ARTICLE 5 V. to on et al. was (Liro (Ciferri 1928), Key words: Basidiomycota Cryptic species Molecular analysis Muscari Phylogeny Plant pathogens Smut fungi , was established for ) Muscari sp. nov. sp. nov. A. vaillantii (Ciferri 1931) or U. urgineae IMA FUNGUS · VOLUME 4 · NO 1: 5–19 NO 1: 4 · · VOLUME FUNGUS IMA Urocystidales , on Muscari botryoides ) on M. botryoides, and (Ershad 2000). Molecular analyses revealed that (Vánky 2009). In addition to describing a new Floromycetaceae ( were analysed by rDNA sequences were analysed by rDNA " U. muscari-botryoidis U. scillae on Scilla bifolia Charybdis maritima (Maire 1931), were based mostly on small smut researchers and received moderate acceptance by recognised anther (Zundel 1953). In general, most authors smuts on monocots as one species Ustilago vaillantii was adopted until 1924, Vánky 1985) and such an approach very recently (Vánky 1994). and molecular phylogeny (Bauer et al. 1997, Begerow 1997, Bauer et al. 2001) restricted the genus Ustilago Ustilago vaillantii species on Poaceae. Consequently, moved to the genus Vankya ornithogali the anther smuts on monocots were only distantly related to , and a new genus the foliicolous representatives of Vankya Antherospora The genus Vankya the former group (Bauer et al. 2008). is currently restricted to host plants in Liliaceae subfam. Lilioideae genus, Bauer et al. (2008) demonstrated that the evolution of the anther smuts on monocots is related to host taxonomy,
, Antherospora U. anther smuts and co-evolution as a driver of Muscari anther smuts and co-evolution as a driver of 3 became . All species on Muscari form a monophyletic group within All species . Ustilago vaillantii comb. nov. (syn. Ustilago muscari-botryoidis comb. nov. was described by M. comosum, and , (syn. Hyacinthaceae Muscari comosum. In , and Scilla maritima. The two , and Arthur O. Chater , and 2 in the following order: Muscari M. botryoides sp. (Sydow & Sydow 1914a), , Muscari comosum by Liro (1924) and , A. muscari-botryoidis Albuca species. In this study, specimens of anther specimens of anther Muscari species. In this study, on different s. lato includes anther smuts
on subfam. Scilloideae , Matthias Lutz , Matthias 1 Scilla anthericoides on M. comosum and s. str.
The anther smut fungi in the ustilaginomycetous genus The anther smut fungi
on Ornithogalum lacteum (Sydow & Sydow 1914b), Muscari armeniacum U. albucae (Fischer von Waldheim tourneuxii on Bellevalia trifoliata (Fischer von Waldheim ; Subsequent to its description, Ustilago vaillantii Muscari armeniacum Asparagaceae ; \ ; $ ; " * # > '+VE?V " ~ ; * % EU >8 $ Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Kraków, Poland; corresponding Poland; Lubicz 46, PL-31-512 Kraków, Academy of Sciences, Polish of Botany, Szafer Institute W. Department of Mycology, Antherospora This indicates a common ancestry of respective host species. Submitted: 27 January 2013; Accepted: 28 February 2013; Published: 4 April 2013. 4 Accepted: 28 February 2013; Published: Article info: Submitted: 27 January 2013; ; A. vaillantii ; lineages that were correlated with host plants, yet had only slight molecular phylogeny revealed three distinct " cryptic species: Antherospora hortensis These lineages are assigned to three morphological differences. on Antherospora vaillantii smuts on that infect monocots, are currently placed in nine species. Against the background of the generally observed high the background of the generally observed Against are currently placed in nine species. that infect monocots, Germany Abstract: author e-mail: [email protected] © 2013 International Mycological Association © 2013 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: species 2 3 1 # = complex on complex vaillantii the Antherospora diversity in Cryptic Non-commercial: No derivative works: Any of the above conditions can be waived if you get For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. VOLUME 4 · NO. 1 latter names are synonyms of the species currently named The type Charybdis maritima (Speta 1988, Euro+Med 2006-). host was attributed to Ciferri (1928), who reasonably explained this selection. The anther smut fungus Ustilago vaillantii INTRODUCTION mycology brothers at the beginning of systematic Tulasne the " ` " >|LV " (1669–1722), the pre-Linnean botanist Sébastien Vaillant + brothers included three host Tulasne the protologue, the species of Ustilago vaillantii comosum, smut specimens sporulating in the a catch-all for different anthers of monocotyledonous plants that currently are placed in doi:10.5598/imafungus.2013.04.01.02 var. var. 1880), peglerae APG III 2009, Chase et al. 2009). Some attempts to describe separate taxa within this complex, such as 6 ARTICLE
