Anther smuts of Caryophyllaceae: Molecular analyses reveal further new species

mycological research 112 (2008) 1280–1296

journal homepage: www.elsevier.com/locate/mycres

Anther smuts of Caryophyllaceae: Molecular analyses reveal further new species5 Matthias LUTZa,*, Marcin PIA ˛ TEKb, Martin KEMLERc, Andrzej CHLEBICKIb, Franz OBERWINKLERa a

Lehrstuhl fu¨r Spezielle Botanik und Mykologie, Botanisches Institut, Universita¨t Tu¨bingen, Auf der Morgenstelle 1, D-72076 Tu¨bingen, Germany b Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krako´w, Poland c AG Geobotanik, Ruhr-Universita¨t Bochum, Geba¨ude ND 03/171, Universita¨tsstraße 150, 44780 Bochum, Germany

article info

abstract

Article history:

Recent collections of Microbotryum (Pucciniomycotina, Basidiomycota) specimens inhabiting

Received 25 July 2007

anthers of different caryophyllaceous host plants were analysed using LM and electron mi-

Received in revised form

croscopy, as well as molecular phylogenetic analyses using rDNA (ITS and LSU) sequences.

2 April 2008

The phylogenetic relationships of caryophyllaceous anther parasites are discussed. Three

Accepted 4 April 2008

new species, Microbotryum adenopetalae, M. minuartiae, and M. silenes-acaulis, are described

Corresponding Editor:

based on morphological, ecological, and molecular characteristics. New host plants are

David L. Hawksworth

reported for Microbotryum dianthorum (Dianthus jacquemontii and Petrorhagia saxifraga) and

Keywords:

a neotype is selected.

M. lychnidis-dioicae (Cucubalus baccifer and Silene zawadskii). For Microbotryum violaceum, Microbotryum

ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Molecular systematics Plant pathology Pucciniomycotina

Introduction Comparative biology in all its aspects has become a powerful tool in evolutionary studies. For instance, the field of comparative genomics has elucidated a plethora of topics in genome evolution and function (e.g. Liti & Louis 2005). To address questions on different levels of evolution it is necessary to compare lineages of different phylogenetic distances, which can be inferred by phylogenetic systematics. The anther smuts of the Caryophyllaceae in the genus Microbotryum (Pucciniomycotina, Basidiomycota) were investigated in a number of fields in biology, e.g. genomics (Hood

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2005), genetics (Garber & Ruddat 2002), or population ecology (Giraud 2004), all of which would profit much from comparative biology. Thereby, our knowledge of the phylogenetic relationships in this group is still very preliminary as many parasites of the numerous different hosts have not yet been studied using molecular systematics. The current classification of Microbotryum (Chlebicki & Sukova´ 2005; Denchev 2007a, b; Lutz et al. 2005; Pia˛tek 2005; Va´nky 2005a, b, c) includes 22 species on Caryophyllaceae, 13 of which occur in the host’s anthers, the others producing spores in different parts of the gynoecium. For ovaricolous Microbotryum species, Denchev et al. (2006) described the genus

Part 234 in the series ‘Studies in heterobasidiomycetes’ from the Botanical Institute, University of Tu¨bingen. * Corresponding author. Tel.: þ49 7071 2978816. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.04.010

Anther smuts of Caryophyllaceae

Haradaea. However, phylogenetic analyses (Kemler et al. 2006) revealed the only member of this group studied so far (Microbotryum holostei) as sister taxon of M. scabiosae clustering within what commonly is accepted as the genus Microbotryum. This contradicts an earlier study (Almaraz et al. 2002). However, a BLAST search in GenBank reveals that the sequence of the seed parasite used in that study (GenBank accession no. AF287152) seems to originate from a contamination by a Cryptococcus species. Thus, distinctness of the genus Haradaea is quite doubtful. The diversity of Microbotryum species in anthers of Caryophyllaceae is best known in Europe, to a lesser extent also in North America (Thrall et al. 1993). Thereby, most collections on various Caryophyllaceae are referred to as Microbotryum violaceum s. lat. (e.g. Garr et al. 1997; Hood et al. 2001; Rabeler 1993; Zundel 1953). Species delimitation within these anther parasites has long been discussed since Liro (1924), who split Ustilago violacea (i.e. Microbotryum violaceum) into a couple of species based on infection experiments and field observations. These were only in part accepted by later workers, who failed to find morphological distinctions between most of Liro’s species (Durrieu & Zambettakis 1973; Nannfeldt in Lindeberg 1959, p. 142, p. 159; Va´nky 1994). Some authors (e.g. Bucheli et al. 2000; Freeman et al. 2002; Perlin 1996; Perlin et al. 1997) regard some or all the anther-infecting species of Caryophyllaceae as special forms of a single species, Microbotryum violaceum s. str. or s. lat., respectively. Conversely, it has been shown by several authors that genetic isolation exists between Microbotryum species on different host plants (e.g. Bucheli et al. 2000). By definition special forms are specialized groups belonging to the same species, thereby still exchanging genetic material and may be in the process of speciation (for examples see Crous 2005). Therefore, it seems justified to treat monophyletic groups of anther smuts on different hosts that seem to be genetically isolated as different species. In recent studies using molecular phylogenetic approaches (Le Gac et al. 2007; Lutz et al. 2005) several hostspecific, so-called ‘cryptic’ species were revealed in Microbotryum occurring in anthers of Caryophyllaceae, suggesting that the concept of a host-specific species delimitation might reflect the evolution of those anther parasites best. Against the background of their molecular phylogenetic analyses, Lutz et al. (2005) intended to provide a framework for species delimitation in Microbotryum which was in agreement with the principles of phylogenetic systematics (Hennig 1965). They proposed that genetically isolated lineages, which can be distinguished by other features in addition to molecular characteristics, should be treated as different species. In the present study the results of the examination of recent collections of Microbotryum specimens inhabiting anthers of different caryophyllaceous host plants (Cucubalus baccifer, Dianthus jacquemontii, Minuartia recurva, Petrorhagia saxifraga, Silene acaulis, S. adenopetala, S. moorcroftiana, and S. zawadskii) applying LM and electron microscopy, as well as molecular phylogenetic analyses using rDNA (ITS and LSU) sequences are reported. The phylogenetic relationships of caryophyllaceous anther parasites are discussed.

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Materials and methods Sample sources, and nomenclature The Microbotryum specimens examined in this study are listed in Table 1. The nomenclature follows Va´nky (1994, 1998, 2004) and Lutz et al. (2005). According to Va´nky (2005a) and in contrast to recent publications, we use the grammatically correct ending for Microbotryum majus instead of M. major. Descriptions and nomenclatural novelties were deposited in MycoBank (www.MycoBank.org; see Crous et al. 2004).

