Persoonia 29, 2012: 63–77 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158512X660562

Soral synapomorphies are significant for the systematics of the --Macalpinomyces complex ()

A.R. McTaggart1,2,3,5, R.G. Shivas1,2, A.D.W. Geering1,2,5, B. Callaghan2, K. Vánky4, T. Scharaschkin1,3

Key words Abstract The genera Ustilago, Sporisorium and Macalpinomyces are a polyphyletic complex of pathogenic fungi. The four main morphological characters used to define these genera have been considered homoplasious columella and not useful for resolving the complex. This study re-evaluates character homology and discusses the use of maximum likelihood these characters for defining monophyletic groups recovered from a reconstructed phylogeny using four nuclear morphology loci. Generic delimitation of fungi based on their hosts is also discussed as a means for identifying genera peridium within this group. Morphological characters and host specificity can be used to circumscribe genera within the smut fungi Ustilago-Sporisorium-Macalpinomyces complex. spore balls sterile cells Article info Received: 18 May 2012; Accepted: 3 October 2012; Published: 4 December 2012. systematics

INTRODUCTION Within the sorus, columellae form a central axis of fungal and host origin (Vánky 2002); sterile cells, either derived from non- Three genera of smut fungi (subphylum ), sporogenous hyphae or a fungal peridium, are found with the Ustilago, Sporisorium and Macalpinomyces, contain about spores (Langdon & Fullerton 1975, 1978); spore balls appear 530 described species that all infect grasses (Vánky 2012). as either an ephemeral or permanent agglomeration of spores Several phylogenetic studies have demonstrated that Ustilago (Vánky 2002). A peridium is the outer layer of the sorus and can and Sporisorium together form a monophyletic group within be composed of host or fungal material (Vánky 2002). Soral the Ustilaginomycotina (Swann & Taylor 1995, Bauer et al. characters have had different interpretations by mycologists 1997, Begerow et al. 1997, 2004b, 2006, Stoll et al. 2003, (Stoll et al. 2005). For example, the columella in Ustilago po- 2005). Macalpinomyces has an ambiguous position in the Us- rosa was considered absent by Langdon (1962) but present by tilaginales as the type species, M. eriachnes, sits outside the Vánky & Shivas (2001). Similarly, Sporisorium consanguineum Ustilago-Sporisorium group (Begerow et al. 2006). Morphologi- was considered to have a columella by Langdon & Fullerton cal characters have proven inadequate for separation of species (1975), but not by Vánky & Shivas (2008). Subsequently, soral among the three genera. The three genera are polyphyletic morphology has been considered too variable to serve as a (Stoll et al. 2003, 2005), and collectively form an unresolved reliable character that can separate Ustilago, Sporisorium and complex. Morphological studies (Langdon & Fullerton 1975, Macalpinomyces (Piepenbring 2004, Stoll et al. 2005). Vánky 1991, Piepenbring et al. 1998) and molecular phylo- The current study discusses morphological characters in the genetic analyses (Stoll et al. 2003, 2005) have not identified Ustilago-Sporisorium-Macalpinomyces complex. A re-evalua- characters that define monophyletic groups amongst species tion of their homology is provided in light of the phylogenetic within this complex. results obtained. The merits of using host specificity and soral Smut fungi in the Ustilago-Sporisorium-Macalpinomyces com- synapomorphies are discussed as a basis for delimiting genera. plex either partially or completely destroy the inflorescence of grasses, forming a sorus that contains fungal spores. Four Materials and Methods characteristics of the sorus, namely columellae, sterile cells, spore balls and peridia, have been used traditionally to sepa- Taxon selection rate Ustilago, Sporisorium and Macalpinomyces (Vánky 2002). Taxa were selected to represent the main groups recovered in 1 Cooperative Research Centre for National Plant Biosecurity, GPO Box previous studies (Stoll et al. 2003, 2005), with increased sam- 5012, Bruce, ACT 2617, Australia; pling of under-represented groups, for example species of Ma- corresponding author e-mail: [email protected]. calpinomyces and smut fungi occurring on Aristida. In total, this 2 Department of Agriculture, Fisheries and Forestry, Ecosciences Precinct, study included 136 species (14 species of Macalpinomyces, 81 GPO Box 267, Brisbane, Queensland 4001, Australia. 3 EEBS, Faculty of Science and Technology, Queensland University of species of Sporisorium and 38 species of Ustilago), 35 of which Technology, 2 George Street, Brisbane, Queensland 4001, Australia. had not previously been evaluated in systematic studies (Table 4 Herbarium Ustilaginales Vánky (HUV), Gabriel-Biel-Str. 5, D-72076 Tübin- 1). Two distinctive members of the complex, Anomalomyces gen, Germany. panici and Melanopsichium pennsylvanicum, were also included. 5 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Ecosciences Precinct, GPO Box 267, Brisbane, Queensland Moesziomyces bullatus was included as an outgroup to the 4001, Australia. complex based on a relationship reported by Stoll et al. (2005).

© 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. 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. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 64 Persoonia – Volume 29, 2012 – – – – – – – – – – – – – – – – – – – – – – – – – – – – HQ013063 – – – – – – HQ013057 HQ013058 HQ013059 HQ013055 HQ013065 – HQ013056 HQ013061 – HQ013064 HQ013066 HQ013068 HQ013060 HQ013062 – – HQ013067 – – – – – – – – – – – – – – HQ013027 HQ013029 HQ013034 HQ013035 HQ013026 HQ013031 HQ013032 HQ013033 – HQ013036 HQ013030 – – – – – – – – – – – – – – – – – – – – – – – – – 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 GenBank no. – AY740089 AY740090 – AY740109 – – AY740092 – AY740093 AY740153 AY740129 AY740095 AY740097 AY740098 AY740099 AF453943 AY740131 AF453939 AF133580 AY747077 – AY740132 – – – AY740102 – – AY740133 AY740134 DQ459347 HQ013131 FQ013130 HQ013028 – – HQ013122 – HQ013127 – – – HQ013132 HQ013126 HQ013125 LSU EF-1 α GAPDH – –