infect Muscari. including collectionsfromtypehostsofallspeciesknown to microscope examinationofdifferent anthersmutspecimens, sequences wereappliedaswelllightandscanningelectron this goalmolecularphylogeneticanalysesusingrDNA morphological and/orecologicalcharacteristics. To achieve vaillantii some hostsmayhaveaddedfurtherconfusion. muscari-botryoidis species complexonMuscari,includingtheidentityof botryoides of Baueretal.(2008),anthersmutcollectionson was describedasadistinctbiologicalspecies.Inthestudy healthy. Consequently, theanther smutonMuscaribotryoides still infectedwhileM.comosumand racemosum with non-infected observation ofaco-cultivationinfectedMuscaribotryoides was healthy. Subsequently, Ciferri(1928)madeathree-year Italy.co-occurring The anther-infected populationof of molecularmethods.In1921,Ciferri(1928)discoveredan working atthebeginningof20 descriptions ofdifferent biological speciesbytaxonomists muscari-botryoidis 2001). The historyofdiscovery anddescriptionofUstilago Scholz &1988,Karatygin Azbukina 1989,Denchev a synonymoftheformerspecies(Vánky1985,1994,2012, botryoides on comosum, asecondspecieswasdescribedforanthersmuts Indeed, inadditionto more thanonespeciesmayhaveradiatedonMuscarispecies. specialization maybehigherthantothegenuslevelandthat Muscari species(Bauer genetic divergencesbetweensmutcollectionsfromdifferent species (Vánky2012).However, molecularanalysesrevealed narrowed 2012, 2013)aremoreoftencollected. 2009, Hoodetal.2010,Kemler E??} E?>U = dicots (Lutz Bellevalia, Muscari).Incomparison,theanthersmutson hidden bytheperianths,atleastinsomehostgenera(e.g. for molecularanalyses. The anthersmutsonmonocotsare was warranted,despitelimitedaccesstofreshspecimens and consequentlyanarrowspeciesconceptwithinthisgroup "$8 * $ " > " ; " voucher specimensaredeposited inGLM,KR,KRAMF, $ % ; complex fromfourdifferent Muscarispecies,originatingfrom This studyisbasedonspecimens ofthe Specimen samplingand documentation MATERIALSMETHODS AND Muscari, namely The presentstudyisaimedatresolvingtheAntherospora The speciesconceptadoptedbyBaueretal.(2008) speciescomplexon Antherospora vaillantii werenotavailableandtheAntherosporavaillantii , althoughinrecentmonographsitwasconsidered ). After thethreeyears,Muscaribotryoides et al.2005,2008,RoetsCurran ; # wellillustratestheapproachleadingto M. comosumandneglectum Ustilago muscari-botryoidis et al.2008),whichsuggestthathost Antherospora vaillantii \ \ Muscari comosum th Muscari botryoides century, priortoestablishment Muscari, andtestingwhether tosmutsinfecting M. neglectum Antherospora vaillantii on on in northern remained Ustilago (asM. Muscari Muscari Muscari Muscari was et al.
(Tutin + ; 8 8 + % < '+VE?V " " Ustilaginales to thepersonalcollectionofKálmánVánky, “Herbarium >U E>UUV $ ; and MicroanalysisattheInstituteofGeologicalSciences, Laboratory ofFieldEmissionScanningElectronMicroscopy distance of a HitachiS-4700scanningelectronmicroscope,withworking with carbonusingaCressingtonsputter-coaterandviewed double-sided transparenttape. The tabsweresputter-coated electron microscopy(SEM).Forthispurpose,dry on 27962), onM.comosum(KRAMF-49438=HAI2857),and Muscari armeniacum with aNikonDS-Fi1camera. The sporesofAntherospora by different mountingmedia. LMmicrographsweretaken measured againinlacticacidtominimizetheerrorcaused in previousworklactophenol(Bauer specimens oftheAntherosporavaillantii include combinedvaluesfromallmeasuredspecimens. The show thedistributionofvalues. The speciesdescriptions 1. The sporesizevaluesare presentedinascatterdiagramto spore measurementsfromeachspecimenareshownin Table μm. The sporesizerange,meanandstandarddeviationof50 The extrememeasurementswereadjustedtothenearest0.5 were measuredusingNIS-ElementsBR3.0imagingsoftware. &>??? ' % examined underaNikonEclipse80ilightmicroscopeat mounted inlacticacid,heatedtoboilingpoint,andthen Dried fungalteliosporesoftheinvestigatedspecimenswere Morphological examination GenBank ofthe followingspecieswereused formolecular sp. on "% !%$ Phylogenetic analyses and Fig.1). GenBank (GenBankaccessionnumberaregivenin Table 1 DNA sequencesdeterminedforthisstudyweredepositedin = E??L data seeLutzetal. of PCRproducts,sequencing,andprocessingtheraw '* !%$ the rDNA includingthe5.8SrDNA (ITS)andthe5´-endof '* "% > "% E For methodsofisolationandcrushingfungalmaterial, DNA extraction,PCR,andsequencing follows theproposalofChakrabarty(2010). MycoBank.org, Crousetal.2004). The genetypeconcept Nomenclatural noveltieswereregisteredinMycoBank(www. $ E>?L comosum is ><|U| "$8 et al. >}|? > % ; $ Muscari spp.obtainedinthisstudy, sequencesfrom ca. 12mm. M. armeniacum Vánky”currentlyheldathishome(Gabriel- (GLM48095)werestudiedusingscanning (KRAMF-49437),on M. neglectum SEM micrographsweretakeninthe ; (3)thespecimenfromGermany M. neglectum isM.botryoides complexmeasured is et al.2008)were et al.(2011). The M. botryoides(KR et al.2008)were M. armeniacum Flora Europaea Antherospora IMA FUNGUS spores were spores were ; (2) on M. .