Morphological examination Fungal teliospores of the investigated specimens were mounted in Hoyer’s Fluid (Cunningham 1972), heated to boiling point, and then cooled. Both a Nikon Eclipse 600 light microscope and an Olympus BX-51 equiped with an oil immersion lens were used for LM and spore measurements (magnifications of 1000 and 2000, via a magnification changer), using Nomarski optics (DIC) in some cases. Mean spore size and standard deviation of at least 25 spore diam measurements are included for investigated specimens in Table 1. In the case of basidia, we counted only basidial cells exposed from teliospores. Spore surfaces of Microbotryum sp. on Minuartia recurva (KRAM F55483), on Silene acaulis (KRAM F55485), on S. adenopetala (KRAM F55201) and M. violaceum on S. nutans (GLM 50283) were studied using SEM. In each case dry spores were dusted on carbon tabs and fixed to an aluminium stub with doublesided transparent tape. The tabs were sputter-coated with carbon using a Cressington sputter coater and viewed with a Hitachi S-4700 scanning electron microscope, with a working distance of ca 12–13 mm.

Germination Germination of Microbotryum sp. on Minuartia recurva (HeMP-15, and its duplicates, see Table 1) and on Silene acaulis (HeMP-13, and its duplicates, see Table 1) were observed only in specimens collected in the field. Germination of Microbotryum sp. on S. adenopetala (KRAM F55201) was obtained from teliospores gathered from the central part of an infected flower and spread thinly on water agar (WA, 16 g l1). Petri dishes were incubated at 23  C, 8 h light/16 h darkness. Observations were noted after 5 d.

DNA extraction, PCR and sequencing We isolated genomic DNA from 41 Microbotryum herbarium specimens (Table 1). For methods of isolation and crushing of fungal material, DNA extraction, amplification, purification of PCR products, sequencing, and processing of the raw data see Lutz et al. (2004, 2005). We determined sequences of the 50 -end of the nuLSU rDNA including the domains D1/D2 (LSU) of 37 specimens and the ITS1/2 region of the rDNA including the 5.8S rDNA (ITS) of 17 specimens

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Table 1 – List of examined Microbotryum specimens with host plants, DNA isolation numbers, GenBank accession numbers, mean spore size and standard deviation when measured in the course of this study, and reference specimens Species

GenBank accession no.a

Mean spore size and standard deviation (mm)

ml881

ITS: DQ366856 LSU: DQ366877

6.350  0.397  5.850  0.375

mk282

ITS*: AY877404 LSU: DQ366878

mk305

ITS*: AY877421 LSU: DQ366883

Dianthus sylvestris

ml328

ITS*: AY588082 LSU: DQ366857

D. monspessulanus

ml333

ITS*: AY588080 LSU: DQ366871

D. carthusianorum

ml354

ITS*: AY588079 LSU: DQ366889

D. superbus

ml622

ITS*: AY588081 LSU: DQ366867

D. jacquemontii

ml911

ITS: DQ366844 LSU: DQ366869

5.967  0.472  5.250  0.341

Petrorhagia saxifraga

mk145

ITS: DQ366845 LSU: DQ366866

7.917  0.755  7.233  0.828

Cucubalus baccifer

ml1168

ITS: DQ640066 LSU: DQ640068

6.683  0.564  6.166  0.634

ml1170

ITS: DQ640064 LSU: DQ640067

6.500  0.587  5.733  0.639

ml215

ITS*: AY588094 LSU: DQ366859

ml518

ITS*: AY877416 LSU: DQ366868

ml62

ITS*: AY588096 LSU: DQ366886

ml326

ITS*: AY588097 LSU: DQ366865 ITS: DQ366847 LSU: DQ366860

Host

DNA isolation no.

Microbotryum bardanense

Silene moorcroftiana

M. chloranthae-verrucosum

S. chlorantha

M. dianthorum

M. lychnidis-dioicae

Silene dioica

S. latifolia ssp. alba

S. zawadskii

ml836

Reference specimensb

India, Jammu and Kashmir state, Himalaya Mts, Bardan, alt. 3888 m.a.s.l.; A. Chlebicki, 9 July 2004; KRAM F54962, PRM 907124, HUV 21088 Germany, Brandenburg, Eisenhu¨ttenstadt; S. Ra¨tzel, 13 June 1999; B 700007571 Germany, Brandenburg, Britz; H. Scholz & I. Scholz, 29 June 2001; B 700006053, HUV 21080

6.883  0.364  6.367  0.490

Slovenia, Bovec; D. Begerow & M. Lutz, 10 Aug 2001; TUB 011800 Slovenia, Bovec; D. Begerow & M. Lutz, 10 Aug 2001; TUB 011802 Germany, Saxony-Anhalt, Halle; M. Lutz, 24 Sep 2001; TUB 011801 Switzerland, Grisons, Sur; M. Lutz & W. Maier, 14 July 2003; M 0098771, TUB 011799 India, Jammu and Kashmir state, Himalaya Mts, Bardan, alt. 3888 m.a.s.l.; A. Chlebicki, 8 July 2004; KRAM F54963, PRM 907125, HUV 21089 Italy, Elba, Fetovia; M. Hendrichs, 15 May 2000; TUB 012106 Germany, Saxony, Bad Du¨ben; H. Jage, 22 Sep 1997; GLM 49244 Germany, Saxony-Anhalt, Dessau, Riesigk; H. Jage, 30 Oct 2000; GLM 47387 Germany, BadenWu¨rttemberg, Reutlingen; M. Lutz, 28 May 2001; TUB 011798 Norway, Kristiansund, Farstad; M. Lutz, 14 Aug 2002; TUB 012114 Italy, South Tyrol, Sarntaler Alpen; E. Bronner & M. Lutz, 9 Aug 2000; TUB 011795 Slovenia, Bovec; M. Lutz, 6 Aug 2001; TUB 011796 Poland, Krako´w, Botanical Garden; M. Pia˛tek, 18 May 2004; TUB 012521, HUV 20797

Anther smuts of Caryophyllaceae

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Table 1 – (continued) Species

Host

DNA isolation no.

GenBank accession no.a

Mean spore size and standard deviation (mm)

Reference specimensb

Microbotryum majus

Silene otites

mk279

ITS*: AY877419 LSU: DQ366858

Germany, Brandenburg, Do¨beritz; V. Kummer et al., 19 June 2002; B 700006042

M. saponariae

Saponaria officinalis

ml317

ITS*: AY588089 LSU: DQ366887

S. pumila

ml337

ITS*: AY588091 LSU: DQ366864

Germany, Bavaria, Gu¨nzburg; M. Lutz, 18 July 2001; M 0098773, TUB 011809 Austria, Carinthia, Maltatal; D. Begerow & M. Lutz, 31 July 2001; TUB 011812

M. scabiosae

Knautia arvensis

Uscab1

ITS*: AY588083 LSU: DQ366861

Germany, BadenWu¨rttemberg, Tu¨bingen; L. Kisimova-Horovitz, 8 Aug 1998; TUB 011789

M. silenes-inflatae

Silene vulgaris

ml605

ITS*: AY588105 LSU: DQ366884 ITS*: AY588106 LSU: DQ366879

Austria, Carinthia, Villach; M. Lutz, 18 June 2003; TUB 011793 Switzerland, Grisons, Sur; M. Lutz & W. Maier, 18 July 2003; TUB 011792

ml620

Microbotryum sp. nov.