1 2 2 2 2 2 2 2 2 2 2 2 8 2 2 2 2 8 8 8 8 8 3 8 2 2 2 2 2 4 8 8 GU014817 AY740171 – HQ013088 AY740157 AY740158 AY344978 HQ013086 – – HQ013094 AY344975 HQ013099 HQ013087 AY740152 AY740164 HQ013090 – – HQ013091 HQ013092 – HQ013093 HQ013095 AY998100 – AY344979 AY740039 HQ013089 ITS AY344973 AY344976 AY740151 AY740044 – AY344972 HQ013096 AY740046 HQ013096 – HQ013098 – AY344974 India AY740036 Australia Unknown Australia Australia India AY344977 Bolivia South Africa India AY740049 Australia AY333940 Nicaragua Australia Australia Country Australia Honduras Australia Australia AY740037 Australia Zimbabwe Bolivia India AY740153 Australia Iran AY344970 Ecuador AY740042 Australia India AY740045 Australia Australia Australia Australia Mexico Australia Cuba AY740159 Taiwan Germany AY740056 South Africa Australia India AY740154

Sri Lanka Romania South Africa Papua New Guinea Australia Nicaragua Australia Australia AY740047 India AY740040 Australia DQ459348 China Australia Australia United States sp. Eragrostiella bifaria Eulalia mackinlayi Eragrostis ferruginea Sporobolus australasicus Chrysopogon fallax Dimeria ornithopoda Elionurus muticus Themeda triandra Hackelochloa granularis Chrysopogon fallax Andropogon angustatus Arundinella nepalensis Arundinella nepalensis Host Cenchrus elymoides Andropogon gerardii bombycinus Eriachne aristidea Sarga timorense Loudetia simplex Loudetiopsis chrysothrix Paspalum distichum Schizachyrium fragile Schismus arabicus Bothriochloa saccharoides Themeda triandra Apluda mutica Aristida jerichoensis Dichanthium sericeum Heteropogon contortus Aristida queenslandica Tripsacum Bothriochloa ewartiana Andropogon virginicus Panicum repens Setaria pumila dregeana Trichopteryx Sporobolus actinocladus Themeda quadrivalvis

Chrysopogon fulvus Panicum miliaceum Loudetia flavida Pseudoraphis spinescens Aristida sp. Cenchrus pilosus Aristida sp. Capillipedium spicigerum Polygonum glabrum Panicum trachyrachis Arthraxon lanceolatus Aristida hygrometrica Aristida hygrometrica Sorghum halepense Ust. Exs. 960 52549 BRIP 13634 H.U.V. 51858 BRIP 51865 BRIP Ust. Exs. 472 2601 MP M 55602 Ust. Exs. 849 27687 BRIP 1976 MP BRIP 47958 BRIP 51868 BRIP Specimen no. BRIP 26491 BRIP 2060 MP 52511 BRIP M 54573 51818 BRIP M 56577 2630 MP Ust. Exs. 833 49648 BRIP Ust. Exs. 756 M 56588 49775 BRIP M 56590 26930 BRIP 51819 BRIP 28043 51854/BRIP BRIP 42670 BRIP 20560 H.U.V. 49669 BRIP 2270 MP Ust. Exs. 688 RB 2056 M 56578 49133 BRIP Ust. Exs. 844

Ust. Exs. 407 Ust. Exs. 472 M 56576 39176 BRIP 51871 BRIP 1974 MP 52755 BRIP M 56595 H.U.V. 17548 H.U.V. BRIP 46421 BRIP M 56592 51839 BRIP 27723 BRIP Ust. Exs. 687 Macalpinomyces eragrostiellae Macalpinomyces mackinlayi Macalpinomyces spermophorus Macalpinomyces tubiformis Sporisorium dimeriae-ornithopodae Sporisorium elionuri Sporisorium enteromorphum Sporisorium erythraeense Sporisorium fallax Sporisorium fastigiatum Macalpinomyces arundinellae-setosae Table 1 Isolates of Ustilaginaceae included in this study. Table Species Sporisorium cenchri-elymoidis Sporisorium culmiperdum Sporisorium cymbopogonis-bombycini Macalpinomyces eriachnes Macalpinomyces ewartii Macalpinomyces simplex Macalpinomyces tristachyae Moesziomyces bullatus Sporisorium absconditum Sporisorium aegyptiacum Sporisorium andropogonis Sporisorium anthistiriae Sporisorium apludae-aristatae Sporisorium aristidicola Sporisorium bothriochloae Sporisorium caledonicum Sporisorium confusum Sporisorium dietelianum Sporisorium doidgeae Sporisorium everhartii Sporisorium formosanum Macalpinomyces neglectus Macalpinomyces trichopterygis Macalpinomyces viridans Macalpinomyces bursus

Sporisorium chrysopogonis Sporisorium destruens Macalpinomyces loudetiae Sporisorium anthracoideisporum Sporisorium cenchri Melanopsichium pennsylvanicum Anomalomyces panici Sporisorium arthraxonis Sporisorium consanguineum Sporisorium cruentum A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 65 5 5 5 – – – – – – – – – – HQ013071 – – – – – HQ013070 – – – – – – – – – – – – – – – – – – – – – – – – – – HQ013072 – HQ013076 HQ013073 – HQ013069 HQ013077 HQ013075 HQ013078 HQ013079 – DQ352815 DQ352817 DQ352816 5 5 5 – – – – – – – HQ013039 HQ013040 HQ013041 DQ352829 – – – – – – – HQ013043 HQ013046 HQ013044 HQ013045 DQ352827 HQ013037 HQ013047 HQ013048 HQ013049 – – – HQ013042 – – – – – – – – – – – – – – – – – – – – –

7 – – – 2 2 2 6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 AY740103 AF453941 AY740104 AY740105 AY740135 AY740106 AY740112 – AY740136 AY740107 – AY740108 AY740137 AJ236136 AJ236137 AY740088 AF453944 AY740130 AY740138 AY747076 AY740139 – AY740163 AY740147 AF009872 AY740140 AY740141 AY740113 AY740128 AY740111 AY740114 – – – – GU139171 – – – – – DQ352828 – HQ013135 HQ013134 – – HQ013124