Antherospora on Muscari
phylogenetic analyses (Bauer et al. E??| = et al. 2011): To elucidate the phylogenetic position of the Antherospora ARTICLE Antherospora scillae, A. tractemae, A. vaillantii, and A. specimens from Muscari "%¥!%$ vindobonensis (GenBank accession numbers are included in sequences were analysed within a dataset covering all Fig. 1). Antherospora species available in GenBank. If present in
Table 1. List of Antherospora specimens examined, with host plants, GenBank accession numbers, spore size range, mean spore sizes with standard deviation, and reference specimens.
Species Host GenBank acc. Spore size range Mean spore size Reference specimens1 no. (μm) with standard deviation (μm) A. hortensis M. armeniacum ITS: EF653982 (6.0–)7.0–10.0(–13.0) | / > V & | / 8 +~ !%$ A. hortensis M. armeniacum ITS: KC175333 (6.0–)7.5–10.5(–12.5) | } / > E & V < / 8 +~ !%$ >V VOLUME 4 · NO. 1 7 8 ARTICLE before slashes,estimatesforaposteriori were omittedusing GBlocks0.91b(Castresana 2000)with respectively. Highly divergentportionsofthealignment by Giribet&Wheeler(1999) andGatesyetal.(1993), exclusion ofambiguoussites wereavoidedassuggested manipulation ofthealignment byhandaswellmanual option inMEGA. used forcalculationsaswechosethepair-wisedeletion distances. Gapsordifferent lengthofsequenceswerenot p-distances andreportdistancesaspercentagegenetic using thesoftwareMEGA (Tamura differences andtheestimationofgeneticdistanceswithin L-INS-i option. The resultingalignmentwasusedforboth (Katoh were used. GenBank, thesequencesofrespectivetypespecimens The topologywasrootedwith "%¥!%$ $ ; "/*" >??? Fig. 1.Hypothesisonphylogeneticrelationshipswithinthesampled To ensure reproduciblephylogeneticanalyses, Sequence alignmentwasobtainedusingMAFFT 6.853 et al.2002,2005,Katoh& Toh 2008)usingthe 7/63 8 0.0030 Antherospora scillae 1/- 9 100/70 100/92 9/99 9 et al.2011). We calculated 100/66 probabilities areindicatedafterslashes. A. =Antherospora A. onM.botryoidesKC175332/KC175325 A. onM.tenuiflorumEF653988/EF653970 A. onM.comosum A. onM.tenuiflorumEF653986/EF653968 A. onM.tenuiflorumEF653987/EF653969 A. tractemaeJN204283/JN204279 A. tractemaeJN104589/JN104590 A. onM.botryoidesEF653998/EF653980 , A. onM.armeniacumKC175336/KC175329 A. onM.armeniacumKC175335/KC175328 A. onM.armeniacumKC175337/KC175330 A. onM.armeniacumEF653997/EF653979 A. onM.armeniacumKC175334/KC175327 A. onM.armeniacumEF653982/EF653964 A. onM.armeniacumKC175333/KC175326 A. tractemae,and 100/100 KC175338/KC175331 8/72 9 A. vindobonensis Antherospora 100/87 the generaltime reversiblemodelofDNA substitutionwith each consistingoffourchains, wereimplementedusing (Ronquist as implementedinthecomputer programMrBayes3.2.1 using aMarkovchainMonte Carlotechniquewasused option (Stamatakis ` % ; Michalak 2012),invokingtheGTRCAT andtherapidbootstrap $ E?? % (Felsenstein 1981)wasconductedwiththeRAxML 7.2.6 # Likelihood (ML)andaBayesian Approach (BA).ML analysis U< (73% oftheoriginal1810positions)numbervariablesites: to 5and‘AllowedGapPositions’ to‘Withhalf’. Non-conserved Positions’ to8,‘MinimumLengthofaBlock’ for aFlankPosition’ to10,‘MaximumNumberofContiguous Conserved Position’ to10,‘MinimumNumberofSequences the followingoptions:‘MinimumNumberofSequencesfora A. scillaeEF653992/EF653974 A. scillaeEF653983/EF653965 For BA aBayesianapproachtophylogeneticinference The resultingalignment[newnumberofpositions:1336 A. vindobonensisEF653995/EF653977 A. vindobonensisEF653989/EF653971 specimensbasedonMaximumLikelihoodanalysisofan et al.2012). Two runs over5000generations, . ML bootstrapsupportvaluesareindicatedonbranches ! MUSCARI BOTRYOIDIS ! VAILLANTII et al.2008)with1000replicates. , M.= ! HORTENSIS Muscari. IMA FUNGUS Antherospora on Muscari gamma distributed substitution rates and estimation of The mean spore length was variable between collections of ARTICLE invariant sites, random starting trees and default starting Antherospora on Muscari armeniacum, ranging from 7.8–9.0 parameters of the DNA substitution model as recommended μm, less variable in specimens on M. botryoides (7.7–8.5 by Huelsenbeck & Rannala (2004). Trees were sampled μm), and quite uniform in specimens on M. comosum and every 100th generation, resulting in a sampling of 50 001 (7.9–8.3 μm). Likewise, the mean spore width E< © was variable between collections of Antherospora on Muscari each run were discarded (burnin). The remaining trees were armeniacum, ranging from 6.5–7.6 μm, and less variable in used to compute a 50% majority rule consensus tree to specimens on M. botryoides (6.3–7.2 μm), and in