Minuartia recurva

Silene acaulis

ml835

ITS: DQ366852 LSU: DQ366863

7.950  0.634  7.267  0.537

ml859

ITS: DQ366853 LSU: DQ366862

8.450  0.922  7.483  0.760

mk432

ITS: DQ366850

7.717  0.625  7.017  0.359

ml827

ITS: DQ366846 LSU: DQ366870

7.150  0.494  6.700  0.502

ml828

ITS: DQ366851

7.783  0.665  6.833  0.577

ml829

ITS: DQ366855

7.017  0.382  6.667  0.355

Romania, Muntxii Bucegi, northwestern slopes of Vf. Cosx tila, alt. 2407 m.a.s.l.; A. & M. Ronikier, 26 July 2004; KRAM F55484, TUB 012518, HeMP- 15, HUV 21163 Romania, Muntxii Bucegi, Muntele Caraiman, eastern edge of the plateau towards  , crevices of Valea Alba rocks, alt. 2385 m.a.s.l.; A. & M. Ronikier, 26 July 2004; KRAM F55483dholotype, TUB 012519, HeMP- 31, HUV 20799disotypes Switzerland, Grisons, Sur; M. Kemler & M. Lutz, 15 June 2005; KRAM F55488, TUB 012516 Poland, Tatra Mts, 300 m north from the top of Kopa Kondracka Mt., alt. 1925 m.a.s.l.; M. Pia˛tek & J. Cabała, 25 June 2005; KRAM F55485dholotype, K(M) 137316, LBL M-8583, TUB 012522, HeMP- 18, HUV 21161disotypes Poland, Tatra Mts, southern slopes of Giewont Mt.., alt. 1800 m.a.s.l.; M. Pia˛tek & J. Cabała, 25 June 2005; KRAM F55487, TUB 012520, HeMP- 19, HUV 21162 Poland, Tatra Mts, Hala Ga˛sienicowa glade, alt. 1520 m.a.s.l.; M. Pia˛tek & J. Cabała, 4 June 2004; KRAM F55486, TUB 012517, HeMP13, HUV 20800 (continued on next page)

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Table 1 – (continued) Species

Mean spore size and standard deviation (mm)

ml855

ITS: DQ366854 LSU: DQ366888

7.817  0.713  7.083  0.696

ml875

ITS: DQ366849 LSU: DQ366885

7.150  0.589  6.500  0.415

S. adenopetala

ml877

ITS: DQ366848 LSU: DQ366876

7.200  0.610  6.567  0.537

Stellaria graminea

ml507

ITS*: AY588108 LSU: DQ366873 ITS*: AY588109 LSU: DQ366872

Norway, Oslo; M. Lutz, 15 Aug 2002; TUB 011808 Norway, Molde; M. Lutz, 14 Aug 2002; TUB 011807

ml344

ITS*: AY588099 LSU: DQ366880

ml1167

ITS: DQ640071 LSU: DQ640069

7.233  0.703  6.586  0.683

ml1169

ITS: DQ640065 LSU: DQ640070

7.433  0.762  6.816  0.622

Germany, BadenWu¨rttemberg, Anhausen; G. M. Kovics, 9 June 2001; TUB 011818 Germany, Saxony, Altengroitzsch; H. Jage, 15 June 1997; GLM 49283 Germany, Saxony-Anhalt, Gniest; H. Jage, 19 June 1998; GLM 50283dneotype

S. pusilla

mk292

ITS*: AY877411 LSU: DQ366881

S. rupestris

ml602

ITS*: AY588100 LSU: DQ366874

M. violaceo-irregulare

S. vulgaris

ml50

ITS*: AY588104 LSU: DQ366875

Switzerland, Grisons, Maloja; M. Lutz, 25 July 1998; TUB 011816

M. violaceo-verrucosum

S. viscosa

ml49

ITS*: AY588103 LSU: DQ366882

France, Loze`re, St. Chely du Tarn; D. Begerow & M. Lutz, 19 May 1998; TUB 011815

M. stellariae

Silene acaulis

DNA isolation no.

Reference specimensb

GenBank accession no.a

Microbotryum sp. nov.

Host

ml512

M. violaceum

M. aff. violaceum

Silene nutans

Austria, Salzburger Land, Leoganger Steinberge, alt. 1950 m.a.s.l.; G. Kost & K. Va´nky, 16 July 1987; HUV 13955 Sweden, Lapland, Kiruna, Vistas Valley, alt. 640 m.a.s.l.; M. Koltzenburg, 29 Aug 2005; KRAM F55489, TUB 012515 Kazakhstan, Tian Shan, Zailijskij Alatau Mts, valley of Issyk river, above the point where the Issyk divides into two brooks, alt. 3288 m.a.s.l., N 43 080 0300 , E 77 300 1100 ; A. Chlebicki & M. Sukova´, 29 July 2005; KRAM F55201dholotype, PRM, HUV 21138disotypes

Austria, Salzburgerland, Rauris; V. Kummer, 8 July 2002; B 700006060 Switzerland, canton Bern, Sustenpaß; W. Maier & M. Lutz, 12 June 2003; TUB 011817

a Sequences labelled with an asterisk were taken from GenBank. b Acronyms: B, Herbarium of the Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freien Universita¨t Berlin, Germany; GLM, Herbarium of the Staatliches Museum fu¨r Naturkunde Go¨rlitz, Germany; HeMP, Herbarium Marcin Pia˛tek, Krako´w, Poland; HUV, Herbarium Ustilaginales Va´nky, Tu¨bingen, Germany; K, Herbarium of the Royal Botanic Gardens, Kew, UK; KRAM, Herbarium of the Institute of Botany, Polish Academy of Sciences, Krako´w, Poland; LBL, Herbarium of the M. Curie-Sk1odowska University, Lublin, Poland; M, Botanische Staatssammlung Mu¨nchen, Germany; PRM, Herbarium of the National Museum, Prague, Czech Republic; TUB, Herbarium of the Spezielle Botanik & Mykologie, Eberhard-Karls-Universita¨t Tu¨bingen, Germany.

(see Table 1). The LSU was amplified using the primer pair NL1 and NL4 (O’Donnell 1992, 1993) or LR6 (Vilgalys & Hester 1990), respectively. The ITS was amplified using the primer pair ITS1 and ITS4 (White et al. 1990). For

amplification of both regions we adjusted the annealing temperature to 45  C. DNA sequences prepared in the course of this study were deposited in GenBank; accession numbers are listed in Table 1.