7 2 8 2 2 8 2 2 3 8 2 2 2 8 2 2 2 8 8 8 8 8 2 2 2 2 8 8 2 8 2 8 HQ013102 – – – HQ013104 AF453940 HQ013107 – HQ013108 HQ170519 – HQ013112 AY740050 HQ013109 – HQ013110 HQ013111 GU139172 AY740070 AY740051 HQ013105 – AY344971 – HQ013100 – HQ013103 AY740053 HQ013106 AY740110 AY740019 AY344988 AY344989 AY740162 – AY740060 AY740058 HQ013101 AY998101 AY344985 HQ13113 HQ013114 AY344969 AY344993 AY344991 – HQ013115 AY740021 HQ013116 – HQ013117 Australia Australia Australia Brazil India AY344983 Australia/Indonesia Australia Australia Mexico AY740020 Australia Australia Australia AY740035 Canary Islands Australia Australia Australia China Costa Rica Zimbabwe Australia India AY740055 Canary Islands Indonesia Australia India AY740052 Australia South Africa India AY740059 Australia AY740054 India AY740161 Australia Unknown Cuba AY344982 Greece AY344987 United States Indonesia Yugoslavia United States Cuba AY344992 Venezuela Canary Islands Australia Australia AY344981 Mexico United States Australia Australia Sri Lanka Greece AY740163 Costa Rica Indonesia Australia Panama AY344980 Australia Nicaragua Australia Australia sp. sp. sp. sp. sp. sp. Apluda mutica Heteropogon contortus Sehima nervosum Eulalia aurea Urochloa fasciculata caespitosum Bothriochloa decipiens Digitaria brownii Eremopogon foveolatus Sarga timorense Sehima nervosum Setaria surgens Capillipedium parviflorum Sporobolus panicoides Cymbopogon refractus Heteropogon melanocarpus Pennisetum setaceum Sorghum sp. Aristida nitidula Iseilema Iseilema Coix lacryma-jobi Cymbopogon ambiguus Loudetia pedicellata Sacciolepis indica Iseilema Enneapogon avenaceus Isachne globosa Sehima nervosum Rottboellia cochinchinensis Paspalum notatum Andropogon distachyos Andoropogon gerardii Pseudechinolaena polystachya Saccharum strictum Sorghum sp. Saccharum plumosus Trachypogon plumosus Trachypogon Hyparrhenia hirta Heteropogon contortus Hemarthria uncinata Andropogon Andropogon gerardii Eulalia trispicata Sarga plumosum Chrysopogon aciculatus Sorghum halepense Panicum viscidellum Themeda arguens Saccharum Setaria surgens Andropogon bicornis Whiteochloa semitonsa Sorgum bicolor Sarga leiocladum laniflora Ust. Exs. 967 52504/Ust. Exs. 851 BRIP 49706 BRIP 49134 BRIP MP 1871 MP 43942 BRIP 48629 BRIP DAR 58832a MP 2365 MP 49713 BRIP 49671 BRIP 49636 BRIP HMAS 193085 M 56607 44111 BRIP M 56621 2367 MP CBS 104.17 BRIP 49668 BRIP 51870 BRIP 52517 BRIP M 56611 46819 BRIP M 56615 Ust. Exs. 854 52538 BRIP M 56617 M 56618 27019/M 56622 BRIP HB 20 2101 MP Ust. Exs. 690 Ust. Exs. 759 Ust. Exs. 853 M 56627 2463 MP M 56635 2372 MP BRIP 51822 BRIP Ust. Exs. 966 H.U.V. 20498 H.U.V. Ust. Exs. 758 47730 BRIP 49748 BRIP Ust. Exs. 231 Ust. Exs. 527 MP 735 MP Ust. Exs. 855 MP 541 MP 26910 BRIP MP 1271 MP BRIP 51860 BRIP MP 2036a MP BRIP 27640 BRIP BRIP 49682 BRIP Sporisorium mishrae Sporisorium moniliferum Sporisorium queenslandicum Sporisorium rarum Sporisorium ovarium Sporisorium panici Sporisorium tenue Sporisorium panici-leucophaei Sporisorium setariae Sporisorium foveolati Sporisorium ryleyi Sporisorium sehimatis Sporisorium spinulosum Sporisorium hwangense Sporisorium mutabile Sporisorium nealii Sporisorium penniseti Sporisorium fraserianum Sporisorium iseilematis-ciliati Sporisorium lacrymae-jobi Sporisorium lanigeri Sporisorium loudetiae-pedicellatae Sporisorium manilense Sporisorium mitchellii Sporisorium modestum Sporisorium monakai Sporisorium nervosum Sporisorium ophiuri Sporisorium paspali Sporisorium polliniae Sporisorium provinciale Sporisorium pseudechinolaenae Sporisorium pulverulentum Sr326 Br532 Sporisorium trachypogonicola Sporisorium trachypogonis-plumosi Sporisorium vanderystii Sporisorium heteropogonicola Sporisorium lepturi Sporisorium mexicanum Sporisorium occidentale Sporisorium trispicatae Sporisorium vermiculum Sporisorium tumefaciens Sporisorium reilianum Sporisorium veracruzianum Sporisorium themedae-arguentis Sporisorium scitamineum Sporisorium holwayi Sporisorium whiteochloae Sporisorium wynaadense Sporisorium xerofasciculatum 66 Persoonia – Volume 29, 2012 5 5 – – HQ013080 – – – – – – – – – – – HQ013081 – – – – – – – – – – – – – – HQ013085 – – – – – – – – – HQ013084 HQ013082 – HQ013083 DQ352820 DQ352818 5 5 – – – – – – – – – – – – – – – – – DQ352832 HQ013052 HQ013051 – – – – – HQ013050 – – – – – – – – – – – – – – HQ013053

