 | 1.17 | 14 | 13 | 1.08
39 | 9.5 | 9 | 1.06 | 10.5 | 10 | 1.05 | 11 | 10 | 1.1 | 10.5 | 10.5 | 1 | 13 | 10 | 1.3 | 14 | 13.5 | 1.04
40 | 10 | 8.5 | 1.18 | 12.5 | 10.5 | 1.19 | 10.5 | 9.5 | 1.11 | 10 | 9 | 1.11 | 11.5 | 11 | 1.05 | 13 | 13 | 1
41 | 10.5 | 9.5 | 1.11 | 11 | 9.5 | 1.16 | 10 | 9.5 | 1.05 | 10 | 9 | 1.11 | 13.5 | 10.5 | 1.29 | 14.5 | 12.5 | 1.16
42 | 11.5 | 10.5 | 1.1 | 11.5 | 11 | 1.05 | 10 | 10 | 1 | 10.5 | 10 | 1.05 | 12.5 | 9.5 | 1.32 | 13 | 12 | 1.08
43 | 11 | 10.5 | 1.05 | 10 | 10 | 1 | 10.5 | 9.5 | 1.11 | 10.5 | 9.5 | 1.11 | 13.5 | 11 | 1.23 | 13.5 | 11.5 | 1.17
44 | 10 | 9 | 1.11 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 10 | 10 | 1 | 14 | 12 | 1.17 | 14 | 11.5 | 1.22
45 | 10.5 | 8.5 | 1.24 | 11.5 | 10 | 1.15 | 10.5 | 9.5 | 1.11 | 12 | 10 | 1.2 | 13.5 | 10.5 | 1.29 | 13 | 11.5 | 1.13
46 | 10.5 | 8 | 1.31 | 11.5 | 11.5 | 1 | 10.5 | 9.5 | 1.11 | 10.5 | 10 | 1.05 | 14 | 12 | 1.17 | 13.5 | 12 | 1.13
47 | 12.5 | 10.5 | 1.19 | 11 | 10.5 | 1.05 | 9.5 | 8.5 | 1.12 | 9.5 | 9.5 | 1 | 12 | 11.5 | 1.04 | 12.5 | 11.5 | 1.09
48 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 10 | 10 | 1 | 11.5 | 10 | 1.15 | 13.5 | 12 | 1.13 | 13.5 | 11.5 | 1.17
49 | 11 | 9.5 | 1.16 | 11.5 | 9.5 | 1.21 | 10.5 | 9.5 | 1.11 | 10.5 | 10 | 1.05 | 13 | 11.5 | 1.13 | 13 | 12 | 1.08
50 | 10 | 9.5 | 1.05 | 11.5 | 9.5 | 1.21 | 11 | 10.5 | 1.05 | 10.5 | 8 | 1.31 | 13.5 | 12 | 1.13 | 13 | 12.5 | 1.04
51 | 10 | 9.5 | 1.05 | 10 | 8.5 | 1.18 | 10 | 9 | 1.11 | 11 | 9 | 1.22 | 14 | 11.5 | 1.22 | 12.5 | 10.5 | 1.19
52 | 10.5 | 8.5 | 1.24 | 10.5 | 8 | 1.31 | 10 | 11 | 0.91 | 10.5 | 10.5 | 1 | 13.5 | 10.5 | 1.29 | 14.5 | 12 | 1.21
53 | 12 | 9.5 | 1.26 | 10 | 8.5 | 1.18 | 9 | 9 | 1 | 11.5 | 10 | 1.15 | 14 | 11.5 | 1.22 | 14 | 12 | 1.17
54 | 10.5 | 10.5 | 1 | 11 | 8.5 | 1.29 | 10 | 9.5 | 1.05 | 10.5 | 9 | 1.17 | 13 | 12 | 1.08 | 13.5 | 12 | 1.13
55 | 10.5 | 10.5 | 1 | 12 | 9.5 | 1.26 | 10 | 9.5 | 1.05 | 10 | 10 | 1 | 12 | 11 | 1.09 | 14 | 12 | 1.17
56 | 11 | 10 | 1.1 | 9.5 | 9 | 1.06 | 9.5 | 9.5 | 1 | 10.5 | 10.5 | 1 | 12 | 12 | 1 | 13 | 13 | 1
57 | 10.5 | 9 | 1.17 | 10 | 8.5 | 1.18 | 11.5 | 10.5 | 1.1 | 11 | 9.5 | 1.16 | 13 | 10.5 | 1.24 | 14 | 13 | 1.08
58 | 10 | 10 | 1 | 11.5 | 9.5 | 1.21 | 10 | 9 | 1.11 | 10.5 | 10 | 1.05 | 14.5 | 10.5 | 1.38 | 13 | 12.5 | 1.04
59 | 11 | 10 | 1.1 | 11 | 10 | 1.1 | 10 | 7.5 | 1.33 | 10.5 | 9.5 | 1.11 | 13 | 11.5 | 1.13 | 13.5 | 12 | 1.13