Anther smuts of Caryophyllaceae

Phylogenetic analyses To elucidate the phylogenetic position of the collected Microbotryum specimens, we followed two strategies: (1) we analysed their ITS sequences together with all available Microbotryum ITS sequences of adequate length from caryophyllaceous anther parasites (GenBank accession numbers are given in Fig 1); and (2) we analysed ITS and LSU sequences of the collected Microbotryum specimens within a dataset reduced to few specimens per species. Sequences were aligned for both datasets with MAFFT 5.861 (Katoh et al. 2002, 2005) using the L-INS-i option. Both alignments [length: 537 bp (ITS), 1269 bp (ITS þ LSU); variable sites: 154 (ITS), 208 (ITS þ LSU)] were used throughout their length. We avoided both manipulation of the alignment by hand and manual exclusion of any positions as recommended by Giribet & Wheeler (1999) and Gatesy et al. (1993), respectively. To estimate phylogenetic relationships, we applied a Bayesian approach of phylogenetic inference on both datasets using a MCMC technique as implemented in the computer program MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003) using the general time reversible model of DNA substitution with gamma distributed substitution rates (Gu et al. 1995; Rodrı´guez et al. 1990) and estimation of invariant sites, random starting trees and default starting parameters of the DNA substitution model (Huelsenbeck & Ronquist 2001). Trees were sampled every 100th generation resulting in an overall sampling of 20 001 trees for each alignment. From these, the first 2001 trees were discarded (burn in ¼ 2001). The trees sampled after the process had reached stationarity (18K trees) were used to compute a 60 % majority rule consensus tree to obtain estimates for the PPs of groups of species. This Bayesian approach of phylogenetic analysis was repeated five times to test the independence of the results from topological priors (Huelsenbeck et al. 2002). Based on the results of Freeman et al. (2002), Kemler et al. (2006), and Lutz et al. (2005) the trees were rooted with Microbotryum scabiosae (ITS þ LSU dataset) and the North American M. aff. violaceum specimens AY014239, AY014238, AY014236, AY014235 (ITS dataset), respectively. Pairwise relative base-pair differences were calculated from the MAFFT alignments with PAUP version 4.0b10 using the PAIRDIFF command (Swofford 2001). Note that gaps are not taken into account by this computation and the results are, therefore, not always fully compatible to branch lengths in a tree (Figs 1 and 2), even if pairwise differences are zero.

Results Morphology Spore surface ornamentation was irregularly verrucosereticulate in Microbotryum bardanense, and reticulate in all remaining Microbotryum specimens examined. Spores of Microbotryum sp. on Minuartia recurva (Fig 3D–F), on Silene acaulis (Fig 4A–C), on S. adenopetala (Fig 3A–C), and Microbotryum violaceum on S. nutans (Fig 4D–F) viewed by SEM were reticulate and very variable in respect to shape of meshes,

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from almost rounded and regular to strongly polyangular and sinuose. The spores in some species showed subtle but rather constant morphological differences. In contrast to the remaining species examined, the muri of Microbotryum on Minuartia recurva were often distinctly coronate. The spores of Microbotryum on S. adenopetala were more regular than in the remaining species, and interspaces were often more or less rugulose or even verruculose, not almost smooth as in most other anthericolous Microbotryum species.

Germination Germinating teliospores of Microbotryum sp. on Minuartia recurva and on Silene acaulis were observed only in the specimens collected in the field (in situ germination), while germination of teliospores of Microbotryum sp. on S. adenopetala was observed in the laboratory on WA. The teliospores of Microbotryum sp. on Minuartia recurva (Fig 5B) germinated with 2–4celled phragmobasidia on which few basidiospores without sterigmata were observed; 2-celled basidia predominated. Adjacent basidial cells often fused by a short copulation bridge. The teliospores of Microbotryum sp. on S. acaulis (Fig 5C) germinated with 3–4-celled phragmobasidia, mostly 3-celled, producing basidiospores emerging laterally and terminally without sterigmata. The germination of teliospores of Microbotryum sp. on S. adenopetala (Fig 5A) resulted in 2–4-celled phragmobasidia, with numerous basidiospores emerging laterally and terminally without sterigmata, often budding to yeast-like cells. No copulation bridges between adjacent cells of basidia were observed in Microbotryum sp. on S. acaulis and on S. adenopetala.

Phylogenetic analyses For both, the ITS and ITS þ LSU dataset, the different runs of Bayesian phylogenetic analyses yielded consistent topologies. For each dataset, we present the consensus tree of one run to illustrate the results (Figs 1 and 2). A comparison of the concatenated ITS þ LSU dataset with the ITS dataset showed that the combination of the two rDNA regions resulted in a better resolution of the relationships of higher clades. The two-gene approach resulted in a well-supported monophyletic group containing the clades of Microbotryum dianthorum, M. saponariae, M. aff. violaceum, M. violaceo-verrucosum, and M. stellariae, and the resolution for the inter-clade relationship between these groups was well supported. The ITS dataset exhibited these groups as polytomic in the basal part of the phylogeny, and only resulted in a moderately supported sister relationship of M. dianthorum and M. saponariae, as well as a good supported clade containing M. aff. violaceum and M. chloranthae-verrucosum. A comparison of both approaches revealed only one incongruence. In the concatenated alignment the monophylum containing M. bardanense and M. violaceo-irregulare was included in a group of anther smuts that was sister group to M. chloranthae-verrucosum, whereas in the ITS dataset the M. bardanense/M. violaceo-irregulare group was sister group to a clade of anther smuts containing M. chloranthae-verrucosum. In accordance with Lutz et al. (2005), Kemler et al. (2006), and Le Gac et al. (2007) the two-gene approach resulted in a separation of M. dianthorum in two separate lineages with high support values. The parasites on Silene

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AY014224 AY014223 AY014218 AY588114 AY014227 AY014216 92 AY014217 AY014215 AY014219 AY188368 AY014225 AY014228 60 AY877405 98 AY877410 97 M. on Silene zawadskii* M. lychnidis-dioicae AY588097* AY588096* AY588098 M. on Cucubalus baccifer ml1168* M. on Cucubalus baccifer ml1170* AY588095 AF045873 AY877416* 74 AY877409 AY014222 AY014221 68 AY588094* AY588093 AY014220 ml835* M. sp. on Minuartia recurva ml859* 100 AY877421* 100 M. chloranthae-verrucosum AY877404* AY588105* 100 AY588106* M. silenes-inflatae AY877408 92 75 AY014213 AY014214 69 AY877417 M. aff. violaceum 73 AY588101 AY588102 100 98 100 AF038833 AY588099* M. on Silene nutans ml1169* M. violaceum 88 M. on Silene nutans ml1167* M. bardanense* AY588104* M. violaceo-irregulare 90 ml829 mk432 96 ml827* M. sp. on Silene acaulis ml828 100 ml875* 100 72 ml855* AY014226 AY877418 100 AY877419* M. majus M. sp. on Silene adenopetala* M. sp. on Silene adenopetala AY014232 98 AY014231 AY588077 62 AY014229 62 AY014230 M. on Petrorhagia saxifraga* M. dianthorum 68 AF038834 79 AY588080* 100 100 AY588081* 100 AY588082* 86 AY588079* 83 M. on Dianthus jacquemontii* AY588078 98 AF038832 AY588091* 100 M. saponariae AF045872 AY588089* AY877414 100 AY588109* M. stellariae AY588108* AY877413 90 93 AY588100* M. aff. violaceum 100 AY877411* AF045874 AY588103* M. violaceo-verrucosum 100 AY014235* 77 AY014236 M. aff. violaceum AY014238 AY014239* 0.05 substitutions/site

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acaulis grouped together with high support values, like for the gtub-gene in Le Gac et al. (2007). Our analyses placed, with moderate support values, Microbotryum on S. acaulis inside a cluster of parasites that contained all parasites on Old World Silene species. Two (on Minuartia recurva and S. acaulis) of the three new parasites with more than one specimen available clustered in highly supported monophyletic groups, only the relationship of specimens from Microbotryum lychnidis-dioicae on Cucubalus baccifer was not resolved in our analyses. The parasite on S. zawadskii clustered within M. lychnidis-dioicae. Microbotryum on S. adenopetala did not cluster with any other parasite in this study, but formed part of a basal polytomy in the group of parasites on Old World Silene species and showed a great genetic distance to all other species studied. The recently described M. bardanense (Chlebicki & Sukova´ 2005) clustered together with the specimen of M. violaceo-irregulare.