12 – 2 2 2 2 2 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 GenBank no. AF133581 – AY740115 AF453933 – AY740118 AF453935 AY740119 AY740143 HQ013123 AF009881 – – AY740144 AF453937 AY740120 AF4539432 AY740121 – AJ236139 AY740145 AY740146 – DQ875375 AY740123 AY740124 AY740148 AY740126 AF453938 – HQ013136 – AF453938 – DQ352830 AF453939 HQ013054 – HQ013128 LSU EF-1 α GAPDH – HQ013129

– –

AF453936 AF453937 10 13 8 2 2 8 2 2 8 2 8 2 8 2 2 8 8 2 8 2 8 2 8 8 8 11 2 2 2 2 2 2 2 2 – AY740064 AY740170 – – AY772736 – HQ013118 AY740067 AY740167 AY740065 AY740169 – – AY345002 – HQ013119 AY740072 – AF135424 HQ13120 AY344998 AY740061 ITS – AY344995 HQ013121 AY345006 Australia Argentina Mexico AY345000 South Africa Germany AY345001 Canada Germany South Africa Germany AY740172 Iran AY345005 Country Australia Greece AY740069 Costa Rica Australia AY740166 La Réunion Bulgaria Argentina Germany AY740066 Brazil India AY345011 Australia AY345012 Australia Australia AY740165 Taiwan Iran AY345003 Australia DQ139961 Turkey India AY345008 Zimbabwe Cuba AY345009 Australia Canada Germany AY345004 Iran AY345011 Australia Germany AY344997 Canary Islands India AY344999 Zimbabwe India AY740071

Australia AY740074 Australia Costa Rica Australia Costa Rica sp. sp. sp. Triodia Triodia Bromus catharticus Cynodon dactylon Digitaria tricholaenoides Phalaris arundinacea Hordeum Zea mays Sporobolous pyramidalis Alopecurus pratensis Bromus gracillimus Host longiloba Triodia Hordeum leporinum Ixophorus unisetus Triodia pungens Triodia Stenotaphrum dimidiatum Calamagrostis epigeios Glyceria multiflora Glyceria fluitans Zea mays zizanioides Vetiveria Xerochloa imberbis Xerochloa barbata Tripogon loliiformis Tripogon Hordeum vulgare epactia Triodia Elymus trachycaulus Dactyloctenium radulans berteronianus Tragus Echinochloa colona Echinolchloa utilis aestivum Triticum Zea mays Eremopyrum distans Sarga timorense Avena barbata Avena Bromus diandrus Setaria italica Enneapogon cenchroides Digitaria ternata

Triodia microstachya Triodia Enneapogon polyphyllus Stenotaphrum secundatum Enneapogon Paspalum paniculatum Stoll et al. (2005) Shivas et al. (2004) Begerow et al. (1997) Stoll et al. (2003) Berner et al. (2007) Begerow et al. (2006)

2 4 6 8 10 12 BRIP 52543 BRIP 19322 H.U.V. 51207 1838/BRIP MP M 56523 Ust. Exs. 540 MS 115 39706 BRIP H.U.V. 18286 H.U.V. Ust. Exs. 887 Specimen no. 49123 BRIP 17782 H.U.V. MP 2194 MP Ust. Exs. 418 M 56517 M 56518 19252 H.U.V. RB 3011 17954 H.U.V. Ust. Exs. 1000 49820 BRIP M 56514/BRIP 26929 M 56514/BRIP Ust. Exs. 590 Ust. Exs. 784 46795 BRIP BPI 871725 Ust. Exs. 892 M 56562 2473 MP 49159 BRIP not given RB 3093 H.U.V. 23 H.U.V. BRIP 51842 BRIP DB 559 2363 MP Ust. Exs. 995 M 56516 Ust. Exs. 998

H.U.V. 17662/BRIP 49124 17662/BRIP H.U.V. BRIP 26906 BRIP G. Rivera s.n. BRIP 51848 BRIP Ust. Exs. 887 Vánky et al. (2006) Cunnington et al. (2005) Munkacsi et al. (2007) & Lutz unpubl. data Vanky Piepenbring et al. (2002) Cunnington & Shivas (2006) Bakkeren et al. (2000)

Table 1 (cont.) Table Ustilago bromivora Ustilago cynodontis Ustilago drakensbergiana Ustilago echinata Uh362 Ustilago sporoboli-indici Ustilago striiformis Ustilago pamirica Species Ustilago inaltilis Ustilago nuda 1 3 5 7 9 11 13 Ustilago ixophori Ustilago altilis Ustilago bouriqueti Ustilago calamagrostidis Ustilago davisii Ustilago filiformis B-Pbi-4-1-4 Ustilago vetiveriae Ustilago xerochloae Ustilago curta Ustilago hordei Ustilago lituana Ustilago phrygica Ustilago sparsa Ustilago tragana Ustilago trichophora Ustilago tritici Ustilago maydis Ustilago turcomanica Ustilago porosa Ustilago bullata Ustilago crameri Ustilago austro-africana Ustilago syntherismae