specimens obtain estimates for the a posteriori probabilities of groups on M. comosum and (6.3–7.0 μm) (Table 1). of species. This Bayesian approach to phylogenetic analysis The combined mean spore length and width were similar in ; Antherospora on Muscari botryoides and Antherospora on M. results from topological priors (Huelsenbeck et al. 2002). comosum+ , while both values were somewhat Trees were rooted with Antherospora scillae, A. tractemae, larger in Antherospora on M. armeniacum. The detailed and A. vindobonensis. morphological characteristics of the anther smuts on Muscari spp. are included in the species descriptions and depicted in Figs 3–5. RESULTS Phylogenetic analyses TAXONOMY % Antherospora hortensis # = ` # ! sp. nov. "%¥!%$ '* MycoBank MB803427 given in Table 2. (Fig. 3) The different runs of BA that were performed and the ML analyses yielded consistent topologies. To illustrate the Etymology: In reference to the occurrence of this fungus results, the phylogenetic hypothesis resulting from the ML on Muscari armeniacum in cultivated gardens, where all analysis is presented in Fig. 1. ML bootstrap support values specimens examined in this study were collected. are indicated on branches before slashes, estimates for a posteriori probabilities are indicated after slashes. Type: UK: Wales ! $ ; In all analyses, the Antherospora species included in campus, on Muscari armeniacum, 20 Apr. 2010, A.O. Chater previous studies (Bauer et al. E??| = et al. 2011) (KRAM F-49437 – holotype) were inferred with high support values, and phylogenetic relationships were as in Bauer et al. E??| = et al. Description: Parasitic on Muscari armeniacum. Sori in the (2011), and Vánky et al. (2008). All Antherospora specimens anthers, producing a dark olive-brown, powdery mass of from Muscari spp. clustered together forming three well spores inside the pollen sacs, enclosed by the perianths. supported subgroups: one contained all sampled specimens \ from M. botryoides, another all specimens from M. \ % armeniacum, and the third specimens from both M. comosum broadly ellipsoidal, sometimes allantoid, tear-shaped or and . The former two subgroups were revealed < VOLUME 4 · NO. 1 9 10 ARTICLE A. vaillantii Fig. 2. A scatterdiagramshowingthesporesizedistribution inAntherosporahortensis(bluesquares),A.muscari-botryoidis A. scillae A. tractemae A. vindobonensis A. vaillantii A. muscari-botryoidis A. hortensis ! "# "$ %'* + Antherospora vaillantii subgen. Host anddistribution:OnMuscariarmeniacum A.O. Chater(KRAMF-49436). " schliemannii for thesourceofthisrecord. The anthersmutonMuscari ; E?>E Garden (Magnus 1896,Scholz&1988) andonM. 10 0 1 2 3 4 5 6 7 8 9 02468101214 (yellowtriangles).Notethatonepointmayincludemeasurements oftherespectivelength/widthvaluefrommorethanonespore. Botryanthus, (syn. M.armeniacum .-. . . 1.9 1 1.9 0.9 A.scillae 0.4-0.5 A.tractemae A.vindobonensis A.vaillantii 0 A.muscari-botryoidis A. hortensis Asparagaceae s. latoonM.armeniacum M. armeniacum ) fromtheBerlinBotanical . . .-. .-. 1.8-1.9 1.1-1.2 1.8-1.9 0.7 0.1 M $ , 15 Apr. 2010, ( Muscari was . . 1.8 1.1 1.8 0 cyaneoviolaceum "¥% UEMU< positions in16 loci. Among thesequence differences there "%¥!%$ vaillantii and A. in (longer andwider)average size ofsporesandhostplant Antherospora vaillantii Comments: This speciesismorphologicallysimilarto belongs tothisspecies. Botanic GardensKew(Kirk&Cooper2013)mostprobably Muscari subgen.Botryanthus . . 0.3 1.7 0 (syn.M.armeniacum s. str., differing byasomewhatlarger 1.6 0 Antherospora hortensis ) fromtheRoyal (blackspots),and IMA FUNGUS 0 Antherospora on Muscari ARTICLE Fig. 3. Antherospora hortensis sp. nov. on Muscari armeniacum. A. General habit of infected plant. B–C. Healthy (to the left) and infected (to \ D. Close-up of the anthers with fungal spores. Note that ovary is not destroyed. E–I. % !# ; /*# +L}LUV J–L. Ornamentation of spores seen by SEM (KRAM F-49437). Bars: A–C = 5 mm, D = 1 mm, E–I = 10 μm, J–L = 5 μm. VOLUME 4 · NO. 1 11 12 ARTICLE 1311 inthealignmentfor >}E >}U LUV !%$ are autapomorphicnucleotidedifferences intheITSat SEM (KR27962). Bars:B–C=5mm,D–H10μm, I–J=5μm. fungus sporulatingintheanthers. Fig. 4. Antherospora muscari-botryoidis Antherospora hortensis D–G. % !# ; / EV}E comb.nov. onMuscaribotryoides . . (Fig. 4) MycoBank MB803428 M. Lutz, Antherospora muscari-botryoidis # = ` A. Generalhabitofinfectedplant. B–C. \ comb. nov. H–J. Ornamentationofsporesseenby IMA FUNGUS Antherospora on Muscari Basionym: Ustilago muscari-botryoidis Cif., Ann. Mycol. 26: "%¥!