Taxonomy Microbotryum adenopetalae M. Lutz, Kemler & Chleb., sp. nov. (Figs 3A–C, 5A) MycoBank no.: MB 510817 Etym.: The name refers to the host plant species, Silene adenopetala. Species characteribus generis. Sori indeterminati, teliosporas in calyce efferentes, antheras omnino destruentes, mox pulverulenti, in Silene adenopetala (Caryophyllaceae). Massa sporarum brunneo-violacea. Teliosporae globosae vel ovoideae, 6–8(–8.5)  6–7(8) mm diametro, pariete reticulo denso decorato, inter reticuli cristas laevi vel subtiliter aspero. Sequentia acidi nucleici ITS typi in collectione sequentiarum acidi nucleici NCBI (GenBank) numero DQ366848 deposita est. Typus: Kazakhstan: Tian Shan: Zailijskij Alatau Mts, valley of Issyk river, above the point where the Issyk divides into two brooks, alt. 3288 m.a.s.l., 43 080 0300 N, 77 300 1100 E, on Silene adenopetala, 29 July 2005, A. Chlebicki & M. Sukova´ (KRAM F55201dholotypus; HUV 21138, PRMdisotypi).

Sori in deformed and swollen flowers, teliospores filling the area around the ovary delimited by the calyx, anthers completely destroyed. Infection systemic. Spore mass powdery, brownish violet. Teliospores pale violet, globose, subglobose to ovoid, 6–8(–8.5)  6–7(–8) mm; wall densely reticulate, 5–7 meshes per spore diameter, interspaces smooth, finely rugulose or verruculose. Spore germination results in 2–4-celled basidia, mostly straight, very rarely branched, 10–24  4–5 mm, slightly constricted at the septa;

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basidiospores elliptical, without sterigmata, emerged laterally and terminally, 5–7(–8)  2–3 mm, budding like yeasts. The ITS type sequence from DNA isolation ml877 from the holotype (KRAM F55201) is deposited in GenBank as DQ366848. Host range and distribution: On subfam. Silenoideae: Silene adenopetala; Asia (Kazakhstan), known only from the type locality. Microbotryum minuartiae M. Lutz, Pia˛tek & Kemler, sp. nov. (Figs 3D–F, 5B, 6A) MycoBank no.: MB 510816 Etym.: The name refers to the host plant genus, Minuartia. Species characteribus generis. Sori in antheris Minuartiae recurvae (Caryophyllaceae). Massa sporarum brunneo-violacea. Teliosporae globosae, (7–)8–9(–10)  7–9(–9.5) mm, pariete reticulo denso decorato, inter reticuli cristas laevi. Sequentia acidi nucleici ITS typi in collectione sequentiarum acidi nucleici NCBI (GenBank) numero DQ366853 deposita est. Typus: Romania: Muntxii Bucegi: Muntele Caraiman, eastern edge of  crevices of rocks, alt. 2385 m.a.s.l., the plateau towards Valea Alba on Minuartia recurva, 26 July 2004, A. & M. Ronikier (KRAM F55483d holotypus; HeMP- 31, HUV 20799, TUB 012519disotypi).

Sori in anthers. Infection systemic, all anthers of a plant being infected. Spore mass powdery, brownish violet. Teliospores hyaline to pale violet, globose to subglobose, (7–)8–9(–10)  7–9(–9.5) mm; wall densely reticulate, 5–7 meshes per spore diameter, interspaces smooth. Spore germination without resting period, results in 2–4 celled basidia, mostly 2-celled, 10–20  2.5–3.5 mm, usually about 13  3 mm, adjacent basidial cells often fuse by a short copulation bridge; only few observed ovoid basidiospores emerged laterally. The ITS type sequence from DNA isolation ml859 from the holotype (KRAM F55483) is deposited in GenBank as DQ366853. Host range and distribution: On subfam. Alsinoideae: Minuartia recurva; Europe (Romania), hitherto known only from two locations in Muntxii Bucegi. Additional specimens examined: Romania: Muntxii Bucegi: NW slopes of Vf. Cosx tila, alt. 2407 m.a.s.l., on Minuartia recurva, 26 July 2004, leg. A. & M. Ronikier (HeMP- 15, HUV 21163, KRAM F55484, TUB 012518).

Fig 1 – Bayesian inference of phylogenetic relationships of the sampled Microbotryum specimens: MCMC analysis of an alignment of base sequences from the ITS1/2 region of the nuc-rDNA including the 5.8S rDNA using the GTRDG model of DNA substitution with gamma distributed substitution rates, random starting trees and default starting parameters of the DNA substitution model. A 60 % majority-rule consensus tree computed from 18K trees that were sampled after the process had reached stationarity is shown. The topology was rooted with the North American M. aff. violaceum specimens AY014239, AY014238, AY014236, AY014235. Numbers on branches are estimates for PPs. Branch lengths were averaged over the sampled trees. They are scaled in terms of expected numbers of nucleotide substitutions per site. Assignment to species after Lutz et al. (2005) is indicated. Specimens labelled with an asterisk were included in the ITS D LSU dataset. M. [ Microbotryum.

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M. lychnidis-dioicae ml326 99

M. lychnidis-dioicae ml62 M. lychnidis-dioicae on Silene zawadskii

100 M. lychnidis-dioicae on Cucubalus baccifer ml1170 100

M. lychnidis-dioicae on Cucubalus baccifer ml1168 M. lychnidis-dioicae ml215

82

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M. lychnidis-dioicae ml518 M. sp. on Minuartia recurva ml835

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M. sp. on Minuartia recurva ml859 M. silenes-inflatae ml620

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M. silenes-inflatae ml605

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M. violaceum ml344 95

M. violaceum on Silene nutans ml1167

64 100

M. violaceum on Silene nutans ml1169

M. bardanense ml881

83 87

M. violaceo-irregulare M. chloranthae-verrucosum mk282

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M. chloranthae-verrucosum mk305 M. sp. on Silene adenopetala ml877 86 95 100

M. sp. on Silene acaulis ml875 M. sp. on Silene acaulis ml827 M. sp. on Silene acaulis ml855

M. majus 100 80 100 100 100

M. dianthorum ml354 M. dianthorum ml328 M. dianthorum ml333 M. dianthorum ml622 M. dianthorum on Dianthus jacquemontii

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M. dianthorum on Petrorhagia saxifraga

M. saponariae ml337 88

100 98

M. saponariae ml317 M. aff. violaceum mk292 M. aff. violaceum ml602

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M. violaceo-verrucosum M. stellariae ml512

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M. stellariae ml507 100

M. aff. violaceum* AY014239 M. aff. violaceum* AY014235

M. scabiosae 0.01 substitutions/site

Fig 2 – Bayesian inference of phylogenetic relationships of the sampled Microbotryum specimens: MCMC analysis of an alignment of base sequences from the ITS1/2 region of the nu-rDNA including the 5.8S rDNA and from the 50 -end of the nuLSU rDNA using the GTRDG model of DNA substitution with gamma distributed substitution rates, random starting trees and default starting parameters of the DNA substitution model. A 60 % majority-rule consensus tree computed from 18K trees that were sampled after the process had reached stationarity is shown. The topology was rooted with Microbotryum scabiosae. Numbers on branches are estimates for PPs. Branch lengths were averaged over the sampled trees. They are scaled in terms of expected numbers of nucleotide substitutions per site. For specimens labelled with an asterisk only ITS sequences were analysed.