Ustilago triodiae Ustilago schmidtiae Ustilago affinis

Ustilago schroeteriana A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 67

Morphological data Phylogenetic analyses Character and character state selection were based on taxo- Two phylogenetic assessment criteria were implemented: Bayes- nomic descriptions in monographs of the Ustilaginomycotina ian inference using MrBayes (Huelsenbeck & Ronquist 2001, (Vánky 1994, 2012, Vánky & Shivas 2008) and from direct Ronquist & Huelsenbeck 2003) and maximum likelihood using observation of 61 Australian species. Columellae were scored RAxML (Stamatakis 2006) and PhyML 3.0 (Guindon et al. 2010). as either absent, stout or filiform. Spore states were classified Resulting trees were observed with FigTree (available at http:// as single spores, permanent spore balls, ephemeral spore www.tree.bio.ed.ac.uk/software/figtree/). Data and command balls or dimorphic spores. Sterile cells were scored as present files for both Bayesian and RAxML analyses and the resulting or absent. The peridium was classified as either host derived, trees are available at TreeBASE (http://purl.org/phylo/treebase/ hypertrophied-host derived or fungal derived. These characters phylows/study/TB2:S11013). The four loci were included as were mapped onto the final tree topology using MacClade v. separate partitions in the maximum likelihood and Bayesian 4.08 (Maddison & Maddison 2001). analyses so that each locus could be run under different optimal model parameters. DNA extraction Maximum likelihood analysis — Maximum likelihood was im­ DNA was extracted from 120 smut specimens representing plemented as a search criterion in RAxML (Stamatakis 2006) and 92 taxa, by a combination of enzymatic and mechanical lysis. PhyML 3.0 (Guindon et al. 2010). GTRGAMMA was specified as Smut sori or spores were mechanically lysed using a QIAGEN the model of evolution in both programs. The RAxML analyses TissueLyser with 0.5 mm stainless steel beads, then shaken were run with a rapid Bootstrap analysis (command -f a) using at 55 °C overnight in SNES buffer (0.01 M sodium phosphate a random starting tree and 1 000 maximum likelihood bootstrap pH 7.6, 0.15 M sodium chloride, 0.005 M EDTA, 1 % SDS) replicates. The PhyML analyses were implemented using the containing proteinase K at a final concentration of 0.8 µg/mL. ATGC bioinformatics platform (available at: http://www.atgc- The purification was then completed using the QIAGEN Gentra montpellier.fr/phyml/), with SPR and NNI tree improvement, Puregene kit according to the manufacturer’s instructions. and support obtained from an approximate likelihood ratio test (Anisimova et al. 2011). PCR and sequencing Genomic DNA was amplified by PCR with high fidelity Phusion® Bayesian analysis — MrBayes was used to conduct a Markov DNA Polymerase (Finnzymes) using the manufacturer-specified Chain Monte Carlo (MCMC) search in a Bayesian analysis. Four cycling and reaction conditions. The ITS region was amplified runs, each consisting of four chains, were implemented until the with primers M-ITS1 (Stoll et al. 2003) and ITS4 (White et al. standard deviation of split frequencies were 0.02. The cold chain 1990) at 58 °C; the LSU region was amplified with primers was heated at a temperature of 0.25. Substitution model param- LROR and LR5 (Vilgalys & Hester 1990) at 58 °C; the GAPDH eters were sampled every 50 generations and trees were saved locus was amplified with GAPDH-F (CGGTCGTATCGGMCG- every 5 000 generations. Convergence of the Bayesian analysis TATC) and GAPDH-R (GTARCCCCACTCGTTGTCGTA) at was confirmed using AWTY (Nylander et al. 2008) (available 65 °C; the EF-1α locus was amplified with primers EF-1αF at: ceb.csit.fsu.edu/awty/). Convergence was not reached even (GCCCTMTGGAAGTTCGAGACYCCCA) and EF-1αR (GAY- after 40 million generations with all datasets. A user-defined tree ACCGACAGCRACGGTCTG) at 62 °C. PCR products were obtained from the maximum likelihood analyses was used as a purified by ethanol precipitation using standard methods (Ma- starting point for all of the Bayesian analyses, which helped to niatis et al. 1982). Purified PCR product was sent to Macrogen improve convergence of the four runs. A burn-in was not used Korea or the Australian Genome Research Facility, Queensland to summarize the values that were created with a user-defined for sequencing using the forward and reverse primers from tree. amplification. ABI sequence trace files were assembled using ContigExpress® (Invitrogen™). The 165 novel sequences have RESULTS AND DISCUSSION been deposited in GenBank (Table 1). Eight clades were consistently recovered in a phylogenetic ana­ Alignment of sequences lysis of four molecular loci (Fig. 1). The major clades recovered Sequences were aligned using the Muscle algorithm (Edgar in this study were similar to those obtained in previous molecular 2004) included in the MEGA5 software package (Kumar et al. phylogenetic analyses using different assessment criteria. For 2008). Alignments of protein-coding loci (GAPDH and EF-1α) example, several phylogenetic studies have reconstructed two were converted to amino acid sequences in MEGA. The origi- monophyletic groups in Sporisorium (Stoll et al. 2003, 2005, nal and curated nucleotide alignments have been deposited Cunnington et al. 2005, Vánky et al. 2006, Vánky & Lutz 2011), as Nexus files in TreeBASE (http://purl.org/phylo/treebase/ but these studies were not able to separate the two groups using phylows/study/TB2:S11013). The super-matrix consisted of morphological characters. The structure of columellae (Fig. 2), ITS sequences for 134 taxa, LSU sequences for 91 taxa, EF-1α the presence or absence of sterile cells (Fig. 3) and the pres- sequences for 32 taxa and GAPDH sequences for 35 taxa. ence or absence of spore balls (Fig. 4) were traced onto the topology. A discussion of the homology of these characters and Curation of alignments their use in identifying the clades of the Ustilago-Sporisorium- Macalpinomyces complex follows. Alignments were uploaded to Phylogeny.fr (available at http:// www.phylogeny.fr/) (Dereeper et al. 2008) and curated in Clade 1 Gblocks to remove poorly aligned positions and divergent re- gions (Talavera & Castresana 2007). Alignments were trimmed Clade 1 includes S. sorghi, the type of Sporisorium. The mem- as follows: ITS from 1 140 nucleotides, including gaps, to 448 bers of this clade share a number of characters. nucleotides with no gaps; LSU from 609 to 593 nucleotides; 1. A hardened or stout columella that either replaces the entire EF-1α from 935 to 926 nucleotides; GAPDH from 1 158 to inflorescence, for example in Sporisorium andropogonis, 769 nucleotides. The final curated super-matrix consisted of S. doidgeae and S. scitamineum (Fig. 5b), or that occurs in 2 736 nucleotides, which was composed of approximately all of the ovaries or spikelets of an inflorescence, for example 47 % missing data. in S. ryleyi, S. sorghi (Fig. 5d) and S. rarum (Fig. 5e). 68 Persoonia – Volume 29, 2012