%$ EEME >>M>E ARTICLE 14 (1928). Among the sequence differences there is an autapomorphic !%$ >?V| Type: Italy: Province of Cuneo: Alba, Moretta, towards alignment for Antherospora muscari-botryoidis. Santa Rosalia, on Muscari botryoides, [year and collector The name “Antherospora neglecta Vánky, in prep.” was >}E> / ; assigned by Vánky to the Slovenian specimen in the checklist not located, probably does not exist). – Germany: Baden- of smut fungi from Slovenia (Lutz & Vánky 2009). This name Württemberg: Hechingen, B32 in Richtung Burladingen, is a nomen nudum as it was never validated. The host plant 48º20’55”N, 09º00’12”E, on Muscari botryoides, 22 Apr. 2011, Muscari neglectum. However, its M. Lutz (KR 27962 – neotype designated here). + ; Muscari botryoides \ ±globose, not elongated). Consequently, Antherospora Synonym: Antherospora neglecta Vánky, in Lutz & Vánky, neglecta is listed as a synonym of A. muscari-botryoidis. Lidia 7(2–3): 35 (2009), nom. nud. Antherospora vaillantii (Tul. & C. Tul.) R. Bauer et Original material: Slovenia: Kras, 7 km ESE from Sezana, on al., Mycol. Res. 112: 1304 (2008). Muscari botryoides (as M. neglectum), 30 Apr. 2006, C. & K. (Fig. 5) Vánky $ E>UUV Basionym: Ustilago vaillantii Tul. & C. Tul., Ann. Sci. Nat., Bot., sér. 3, 7: 90 (1847). Parasitic on Muscari botryoides. Sori in the anthers, producing Synonyms: Yenia vaillantii (Tul. & C. Tul.) Liou, Contrib. Inst. a dark olive-brown, powdery mass of spores inside the pollen Bot. Natl. Acad. Peiping 6: 45 (1949). sacs, enclosed by the perianths. Infection systemic, all Vankya vaillantii (Tul. & C. Tul.) Ershad, Rostaniha 1: 69 \ \ (2000). Spores globose, subglobose, broadly ellipsoidal, sometimes + < ?M ?M>? < M>> ? & L Comments: This species shares with Antherospora hortensis Specimens examined: Germany: Sachsen-Anhalt: Saalkreis, the systematic position of its host plant in Muscari subgen. ! % , 25 May 2000, Botryanthus, but differs by somewhat shorter spores and a H. Jage !# LVU} % ! % M. different host species (M. botryoides vs. M. armeniacum). , 29 May 1999, U. Richter (GLM 48095); Saalkreis, Antherospora muscari-botryoidis and A. hortensis "%¥!%$ ! % , 24 May 1999, W. Durka sequences differ in 11–12 positions in 8 loci. Among the (GLM 50411). – Israel: Haifa district: Carmel National Park, on M. sequence differences there is an autapomorphic nucleotide comosum, 2 Feb. 2011, K.G. Savchenko (HAI 2857, KRAM F-49438 !%$ >?V| – representative specimens). Antherospora muscari-botryoidis. This species differs from Antherospora vaillantii s. str. Hosts and distribution: On Muscari comosum and M. by somewhat shorter spores and its host plant in Muscari (Muscari subgen. Leopoldia, Asparagaceae), subgen. Botryanthus. The host plants of Antherospora France, Germany, and Israel. – The type material was vaillantii s. str. belong to Muscari subgen. Leopoldia. collected in France (Tulasne & Tulasne 1847), and the Antherospora muscari-botryoidis and A. vaillantii s. str. sequenced specimens are from Israel (Savchenko et al. VOLUME 4 · NO. 1 13 14 ARTICLE Croatia, “Czechoslovakia”, France,Germany, Hungary, (Austria, Belgium,Bosnia and Herzegovina,Bulgaria, anther smutreportson belongtoAntherosporavaillantii numerous 2011) andGermany (Jage&Richter2011). Mostlikelythe Fig. 5. spores seenbySEM(G–from ,GLM48095,H–J M. comosum,KRAMF-49438).Bars: A–F =10μm,G–J5μm. comosum, KRAMF-49438). Antherospora vaillantiis.str. Antherospora reportsonMuscaricomosum and Muscari comosumarefromEurope C–F. % !# ; on Muscari comosumand s. str. The Heluta 2012), from Africa (Algeria, Egypt,andMorocco) Denchev 2001,Zwetko& Blanz 2004,Savchenko& 1955, Scholz&1988, Karatygin& Azbukina 1989, >}}VU >}|< %;; % ; % $ ! >} (Zundel 1953, Zambettakis 1970), and from North America DISCUSSION ARTICLE $%* M # « >}}}}<< ` # >}VU specimen sampling covers four of the six Muscari species 1985, Scholz & Scholz 1988, Karatygin & Azbukina 1989, reported as hosts for Antherospora vaillantii s. lato in the Denchev 2001, Zwetko & Blanz 2004). The records from recent world monograph of smut fungi (Vánky 20121), outside the natural range of the two Muscari species, for including the type hosts for both species that were described $%* 8 M. comosum) or Poland (M. for Muscari anther smuts, namely Ustilago vaillantii (M. ), are the result of the introduction of host plant comosum) and U. muscari-botryoidis (M. botryoides). and fungus. In accordance with previous molecular phylogenetic analyses based on a smaller sampling of Muscari anther Comments: The type material of Ustilago vaillantii could not smuts (Bauer et al. E??