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Fig 3 – SEM micrographs of teliospores. (A–C) Microbotryum adenopetalae (holotype, KRAM F55201). Note spores with verruculose interspaces indicated by arrows. (D–F) M. minuartiae (holotype, KRAM F55483). Note two germinating spores and two spores, indicated by arrows, with distinctly coronate muri.

Microbotryum silenes-acaulis M. Lutz, Pia˛tek & Kemler, sp. nov. (Figs 4A–C, 5C, 6B) MycoBank no.: MB 510815 Etym.: The name refers to the host plant species, Silene acaulis. Species characteribus generis. Sori in antheris Silenes acaulis (Caryophyllaceae). Massa sporarum atro-violacea. Teliosporae globosae, 6–8.5(–10)  (5–)6–8 mm, pariete reticulo denso decorato, inter reticuli cristas laevi. Sequentia acidi nucleici ITS typi in collectione sequentiarum acidi nucleici NCBI (GenBank) numero DQ366846 deposita est. Typus: Poland: Tatra Mts, 300 m north from the top of Kopa Kondracka Mt., alt. 1925 m.a.s.l., on Silene acaulis, 25 June

2005, leg. M. Pia˛tek & J. Cabała (KRAM F55485dholotypus; HeMP- 18, HUV 21161, K(M) 137316, LBL M-8583, TUB 012522disotypi).

Sori in anthers. Infection systemic, all anthers of a plant being infected. Spore mass powdery, dark brownish violet. Teliospores hyaline to pale violet, globose, subglobose, ovoid to elongated, sometimes lacrymiform, 6–8.5(–10)  (5–)6–8 mm; wall densely reticulate, 5–8 meshes per spore diam, interspaces smooth. Spore germination without resting period, results in 3–4-celled basidia, mostly 3-celled, 12–18  2.5– 3.5 mm, usually about 15  3 mm, only two observed ovoid basidiospores emerged laterally and terminally, respectively.

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Fig 4 – SEM micrographs of teliospores. (A–C) Microbotryum silenes-acaulis (holotype, KRAM F55485). (D–F) M. violaceum (neotype, GLM 50283).

The ITS type sequence from DNA isolation ml827 from the holotype (KRAM F55485) is deposited in GenBank as DQ366846. Host range and distribution: On subfam. Silenoideae: Silene acaulis; Europe (Austria, Poland, Sweden, Switzerland), North America (Canada), certainly more widely distributed as presumably numerous worldwide collections of Microbotryum violaceum on S. acaulis belong to this species. Additional specimens examined: Austria: Salzburger Land, Leoganger Steinberge, alt. 1950 m.a.s.l., on Silene acaulis, 16 July 1987, G. Kost & K. Va´nky (HUV 13955).dPoland: Tatra Mts, southern slopes of Giewont Mt., alt. 1800 m.a.s.l., on Silene acaulis, 25 June 2005, M. Pia˛tek & J. Cabała (HeMP- 19, HUV 21162, KRAM F55487, TUB 012520); Tatra Mts, Hala Ga˛sienicowa glade, alt.

1520 m.a.s.l., on Silene acaulis, 4 June 2004, M. Pia˛tek & J. Cabała (HeMP- 13, HUV 20800, KRAM F55486, TUB 012517).dSweden: Lapland, Kiruna, Vistas Valley, alt. 640 m.a.s.l., on Silene acaulis, 29 Aug 2005, M. Koltzenburg (KRAM F55489, TUB 012515).dSwitzerland: Grisons, Sur, on Silene acaulis, 15 June 2005, M. Kemler & M. Lutz (KRAM F55488, TUB 012516).

Microbotryum lychnidis-dioicae (DC. ex Liro) G. Deml & Oberw., Phytopathol. Z. 104: 353 (1982). Sori in anthers. Infection systemic, all anthers of a plant being infected. Spore mass powdery, light brownish violet. Teliospores hyaline to pale violet, globose, subglobose to ovoid, 6–8(–9)  6–7(–8) mm on Silene zawadskii and 5.5–7 (–8)  5–7(–7.5) mm on Cucubalus baccifer; wall densely reticulate, 5–7(–8) meshes per spore diam, interspaces smooth.

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Fig 5 – Line drawings of germinating teliospores. (A) Microbotryum adenopetalae on Silene adenopetala (germination on WA, KRAM F55201). (B) M. minuartiae on Minuartia recurva (in situ germination, HeMP-15). Note copulation bridges indicated by arrows. (C) M. silenes-acaulis on Silene acaulis (in situ germination, HeMP-13). Bars [ 10 mm.

Specimens examined: Germany: Saxony, Bad Du¨ben, on Cucubalus baccifer, 22. Sep 1997, leg. H. Jage (GLM 49244); Saxony– Anhalt, Dessau, Riesigk, on Cucubalus baccifer, 30 Oct 2000, leg. H. Jage (GLM 47387).dPoland: Krako´w, Botanical Garden, on Silene zawadskii, 18 May 2004, leg. M. Pia˛tek (HUV 20797, TUB 012521). Microbotryum dianthorum (Liro) H. & I. Scholz, Englera 8: 206 (1988), emend. M. Lutz, Go¨ker, Pia˛tek, Kemler, Begerow & Oberw., Mycol. Prog. 4: 234 (2005). Sori in anthers. Infection systemic, all anthers of a plant being infected. Spore mass powdery, light brownish violet.

Teliospores pale violet, globose, subglobose to ovoid, (5–)5.5– 6.5(–7)  (4.5–)5–5.5(–6) mm on Dianthus jaquemontii and (6.5–)7–9  6–8(–9) mm on Petrorhagia saxifraga; wall densely reticulate, 4–6 meshes per spore diam on Dianthus jaquemontii, 5–7 meshes per spore diam on Petrorhagia saxifraga, interspaces smooth.

Specimens examined: Italy, Elba, Fetovia, on Petrorhagia saxifraga, 15 May 2000, M. Hendrichs (TUB 012106)dIndia, Jammu and Kashmir State, Himalaya Mts, Bardan, alt. 3888 m.a.s.l., on Dianthus jaquemontii, 8 July 2004, A. Chlebicki (KRAM F54963, PRM 907125, HUV 21089).

Fig 6 – Macroscopic appearance of infection by two Microbotryum species. (A) M. minuartiae on Minuartia recurva (photo by A. Ronikier). (B) M. silenes-acaulis on Silene acaulis (photo by M. Pia˛tek).