2. Sterile cells formed from non-sporogenous hyphae that are The infection is usually systemic and destroys either the entire intermixed with spores in the sorus (Fig. 5c, f), except in inflorescence or all of the ovaries or spikelets. Ustilago porosa and Sporisorium culmiperdum. Langdon & Fullerton (1978) examined the soral ontogeny of 3. A peridium derived mainly from host tissue, either from leaf several species included in Clade 1, namely Sporisorium an- sheaths or the ovary wall. dropogonis, S. sorghi and S. vanderystii. They observed that the columella began to form after intercellular hyphae became Taxa in Clade 1 mainly infect grasses belonging to the subfamily confluent and caused the host cells to proliferate. Hyphae at , in one of two tribes, Paniceae or Andropogoneae. the periphery of the columella formed a sheath of elongated,

Moesziomyces bullatus Macalpinomyces eriachnes 100/100 Sporisorium aristidicola Spo. fraserianum CLADE 8 Spo. confusum 1.0 Spo. consanguineum /78 Mac. eragrostiellae Spo. veracruzianum Mac. spermophorus Ustilago austroafricana 97/92 Mac. viridans Ust. ixophori Mac. neglectus CLADE 7 1.0 Ust. crameri Spo. panici Ust. affinis Ust. drakensbergiana Ust. syntherismae Ust. echinata Melanopsichium pennsylvanicum Ustilago esculenta sub-clade Ust. esculenta Ust. schmidtiae Ust. tragana 89/96 Spo. aegyptiacum /75 Spo. modestum Ustilago davisii Ust. filiformis sub-clade Ust. davisii Ust. schroeteriana Ust. trichophora Ust. nuda Ust. avenae Ust. hordei CLADE 6 Ust. phrygica Ust. tritici Ust. bullata Ustilago 84/97 Ust. pamirica sensu stricto Ust. turcomanica 0.96 Ust. bromivora sub-clade Ust. calamagrostidis Ust. striiformis Ust. sporoboli-indici 0.96 Ust. cynodontis Ust. sparsa Ust. xerochloae 80/87 Ust. lituana Ust. triodiae Ust. altilis CLADE 5 0.99 Ust. inaltilis Anomalomyces panici Spo. mexicanum Spo. provinciale Spo. elionuri Spo. loudetiae-pedicellatae Spo. bothriochloae Spo. mishrae Spo. absconditum Spo. cymbopogonis-bombycini Spo. heteropogonicola Spo. everhartii Spo. apludae-aristatae Spo. sehimatis Spo. anthistiriae Spo. mutabile Spo. trispicatae Spo. hwangense 85/90 Spo. chrysopogonis Spo. themedae-arguentis CLADE 4 1.0 Spo. polliniae Spo. caledonicum Spo. setariae Spo. whiteochloae Spo. cenchri-elymoidis Spo. xerofasciculatum Spo. penniseti Spo. anthracoideisporum Spo. enteromorphum Spo. ovarium Spo. cenchri Spo. panici-leucophaei Spo. destruens Spo. formosanum Spo. fallax Spo. tumefaciens

81/95 Mac. bursus Mac. ewartii CLADE 3 0.96 Ust. bouriquetii /84 Ust. maydis Mac. mackinlayi Mac. arundinellae-setosae Ust. vetiveriae CLADE 2 Spo. trachypogonis 100/99 Mac. simplex Systemic Mac. trichopterygis group 1.0 Mac. tristachyae Spo. scitamineum Spo. nealii Spo. andropogonis Spo. occidentale Spo. tenue Spo. holwayi Spo. lanigeri Spo. vermiculum Spo. queenslandicum Spo. trachypogonicola Spo. vanderystii Spo. paspali Spo. porosum Spo. rarum Spo. ryleyi Spo. wynaadense Spo. erythraeense Spo. doidgeae 90/92 Spo. pulverulentum Spo. cruentum CLADE 1 1.0 Spo. moniliferum Spo. sorghi Spo. foveolati Spo. nervosum Spo. mitchellii Spo. iseilematis-ciliati Spo. culmiperdum Spo. spinulosum Spo. dimeriae-ornithopodae Spo. lepturi Spo. monakai Fig. 1 Phylogram obtained from a maximum likelihood analysis in RAxML. Spo. manilense Spo. arthraxonis Maximum likelihood support values (> 70 %) from RAxML 1 000 bootstrap Spo. lacrymae-jobi Spo. pseudechinolaenae replicates and PhyML aRLT values shown above the nodes. Posterior prob- Spo. ophiuri Spo. reilianum abilities (> 0.95) from Bayesian inference shown below the nodes. Spo. fastigiatum A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 69 thick-walled, vacuolate cells. Other hyphae were present inter- though subsequent descriptions of smut fungi referred to them and intracellularly in the tissue of the columella. as sterile cells. The term sterile cells is maintained to differenti- Columellae of species in Clade 1 are stout and woody due to ate between the cells formed by non-sporogenous, partitioning the peripheral formation of thick-walled, vacuolate cells (Fig. 2). hyphae, and the peridial cells formed from the peridium. These columellae are cylindrical and grow vertically. Occasion- ally, more than one columella is present in a sorus, for example Clade 2 in S. reilianum (Fig. 5a). Sometimes columellae are branched, Species within Clade 2 have been described in Ustilago, Spo­ for example in S. doidgeae (Fig. 5b). Stout columellae are a risorium­ and Macalpinomyces. They share two common mor- synapomorphy for species in Clade 1 (Fig. 2) phological characters. Langdon & Fullerton (1978) observed that non-sporogenous 1. The sori are relatively long, twisted and cylindrical, and are hyphae partitioned the sporogenous hyphae in sori of Sporiso- derived from hypertrophied host material, as in Macalpino- rium sorghi. The partitioning hyphae formed groups of hyaline myces tubiformis (Fig 6a), M. mackinlayi and Sporisorium cells that mixed with the spores as the sorus matured. This dietelianum. pattern of development accounts for the chains of sterile cells 2. Sterile cells are usually found within the sori. found in many species of Sporisorium (Fig. 3), for example S. rarum (Fig. 5f), S. ophiuri, S. themedae and S. vermiculum. There are two types of infection in Clade 2: a localized infection Langdon & Fullerton (1978) termed these ‘partitioning cells’, seen in most of the species, or a systemic infection seen in a