| = et al. 2011), the present analyses revealed that all anther smut specimens on Muscari " ` " spp. form a monophyletic group within the Antherospora (1847) included three host species in the following order: clade. The phylogenetic relations within this lineage agree Muscari comosum, Scilla anthericoides, and Scilla maritima, ; but from the text it is apparent that they directly studied only basal group contains anther smuts on two hosts belonging to three smut specimens on Muscari comosum collected by Muscari subgen. Leopoldia (M. comosum, ), Vaillant, Delastre and Léveillé, respectively. The type host while the derived group includes anther smuts on hosts was assigned to Muscari comosum by Liro (1924), and belonging to Muscari subgen. Botryanthus that is further accepted as such by Ciferri (1928). Furthermore, Liro (1924) subdivided into two lineages correlated with the host plant studied the Delastre specimen included in the protologue, species M. armeniacum and M. botryoides, respectively. That which was then deposited in the Persoon Herbarium in indicates a common ancestry of the Muscari anther smuts ! ! + ; ; ; / Muscari comosum. That specimen should be treated as a showing similar patterns in anther smuts on Scilla (Bauer syntype, and cannot be considered as lectotype since it et al. 2008) and Tractema = et al. 2011) support this was not indicated with the term “type” or an equivalent by ; Liro (1924), thus not meeting the criteria of Article 7.10 of of the Muscari anther smuts with a larger sampling of anther the ICN. Liro (1924) only selected the type host of Ustilago smut specimens on diverse host plants, especially on the vaillantii from the three hosts mentioned by the Tulasne related genus Bellevalia, is desirable. brothers. Lindeberg (1959) designated Vaillant’s collection The genetic divergence between three lineages of Muscari as a lectotype of Ustilago vaillantii, but apparently she did anther smuts obtained does not correspond to differences not see this material – the problem is that the specimen in phenotypic characteristics. The morphology of all anther probably does not now exist. smut specimens was quite similar and, without support of Vánky (2012) indicated that he studied the lectotype non-morphological data, the small differences in spore size specimen from PC (he included the symbol “!” at the or average spore sizes observed between them could easily Antherospora vaillantii) selected by be treated as phenotypic variability of one single species. In Lindeberg (1959). However, the examination of all materials contrast to the study of Bauer et al. (2008), where the spore from PC labelled as Ustilago vaillantii made in the course size of one collection on (GLM 47396) of the present study revealed no authentic specimen of this had much larger spores and deviated from the remaining smut fungus matching the data from the protologue (Tulasne specimens on this host, the repeated measurement of the & Tulasne 1847). Likewise, the Delastre syntype specimen ; mentioned in the protologue and later studied by Liro (1924) They were similar to values obtained from the remaining was not found in the Persoon herbarium in L (G. Thijsse, pers. specimens on . The different mounting comm.). It is reasonable to assume that no authentic collection media (lactophenol used by Bauer et al. 2008 vs. lactic of Ustilago vaillantii is currently preserved and that a neotype acid used in this study) cannot explain this discrepancy should be selected for this species. However, the neotype of because the repeated measurements of the remaining Antherospora vaillantii based on the Israeli specimen analysed specimens used in the previous study did not reveal such in the present study is not designated here, as in our opinion it differences. This discordance may have resulted from is better to select the neotype from a fresh European collection, measuring spores from different sori. The examples from which is not available at present (most herbarium collections other studies indicate that in some smut samples, the spores are old and not useful for molecular analyses). Thus, in line measured from different sori within a collection may have a with Hawksworth (2012), the Israeli collection of Antherospora different size or morphology, for example in Anthracoidea vaillantii s. str. on the type host Muscari comosum is referred to as a “representative specimen”. 