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Neotypification of Microbotryum violaceum Microbotryum violaceum (Pers.) G. Deml & Oberw., Phytopathol. Z. 104: 353 (1982). Synonyms: Uredo violacea Pers., Tent. Disp. Meth. Fung.: 57 (1797): Pers., Syn. meth. fung. 225 (1801). Caeoma violaceum (Pers.) Nees, System der Pilze und Schwa¨mme: 14 (1817). Ustilago violacea (Pers.) Roussel, Fl. Calvados, 2nd edn 47 (1806) Typus: Germany: Saxony–Anhalt: Gniest: on Silene nutans, 19 June 1998, H. Jage (GLM 50283 – neotypus hic designatus). Sori in anthers. Infection systemic, usually all anthers of a plant being infected, but sometimes some flowers in inflorescence may escape the infection. Spore mass powdery, dark brownish violet. Teliospores pale violet, globose, subglobose to ovoid, (5.5–)6.5–9  (5–)6–8(–9) mm; wall densely reticulate, 5–7 meshes per spore diam, interspaces smooth. The ITS type sequence from DNA isolation ml1169 from the neotype (GLM 50283) is deposited in GenBank as DQ640065.

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only tentatively considered as taxonomically valuable, because of the still very limited number of available SEM micrographs of anther smuts. In fact, Deml & Oberwinkler (1983) depicted spores of M. silenes-inflatae with small tips at the crossing points of the ridges, similar to those observed in Microbotryum sp. on Minuartia recurva, but they were not as evident as in this latter species. Although the morphology of basidia is influenced by different nutritional conditions, as well as temperature (Hood & Antonovics 1998), and, therefore, might be of limited value for species characterization in the caryophyllaceous anther smuts, the formation of predominantly 2-celled basidia in Microbotryum sp. on Minuartia recurva was quite remarkable, as most other anther smuts produce predominantly 3-celled basidia (e.g. Deml & Oberwinkler 1982, 1983; Ingold 1983). Exceptions from this general pattern of germination of anther smuts are possible, as was already demonstrated by Hood et al. (2001) who found that Microbotryum sp. on Silene caroliniana and on S. virginica in North America produced 4-celled basidia.

Additional specimen examined: Germany: Saxony: Altengroitzsch, on Silene nutans, 15 June 1997, H. Jage (GLM 49283).

Microbotryum on Cucubalus baccifer, Dianthus jacquemontii, Petrorhagia saxifraga, and Silene zawadskiidreport of new hosts

Discussion

The phylogenetic analyses reveal that the parasites on Cucubalus baccifer and Petrorhagia saxifraga being described as Microbotryum violaceum (Va´nky 1994) do not group with the sampled representatives of that species. Rather, the parasite on C. baccifer clusters within the specimens of M. lychnidisdioicae and the parasite on Petrorhagia saxifraga clusters within M. dianthorum. The parasite on Dianthus jacquemontii clusters within M. dianthorum and the parasite on Silene zawadskii clusters within M. lychnidis-dioicae. Therefore, these plants are newly reported host plants for the respective Microbotryum species. The species descriptions of M. dianthorum and M. lychnidis-dioicae are amended accordingly.

The examination of recent collections of Microbotryum specimens inhabiting anthers of different caryophyllaceous host plants reveals new species of smut fungi and new host plant species. It sheds light on the use of morphological characteristics in species delimitation and phylogenetical aspects. The phylogenetic analyses in this study confirm those in other studies (Lutz et al. 2005; Le Gac et al. 2007; Kemler et al., submitted for publication) in that neither single-host nor multi-host specificity is the general pattern in this group of plant parasites.

Morphological characteristics The biometric studies of spores confirm our opinion of previous work (Lutz et al. 2005) that spore size is almost useless for delimitation of Microbotryum species, because spore size overlaps between different species and the intraspecific size variation is too high to characterize a specific group. A notable example for this is the cluster of Microbotryum dianthorum, in which the spores of the specimen infecting Dianthus jacquemontii were small, whereas those of the specimen infecting Petrorhagia saxifraga were rather large (see Table 1). This is not reflected by molecular data, where in the combined analyses (Fig 2) both parasites cluster together, suggesting heterogeneity for this characteristic in the M. dianthorum clade (see also Lutz et al. 2005). Spore surface ornamentation in different reticulate Microbotryum species visible using SEM shows great variability, even within the same specimen. Thus, spore surface ornamentation is not a valuable characteristic for delimitation of species. Some subtle differences in morphology of spores of Microbotryum sp. on Minuartia recurva and Microbotryum sp. on Silene adenopetala must be treated with great caution and

Microbotryum on Minuartia recurva, Silene acaulis, and S. adenopetaladnew species The occurrence of an anther smut on Minuartia recurva in Muntxii Bucegi has already been noted by Magnus (1913). After almost 100 y both host plant and accompanying smut, which is described here as new species Microbotryum minuartiae, are still abundant in this area. In the available literature we found only few other records of anther smuts on Minuartia species, among others on M. laricifolia in ‘Czechoslovakia’ (Va´nky 1994), M. recurva ssp. condensata and M. villarsii in Spain (Pando & Herna´ndez 2002), M. verna in Germany (Scholz & Scholz 1988) and M. pusilla in California (French 1989). All these records except the latter were referred to M. stellariae. Molecular studies are necessary to check whether they belong to M. minuartiae. Anther smuts on Silene acaulis have been reported from various places in North America and in arctic and alpine areas of Europe (e.g. Zundel 1953; Va´nky 1994). These collections have been referred to Ustilago violacea in older literature, more recently to Microbotryum violaceum. The molecular analyses of Lutz et al. (2005), when only one

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sequence of a Microbotryum specimen from S. acaulis collected in Canada was available (GenBank accession no.: AY014226), revealed an isolated position of the specimen clustering as sister taxon to M. majus within the cluster of Microbotryum species on Silene. After having found another Microbotryum specimen from S. acaulis in Switzerland (TUB 012516) and analysing its ITS sequence our idea that the concept of host specific species delimitation in caryophyllacean anther smuts might hold for Microbotryum on Silene acaulis too, was reconfirmed. Both specimens clustered together, as well as further specimens from the same host species from Austria, Poland, and Sweden. Our results confirm those of Le Gac et al. (2007), who demonstrated an independent evolutionary lineage for S. acaulis inhabiting Microbotryum specimens from France and Switzerland based on the analysis of three genes (b-tub, g-tub, Ef1a). Consequently, we propose a new species, M. silenes-acaulis for Microbotryum anther parasites inhabiting S. acaulis. An anther smut on S. adenopetala has not been reported in any literature available. The collection from Tian Shan is very peculiar because the spores completely cover the area around the ovary delimited by the calyx. The sorus morphology made it difficult to recognize which part of the flower was infected. In young flowers sori were present only above ovaries containing young seeds, while anthers were completely destroyed. In other specimens ovaries were mostly small and not fully developed, avoid of seeds, and anthers also being not visible. The infection resembles that of Silene flowers by Microbotryum majus and M. savilei. However, the anther smut on S. adenopetala differs from those species by having a smaller number of meshes per spore diameter and finely rugulose or even verruculose interspaces. Our molecular analyses (Figs 1 and 2) were not able to resolve the phylogenetic relationship between M. majus and Microbotryum sp. on Silene adenopetala. However, the genetic distance between the latter and the two M. majus specimens is considerable (39 bp differ in the ITS). Therefore, and against the background of the knowledge of high host specificity of most Silene parasites, we propose a new species, M. adenopetalae, for Microbotryum inhabiting S. adenopetala. Molecular analyses including all three species are needed to clarify their phylogenetic relationship.