60 | 10.5 | 10.5 | 1 | 12 | 9.5 | 1.26 | 10 | 10 | 1 | 10 | 9.5 | 1.05 | 13 | 12 | 1.08 | 14.5 | 12 | 1.21
61 | 10.5 | 8.5 | 1.24 | 11 | 10 | 1.1 | 10.5 | 10 | 1.05 | 10.5 | 9.5 | 1.11 | 13.5 | 10 | 1.35 | 14.5 | 13 | 1.12
62 | 11.5 | 9 | 1.28 | 10.5 | 10 | 1.05 | 11 | 9 | 1.22 | 10.5 | 10 | 1.05 | 13 | 11.5 | 1.13 | 14.5 | 13 | 1.12
63 | 10.5 | 8.5 | 1.24 | 10.5 | 9.5 | 1.11 | 10.5 | 9.5 | 1.11 | 9.5 | 9.5 | 1 | 12.5 | 12 | 1.04 | 13.5 | 12 | 1.13
64 | 10.5 | 9.5 | 1.11 | 10.5 | 10 | 1.05 | 11 | 10.5 | 1.05 | 10 | 9.5 | 1.05 | 12.5 | 12 | 1.04 | 14 | 12.5 | 1.12
65 | 10 | 10 | 1 | 10 | 8.5 | 1.18 | 10.5 | 10.5 | 1 | 11 | 9 | 1.22 | 14.5 | 10.5 | 1.38 | 13 | 12.5 | 1.04
66 | 10.5 | 8.5 | 1.24 | 11.5 | 11 | 1.05 | 11 | 11 | 1 | 10.5 | 8.5 | 1.24 | 11.5 | 10.5 | 1.1 | 13 | 12.5 | 1.04
67 | 11 | 10.5 | 1.05 | 11 | 9.5 | 1.16 | 11.5 | 10 | 1.15 | 11 | 9.5 | 1.16 | 15 | 12 | 1.25 | 13.5 | 12.5 | 1.08
68 | 10.5 | 8.5 | 1.24 | 11 | 9.5 | 1.16 | 11 | 11 | 1 | 10.5 | 9.5 | 1.11 | 12.5 | 11 | 1.14 | 14 | 13 | 1.08
69 | 10 | 10 | 1 | 11.5 | 10.5 | 1.1 | 11.5 | 11 | 1.05 | 10 | 9 | 1.11 | 14 | 11 | 1.27 | 14.5 | 14 | 1.04
70 | 10 | 10 | 1 | 11.5 | 11 | 1.05 | 10 | 9.5 | 1.05 | 11 | 10 | 1.1 | 12 | 11 | 1.09 | 13.5 | 12.5 | 1.08
71 | 11 | 9 | 1.22 | 11 | 10 | 1.1 | 9 | 9 | 1 | 10.5 | 9 | 1.17 | 13 | 10.5 | 1.24 | 13 | 12.5 | 1.04
72 | 10 | 10 | 1 | 10.5 | 10 | 1.05 | 10 | 9.5 | 1.05 | 11 | 10.5 | 1.05 | 13 | 12 | 1.08 | 13.5 | 12.5 | 1.08
73 | 10 | 10 | 1 | 13 | 10 | 1.3 | 11 | 10 | 1.1 | 9.5 | 9 | 1.06 | 11.5 | 11.5 | 1 | 13.5 | 13 | 1.04
74 | 10.5 | 8 | 1.31 | 11 | 9 | 1.22 | 10.5 | 9.5 | 1.11 | 11 | 9.5 | 1.16 | 14 | 11 | 1.27 | 13.5 | 13 | 1.04
75 | 10 | 9.5 | 1.05 | 11.5 | 10 | 1.15 | 10.5 | 9.5 | 1.11 | 10.5 | 10.5 | 1 | 12 | 10.5 | 1.14 | 15.5 | 13.5 | 1.15
76 | 11.5 | 9 | 1.28 | 12 | 10.5 | 1.14 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 11.5 | 9 | 1.28 | 13 | 12.5 | 1.04
77 | 11 | 10 | 1.1 | 10.5 | 10 | 1.05 | 9 | 9 | 1 | 11.5 | 9.5 | 1.21 | 12.5 | 11 | 1.14 | 14 | 12.5 | 1.12
78 | 11.5 | 9.5 | 1.21 | 10.5 | 10 | 1.05 | 9.5 | 9.5 | 1 | 9.5 | 8.5 | 1.12 | 11 | 10.5 | 1.05 | 14.5 | 12 | 1.21
79 | 11 | 9 | 1.22 | 11.5 | 8.5 | 1.35 | 11 | 10 | 1.1 | 10 | 9 | 1.11 | 13 | 11 | 1.18 | 13.5 | 12 | 1.13
80 | 11.5 | 9.5 | 1.21 | 10.5 | 9.5 | 1.11 | 10 | 8.5 | 1.18 | 11 | 8.5 | 1.29 | 11.5 | 11 | 1.05 | 13.5 | 12.5 | 1.08