CLADE 4

CLADE 3

CLADE 2

Columellae absent

Columellae stout CLADE 1

Columellae filiform

Fig. 2 Structure of the columella mapped onto five clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Macalpinomyces. 70 Persoonia – Volume 29, 2012

CLADE 8

CLADE 7

CLADE 6

CLADE 5

CLADE 4

CLADE 3

CLADE 2

Partitioning cells absent

Partitioning cells present

CLADE 1

Fig. 3 Distribution of sterile cells within clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Macal­ pinomyces. monophyletic group of taxa that destroy the entire inflorescence its uncertain taxonomic position and distinct appearance on or infect the culms of the host. The position of the systemic the host. group was ambiguous and only had data from the ITS and The systemic group of Clade 2 contained four species, Macal- LSU regions. It either formed a well-supported monophyletic pinomyces loudetiae (not included in Fig. 1), M. simplex, M. tri- group within Clade 6, which was also recovered by Stoll et al. chopterygis and M. tristachyae. These smuts infect grasses (2005) using nuclear rDNA loci; or it occurred sister to Clade 2 in the subfamily Arundinoideae, a character first observed by when using nuclear rDNA and protein-coding loci, as was also Stoll et al. (2005). The entire inflorescence or every spikelet in recovered by Vánky & Lutz (2011). The systemic monophyletic the inflorescence is destroyed by tubular sori. Vánky (1995a) group will be discussed separately from Clade 2 because of described Endosporisorium, based on Sorosporium capillipedii A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 71

CLADE 8

CLADE 7

CLADE 6

CLADE 5

CLADE 4

CLADE 3 Spore balls absent

Spore balls permanent

Spore balls ephemeral CLADE 2

CLADE 1

Fig. 4 Presence of spore balls within clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Ma- calpinomyces.

(syn. M. chrysopogonicola), to accommodate smuts with long, to determine if this method of infection is synapomorphic and tubular, host derived sori that contained sterile cells and lacked whether the separation of Endosporisorium from Macalpino- columellae (Vánky 1995a, 2002). Endosporisorium was later myces is warranted. synoymised with Macalpinomyces, as Vánky (1997) preferred The remaining taxa in Clade 2 form tubular sori derived from to have few larger, well-known genera rather than many hypertrophied host material in some ovaries of the inflorescence smaller, unresolved genera. Three other taxa not included in the and have sterile cells in the sori, with the exception of U. maydis. phylo­genetic analysis have a similar appearance to members The model organism U. maydis occurred in Clade 2 and was of the systemic group, namely M. effusus, M. magicus and considered more closely related to Sporisorium than Ustilago M. ugandensis. These taxa should be included in future studies by Piepenbring et al. (2002) and Stoll et al. (2005). 72 Persoonia – Volume 29, 2012

a b c

d e f Fig. 5 Clade 1 character states. a. Columellae in Sporisorium reilianum; b. branched columella destroying entire inflorescence in S. doidgeae; c. spores and sterile cells of S. themedae; d. all ovaries of the inflorescence infected in S. ryleyi; e. all spikelets of the inflorescence infected in S. rarum; f. spores and sterile cells of S. rarum. — Scale bars: a, b, e = 1 cm; c, f = 10 µm.

Brefeld (1912) established Mycosarcoma for Ustilago maydis, Sporisorium dietelianum and S. trachypogonis, which are mem- which he diagnosed as different to Sporisorium sorghi (as bers of Clade 2, were both described as having columellae Ustilago sorghi) for three reasons: i) the incubation time in the (Fig. 2). It is unlikely that these structures are homologous to host; ii) the development of the sorus at the site of penetration the stout and filiform columellae in Clades 1 and 4, which are in the host plant; and iii) the development of aerial conidia. The synapomorphies for these clades. Vánky (2004) combined peridial structure of Ustilago maydis was another character that Sporisorium dietelianum into Lundquistia because he did not Brefeld (1912) considered different to other species of Ustilago. consider the fascicles of host tissue as true columellae. Vánky Two of the characters that Brefeld (1912) described are unique (2012) later re-considered this view, equating these fascicles characters to Clade 2, excluding the systemic group. The hy- with columellae. The columellae of Sporisorium dietelianum pertrophied, host derived peridium and the localized infection are filiform and similar to the columellae of species in Clade 4. sites on the host inflorescence are morphological synapomor- Sporisorium dietelianum can be distinguished from species in phies of these taxa. Furthermore, the localized, hypertrophied, Clade 4 because it does not form either a fungal peridium or often tubular sori mostly contain sterile cells. Piepenbring et al. spore balls, and it possesses sterile cells. (2002) concluded from a molecular phylogenetic analysis that The columella of Sporisorium trachypogonis was described by Ustilago maydis was separate to other Ustilago taxa, and that it Vánky (1995b) as well-formed and central, which is typical to may warrant placement in the genus originally assigned to it by those formed in the taxa of Clade 1. Sporisorium trachypogonis Brefeld (1912). Other taxa that may belong to Clade 2, based can be distinguished from other species in Clade 1 by the on soral characters, are Macalpinomyces elionuri-tripsacoidis, pre­sence of a localized tubular sorus, rather than a systemic M. flaccidus, M. nodiglumis, M. siamensis and M. zonotriches. infection. A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 73

a b Fig. 6 Clade 2 character states. a. Localized spikelets infected by Macalpinomyces tubiformis; b. spores and sterile cells in M. tubiformis. — Scale bars: a = 1 cm; b = 10 µm.