1M. alpinum and M. schliemannii listed in this monograph are excluded here as both are synonyms of other species (Euro+Med 2006-) VOLUME 4 · NO. 1 15 16 ARTICLE smaller spores.However on muscari-botryoidis. Comparedtotheremaining anther smuts >}E| Karatygin & Azbukina 1989,Denchev2001),thepresentstudy vaillantii (e.g.Vánky1985,1994,2012,Scholz&1988, other monographerswhosynonymizeditwith new combinationinthisgenusisintroduced.Incontrast to (Ciferri 1928).Becausethisisaspeciesof which thenameUstilagomuscari-botryoidisisavailable on . The claderepresentedbytwospecimens that thisspeciesisadditionallyabletoinfectthecloselyrelated to the collectiononMuscaricomosumfromIsraelisassigned and anundescribedspecies. The basallineagecontaining stable andtaxonomically informativeforthissmut. to "% !%$ species onMuscariinclude,inadditiontothehostplant 2012), thedescriptionsofthreerecognizedAntherospora on thebasisofmolecularcharacters(Tripp &Lendemer uncertainties concerningtheeffective descriptionofspecies Lutz results obtainedforothersmutfungi(Hendrichs of theirgeographicorigin. This isinagreementwithsimilar (M. comosum+ ) groupedtogether, irrespective (Muscari armeniacum group ofparasites,asthecollectionsfromsamehost plant speciesappeartobegoodtaxonomicmarkersforthis in factrepresentthreedifferent crypticspecies. The host indicate thatthethreelineagesof divergence andstrictcorrelationwithhostspeciestaxonomy Thus, inspiteofweakmorphologicaldifferences, thegenetic lineages pairwisegeneticdistancesrangedfrom0–0.1%). as distinctspecies. They rangedfrom0.4–0.9%(withinthe !%$ alignment. Inaddition,pairwisegeneticdistancesbetween "% lineages. Furthermore,eachlineagehadautapomorphic strongly supportedmonophyleticgroupsformingindependent on specimens onMuscaribotryoides such asdiscussedabove. species, aswellpossibleanomaliesinsporemorphology of morphologicalcharacterswithinspecimensthesame This concept,however, doesnottakeintoaccountvariation morphological differences showingnogeneticdifferences. E??| introduced theterm $ size valuesofanthersmuton example inCalonectria(Crous in polypores(Niemeläetal.2001),andascomycetes,for also knowninothergroupsofbasidiomycetes,forexample (Vánky 2004a).Suchanomaliesbetweencollectionsare hostianae Antherospora vaillantii Antherospora vaillantii Muscari, ,respectively, tobein M. comosumand is referred to a second species, for Muscari botryoidesisreferredtoasecondspecies,for The threelineagesofanthersmutson Molecular phylogeneticanalysesrevealedallanthersmut et al. E??< E??| = = ` E> E?>? # ` = Antherospora muscari-botryoidis hassomewhat pseudomorphospecies , M. botryoides)orcloselyrelatedhosts , more specimens should be analysed , morespecimensshouldbeanalysed s. str. The molecularresultsindicate s. str., et al.2012).Becauseofsome et al.2006). The largespore Ustilago muscari-botryoidis , on Muscari anthersmuts M. armeniacum,and correspond Muscari correspond Farysia ugandana forsmutswith , a Antherospora, a Antherospora Antherospora et al.2005, arenot Ustilago Ustilago Gay”, intheoriginal reportthespeciesname isgivenwithout 2006-). Although Vánky(2012)attributedthis name to“J. of Baker and two nameswiththesameepithet: M.alpinum The identityofMuscarialpinum isuncertainsincethereare (Vánky 2012),namely Antherospora vaillantii discussed above,twofurtherspecieshavebeenreportedfor diagnostic characterof \ neglectum armeniacum neglectum and accordingto Tutin two 2001). These on from thisarea.Inregiontherearereportsofanthersmuts Peninsula, butnosmuttedspecimenshavebeenreported native occurrenceofMuscariarmeniacum new geographicalareas. ongoing climatechangeallowsmanypathogenstospread since 2007. At presentAntherosporahortensis examined byoneofus(MP)foranthersmutseveryyear armeniacum was notfoundinKrakówPolanddespitethat supported bytheobservationthat Antherospora hortensisisasyetlocalized. This couldbealso 1936). The currentdata suggestthatthedistributionof by bulbsoreveninfectedseeds(Massee1914,Kochman by gardeners. The anther smuts onmonocotsmaypropagate \ * $ " " ~ probably onlytheresultofspecialcollectingactivitydirected 8 +~ ; $ 2006-). This indicatesthat cyaneoviolaceum probably alsobelongstoAntherosporahortensis cyaneoviolaceum Likewise, the1931collectionofUstilagovaillantii is asynonymofM.armeniacum probably belongtothesamepathogen.Muscarischliemannii during theyears1892–1897(Scholz&Scholz1988),most Botanical Garden(Magnus1896),whereitwasobserved of least underthisspeciesname.However, theoldrecords reported inanypublicationasahostforanthersmuts,at information inVánky(2012), this species, \ outside thenaturalrangeofhostspecies. This special infected The discoveryofthisspeciesisunexpectedasallanther- armeniacum and Muscari neglectum Ustilago vaillantii Besides thehostplantsincludedincurrentstudyor The originofAntherosporahortensisisunclear. The The thirdlineage,containingspecimenson Muscari armeniacum shouldbere-examined,preferablywithfresh fromtheBalkanPeninsulainfactrepresent M. alpinumSzaferexRacib.,whicharesynonyms . The occurrenceofanther smutsonMuscari iscommonlycultivatedingardensandhasbeen , representsapreviouslyundescribedspecies. Antherospora hortensis.Exceptforthebrief isasynonymofM.armeniacum fromKewGardens(Kirk&Cooper2013) Muscari onMuscarischliemannii M. botryoides,respectively(Euro+Med (Lindtner1950,Zundel1953,Denchev s. latointherecentworldmonograph Muscari alpinum M. neglectum. et al.