81 | 9.5 | 9.5 | 1 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 11.5 | 9 | 1.28 | 11.5 | 11 | 1.05 | 13.5 | 10.5 | 1.29
82 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 11 | 9.5 | 1.16 | 10.5 | 10 | 1.05 | 12.5 | 11 | 1.14 | 13 | 13 | 1
83 | 10.5 | 10.5 | 1 | 11.5 | 9 | 1.28 | 11.5 | 9.5 | 1.21 | 11 | 9.5 | 1.16 | 12.5 | 12 | 1.04 | 14.5 | 13 | 1.12
84 | 11.5 | 10 | 1.15 | 11 | 9 | 1.22 | 10 | 9.5 | 1.05 | 10 | 9.5 | 1.05 | 12 | 9.5 | 1.26 | 13.5 | 12.5 | 1.08
85 | 11 | 10 | 1.1 | 10 | 10 | 1 | 11 | 9.5 | 1.16 | 10 | 8 | 1.25 | 13.5 | 11 | 1.23 | 14 | 13 | 1.08
86 | 11 | 9 | 1.22 | 10.5 | 9.5 | 1.11 | 11.5 | 11 | 1.05 | 9.5 | 7.5 | 1.27 | 14 | 9.5 | 1.47 | 14 | 12 | 1.17
87 | 11 | 10.5 | 1.05 | 10.5 | 9 | 1.17 | 11.5 | 10.5 | 1.1 | 11 | 8.5 | 1.29 | 12.5 | 12 | 1.04 | 15 | 13.5 | 1.11
88 | 11 | 9 | 1.22 | 11 | 9.5 | 1.16 | 10 | 9 | 1.11 | 10 | 9 | 1.11 | 12 | 12 | 1 | 12.5 | 12.5 | 1
89 | 10 | 7.5 | 1.33 | 11.5 | 8.5 | 1.35 | 9.5 | 9 | 1.06 | 10 | 9.5 | 1.05 | 15 | 12.5 | 1.2 | 13.5 | 11.5 | 1.17
90 | 11 | 9.5 | 1.16 | 10 | 9.5 | 1.05 | 11 | 9.5 | 1.16 | 11 | 10 | 1.1 | 14.5 | 12 | 1.21 | 12 | 12 | 1
91 | 10.5 | 9 | 1.17 | 13.5 | 11 | 1.23 | 11 | 9.5 | 1.16 | 11 | 10.5 | 1.05 | 12 | 11.5 | 1.04 | 13.5 | 13 | 1.04
92 | 10.5 | 9 | 1.17 | 13.5 | 10.5 | 1.29 | 9.5 | 9.5 | 1 | 11.5 | 10 | 1.15 | 14 | 11 | 1.27 | 13 | 12 | 1.08
93 | 10 | 8.5 | 1.18 | 13 | 10.5 | 1.24 | 9.5 | 7.5 | 1.27 | 11.5 | 9.5 | 1.21 | 13.5 | 11 | 1.23 | 14 | 12.5 | 1.12
94 | 11 | 9.5 | 1.16 | 11.5 | 10.5 | 1.1 | 11 | 11 | 1 | 10.5 | 9.5 | 1.11 | 13 | 11 | 1.18 | 13.5 | 11.5 | 1.17
95 | 10.5 | 9 | 1.17 | 11 | 11 | 1 | 11 | 10 | 1.1 | 10.5 | 10 | 1.05 | 14.5 | 12 | 1.21 | 14.5 | 12 | 1.21
96 | 10.5 | 7.5 | 1.4 | 12 | 10.5 | 1.14 | 10.5 | 8.5 | 1.24 | 10.5 | 9.5 | 1.11 | 12.5 | 10.5 | 1.19 | 14.5 | 13 | 1.12
97 | 11.5 | 9.5 | 1.21 | 11 | 11 | 1 | 10.5 | 10 | 1.05 | 10.5 | 9.5 | 1.11 | 12.5 | 11.5 | 1.09 | 13 | 11 | 1.18
98 | 11.5 | 9.5 | 1.21 | 11 | 9 | 1.22 | 13 | 11 | 1.18 | 11 | 10 | 1.1 | 11.5 | 11 | 1.05 | 13.5 | 11.5 | 1.17
99 | 13.5 | 11.5 | 1.17 | 10.5 | 10 | 1.05 | 10 | 9 | 1.11 | 10.5 | 9.5 | 1.11 | 12.5 | 11.5 | 1.09 | 14.5 | 13.5 | 1.07
100 | 10.5 | 9.5 | 1.11 | 11 | 10 | 1.1 | 10 | 10 | 1 | 11 | 9.5 | 1.16 | 13.5 | 12.5 | 1.08 | 13.5 | 11.5 | 1.17

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