Microbotryum bardanense Recently, Chlebicki & Sukova´ (2005) reported Silene moorcroftiana, a new host for caryophyllaceous anther smuts, infected by Microbotryum from India. Morphological analyses of the specimen revealed a rather irregular verrucose to reticulate spore ornamentation and the authors decided, based on this morphological feature and the new host, to describe a new species for this specimen, M. bardanense. In our molecular analyses, M. bardanense clustered together with the specimen of M. violaceo-irregulare. Due to the rather small genetic distance further phylogenetic studies with parasite specimens on S. moorcroftiana and M. violaceo-irregulare from S. vulgaris are needed to clarify the species status of M. bardanense. Both parasites, M. bardanense and M. violaceo-irregulare, were found only in high

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elevations (Va´nky 2004; Chlebicki & Sukova´ 2005), which might point at their close relationship.

Phylogeny The combination of LSU and ITS data for the inference of the phylogenetic position of parasites on new hosts resulted in an often well-resolved phylogeny. Especially in the clade containing Microbotryum dianthorum, M. saponariae, M. aff. violaceum, M. violaceo-verrucosum, and M. stellariae, the twogene approach resulted in a fully resolved phylogeny. Even though the rest of the phylogeny was often not resolved in the higher clades, it is apparent that most of the new parasites in this study belong to phylogenetically well-separated groups and, therefore, can be treated as new species. One of these findings is the division of M. stellariae. This species is described as parasitizing Minuartia apart from Stellaria and Myosoton, but our study shows that the parasite on Minuartia is not closely related to Microbotryum stellariae. That might well reflect the relationships within the Caryophyllaceae, as Minuartia is not closely related to Stellaria (Fior et al. 2006). An approach using b-tubulin, g-tubulin, and elongation factor 1a (Le Gac et al. 2007) also resulted in many host specific phylogenetic lineages, where of many can be very well correlated with the species found by this and our previous studies (Lutz et al. 2005; Kemler et al. 2006). A problem that could not be resolved fully by any of these studies is the phylogenetic position of Microbotryum on Silene acaulis. Most genes support a position basal to the clade containing the parasites on Old World Silene species, but this is contradicted by the g-tubulin dataset and further studies are required to resolve the position of this group. Based on the knowledge of high host specificity of most Silene parasites, the high genetic distance from other parasites, the geographic separation from other Silene parasites, and the unique position in the phylogeny, it is justifiable to assign species status to the parasite on S. adenopetala, although it is described from only one specimen. Future phylogenetic investigation involving more specimens is required to verify whether the species status of M. bardanense can be justified or whether the host spectrum of M. violaceo-irregulare needs to be extended. Our studies clearly indicate that there are more species to be expected in the caryophyllaceous anther smuts in the future, and another future study indicates the same pattern for non-caryophyllaceous parasites in the genus Microbotryum (M.K., unpubl.). Part of this host specific pattern stems from findings on new hosts, but many more new species are to be expected as a result of molecular studies and the splitting of polyphagous parasites into monophagous or oligophagous species.

Neotypification of Microbotryum violaceum In the light of the current splitting of Microbotryum violaceum s. lat. into several smaller species, it is necessary to typify M. violaceum s. str. This species has first been described by Persoon (1797) as Uredo violacea, with Silene nutans as the sole host plant. Redescribing the smut, Persoon (1801) included Saponaria

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Accordingly, only specimen C on the latter sheet (Fig 7) that belongs to M. violaceum s.str. might be taken into consideration for typification of Uredo violacea. However, this specimen cannot be treated as authentic material, because on the label there are two names ‘Uredo violacea Pers.’ and ‘Uredo antherarum Dec.’, both written by the same hand, which means that Persoon did not see this material before 1797 when he described U. violacea. He saw this specimen only after 1815 when de Candolle (1815) described his U. antherarum. Therefore, in the lack of any authentic material of U. violacea we are proposing here the neotype of M. violaceum collected on S. nutans in Germany.

Acknowledgements

Fig 7 – A sheet from the Persoon herbarium (L) containing three specimens marked as A, B, C, with Microbotryum lychnidis-dioicae on Silene dioica, M. dianthorum on Dianthus carthusianorum, and M. violaceum on S. nutans, respectively.

officinalis and Silene nutans as host plants. This led some authors (e.g., Liro 1924; Deml & Oberwinkler 1982) to consider Saponaria officinalis as the type host of Uredo violacea. However, Nannfeldt (in Lindeberg 1959) convincingly explained that the host plant of Persoon’s Uredo violacea is Silene nutans. In the Herbarium Persoon in Leiden (L) there are four sheets with specimens labelled as Uredo violacea: 1. ‘Uredo violacea In lichnis dioica’, L 119233. In contrast to the statement on the label, the small fragment of the host plant in this specimen belongs to Saponaria officinalis, not to Silene dioica (syn. Lychnis dioica), and the anther smut is Microbotryum saponariae. 2. ‘Uredo violacea – Syn. fung., Ur. antherarum Dec.’, L 119234. This sheet contains three plants of Stellaria sp. infected by Microbotryum stellariae. 3. ‘Uredo violacea in Lychnide dioicae’, L 119231. This sheet contains five infected plants, of which two on the left side represent indeed Silene dioica infected by Microbotryum lychnidis-dioicae, while two on the right side and one in the envelope glued on the left upper side of the sheet belong to Saponaria officinalis infected by Microbotryum saponariae. 4. ‘A. Lychnidis Dioicae. B. Dianthi Carthusianorum. C. Silenes nutantis, Uredo Violacea? Pers. Syn. 225.? – Uredo antherarum Dec. fl. fr. 6 [further illegible] – 204. Dec.’, L 119232. (Fig 7) This sheet contains three specimens marked as A, B, C, with Microbotryum lychnidis-dioicae on Silene dioica, M. dianthorum on Dianthus carthusianorum, and M. violaceum on Silene nutans, respectively.

We are grateful to Michael Weiß who helped to write the Latin diagnoses, to Jaqueline Go¨tze for technical assistance in the laboratory, to K. Va´nky for the loan of specimens and helpful comments on the manuscript, to Michael Koltzenburg, Anna Ronikier, and Michal Ronikier for their endeavors in the field, to Jolanta Pia˛tek for preparing the line drawings, to Anna qatkiewicz for her help with the SEM pictures, to the curators of GLM and L for the loan of specimens, to the Stiftung ‘Schatzinsel Alp Flix’, which supported several collection trips, to the anonymous reviewer for helpful comments and to the Deutsche Forschungsgemeinschaft and Polish Ministry of Science and Higher Education (grant no. 2 P04 G 019 28 for the years 2005–2008) for financial support.

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