(1980),manyrecordsofM. Antherospora hortensis plantswerefoundingardens Muscari armeniacum Antherospora hortensis (Euro+Med2006-). andM.moschatum. isintheBalkan doesnotpose fromBerlin IMA FUNGUS J.Gayex on (Euro+Med persisted . wasnot Muscari Muscari Muscari Muscari M. Antherospora on Muscari any authorities (Bubák 1916). The anther smut on Muscari 8 >}> ' 8 " 8 ARTICLE moschatum that was reported from Georgia (Karatygin & Archiv für die Naturwissenschaftliche Landesdurchforschung Azbukina 1989), is a putative fourth distinct species within von Böhmen 15(3): 1–81. the Antherospora vaillantii complex, a hypothesis based on Carris LM, Castlebury LA, Huang GM, Alderman SC, Luo JF, Bao XD the isolated position of the host in Muscari subgen. Muscari. (2007) Tilletia vankyi, a new species of reticulate-spored bunt Fresh materials for molecular and morphological analyses fungus with non-conjugating basidiospores infecting species of Festuca and Lolium. Mycological Research 111: 1386–1398. In addition to resolving the species within the Antherospora Castlebury LA, Carris LM (1999) Tilletia walkeri, a new species on vaillantii complex on Muscari, this study gives further and L. perenne. Mycologia 91: 121–131. ; ; ; Castresana J (2000) Selection of conserved blocks from multiple some smut fungi, to a greater degree than morphology. Past alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17: 540–552. host family (Fischer & Shaw 1953), subtribe (Vánky 2004b) Chakrabarty P (2010) Genetypes: a concept to help integrate or, mostly, genus level (Vánky 1994, 2012). Recent molecular molecular phylogenetics and taxonomy. Zootaxa 2632: 67–68. studies indicate that host specialization is, in most cases, at #~ / ; ! # E??} * the species level (Kemler et al. 2009, Lutz et al. 2008, 2012, for the expanded asparagalean families Amaryllidaceae, = et al. 2012, 2013, Savchenko et al. 2013). This is not Asparagaceae and Xanthorrhoeaceae. Botanical Journal of the an absolute rule since some smut species may infect two Linnean Society 161: 132–136. or more host species from the same genus (Castlebury & Ciferri R (1928) Quarta contribuzione allo studio degli Ustilaginales. Carris 1999, Lutz et al. 2005, Curran et al. 2009, Kemler et Annales Mycologici 26: 1–68. al. 2013), or two or more species from different but usually Ciferri R (1931) Quinta contribuzione allo studio degli Ustilaginales. phylogenetically closely related genera (Carris et al. 2007, Annales Mycologici 29: 1–74. ` ! E??V " ; Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) different smut species reported from multiple host species MycoBank: an online initiative to launch mycology into the 21st and genera, including the other members of the genus century. Studies in Mycology 50: 19–22. Antherospora, remains to be tested and is a challenge for Crous PW, Groenewald JZ, Risède JM, Simoneau P, Hyde future studies. KD (2006) Calonectria species and their Cylindrocladium anamorphs: species with clavate vesicles. Studies in Mycology 55: 213–226. ACKNOWLEDGEMENTS Curran HR, Roets F, Dreyer LL (2009) Anther-smut fungal infection of South African Oxalis species: spatial distribution patterns and We thank Michael Weiß, Sigisfredo Garnica, and Robert Bauer impacts on host fecundity. South African Journal of Botany 75: " ; 807–815. Kyrylo G. Savchenko (Haifa, Israel) for sending a material, the Denchev CM (2001) Classis Ustomycetes (Ordines Tilletiales, Curators of herbaria: Herbert Boyle (GLM), Bart Buyck (PC), Markus Ustilaginales et Graphiolales) % % / $ Ershad D (2000) Vankya, a new genus of smut fungi. Rostaniha 1: * %# 65–72. pictures. Gerard Thijsse (Leiden, Netherlands) tried to locate the Euro+Med (2006-) Euro+Med PlantBase - the information resource Delastre collection of Ustilago vaillantii in the Persoon herbarium (L) for Euro-Mediterranean plant diversity. 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