The recently described, monotypic genus, Tubisorus was not Langdon & Fullerton (1978) examined the soral ontogeny of two included in the current study. Vánky & Lutz (2011) recovered species found in Clade 4, S. anthistiriae and S. caledonicum. Tubisorus within a clade congruent to Clade 2. The infection They described the columella of Sporisorium caledonicum of Tubisorus is consistent with other members of Clade 2 that as a vascular bundle surrounded by host parenchyma, with possess long tubular sori. However, Tubisorus is described tissues permeated by inter- and intracellular hyphae. Five to as lacking sterile cells and possessing spore balls, which seven columellae were formed by growth of hyphae in the are two characters considered synapomorphies of Clade 4. parenchyma between the vascular bundles that separated The establish­ment of Tubisorus sets a precedent for creation the central column. Host cells close to intercellular hyphae in of monotypic genera that have an eclectic mix of characters some instances were distorted but there was little destruction within Clade 2. of host tissue. Langdon & Fullerton (1975) also studied the soral ontogeny of Sporisorium cryptum, which had a single Clade 3 columella made of several vascular bundles of parenchyma Macalpinomyces bursus and M. ewartii occur in a strongly and mycelium that did not separate. supported clade separate from other clades recovered in the Species within Clade 4 have filiform or slender columellae (Fig. analysis. Macalpinomyces bursus and M. ewartii are morpho- 2). These columellae are typically flattened in one plane and are logically very similar in appearance and occur on Themeda never cylindrical. They are flexuous and do not grow vertically and Sorghum respectively, which are members of the tribe without support from the sorus as there are no thickened cells Andropogoneae. The sori form hypertrophied galls in the host to sustain vertical growth. Many columellae are present in the ovaries. Sterile cells formed from partitioning hyphae are pre- sorus, for example in Sporisorium caledonicum, S. enteromor- sent in the sori, which never have a columella. The spores are phum and S. fallax. A single, filiform columella comprised of prominently echinulate. These characters are similar to smuts in several vascular bundles is sometimes present, for example Clade 7 that infect grasses in the subfamily Chloridoideae and in Sporisorium bothriochloae and S. cryptum. The columellae the tribe Paniceae. Host classification is the simplest character formed in this fashion are not hardened or woody, although to separate these two clades. Other smut taxa that may occur in they are sufficiently robust to persist in the sorus. this clade are Macalpinomyces bothriochloae, M. ovariicolopsis The presence of a columella was the defining character of and M. pseudanthistiriae. Sporisorium (Link 1825, Langdon & Fullerton 1978, Vánky 2002). Members of Clades 1 and 4 that were examined by Clade 4 Langdon & Fullerton (1975, 1978) possessed two differences Species in Clade 4 either destroy the entire inflorescence, as in in development and structure of columellae. The first difference Sporisorium caledonicum (Fig. 7c) and S. tumefaciens; whole was that peripheral cells of Clade 4 species were not distorted racemes, as in S. enteromorphum; or are localized in the in- or hardened in contrast to the thickened, vacuolated peripheral florescence, as in S. heteropogonicola (Fig. 7a), S. anthistiriae cells in Clade 1 species. The second difference was that the and S. bothriochloae. Species in Clade 4 exhibit a number of central columns were separated into several columellae in common morphological characters. Sporisorium caledonicum or were made of numerous vascular 1. Filiform or slender columellae (Fig. 7a, c). bundles, as in S. cryptum; the columellae of Clade 1 members, 2. Persistent spore balls (Fig. 7d). Two distinct spore types are S. andropogonis and S. sorghi were not separated into vascular usually present within the spore ball, namely inner and outer bundles. Filiform columellae composed of vascular bundles spores. Outer spores are often ornamented and are darker constitute a synapomorphy in species of Clade 4 (Fig. 2). than the inner spores (Fig. 7b). Many species of Sporisorium that possess permanent spore 3. A sorus surrounded by a peridium composed mostly of fungal balls were originally described as members of Sorosporium. tissue. Most of these species belong to Clade 4 (Fig. 4). Langdon & 4. Sterile cells derived from non-sporogenous hyphae are rarely Fullerton (1975) observed spore balls in several Sporisorium present within the sorus. (as Sorosporium) species and described their formation. Coils 74 Persoonia – Volume 29, 2012 of sporogenous hyphae were produced among mycelium that ples of smut fungi that share characters in Clade 4 but occur on grew from the columellae as the sorus elongated. Coils con- chloridoid grasses are S. cynodontis, S. normanensis, S. pa- sisted of two or three intertwined hyphae. Non-sporogenous rodii and S. saharianum. hyphae, present between the spore balls, disintegrated and did not form sterile cells. Spores formed in spore balls were dimor- Anomalomyces panici phic. The peripheral spores developed surface ornamentation in Anomalomyces panici is sister to Clades 1, 2, 3 and 4. In terms the form of warts or spines and the internal spores were smooth. of soral morphology, this species is similar to M. bursus and Sporisorium panici-leucophaei has spore balls and occurs in M. ewartii as it forms globose hypertrophied sori localized in Clade 4. According to Vánky (2001) the spore balls of Lund­ the host ovaries. Anomalomyces infects Panicum trachyrachis quistia fascicularis (syn. S. panici-leucophaei) differentiate from in the tribe Paniceae. The sorus is filled with hardened spore non-concentric, sporogenous hyphae. This differed from the balls formed by coiled sporogenous hyphae (Vánky et al. 2006), mode of formation described for Sporisorium by Langdon & dimorphic spores and sterile cells. Anomalomyces possessed a Fullerton (1975), and was one reason Vánky (2001) established unique combination of characters that warrants a monotypic ge- Lundquistia. The mode of spore ball development in Lundquistia nus within the Ustilago-Sporisorium-Macalpinomyces complex. fascicularis (syn. S. panici-leucophaei) cannot be determined from the images provided by Vánky (2001). The spore balls are Clade 5 not agglutinated by sterile cells, as in Moesziomyces, and if the Four taxa that occur on the arid grass Triodia form a clade sporogenous hyphae are intertwined, as for species in Clade 1, supported in maximum likelihood and Bayesian inference. The then it is unlikely that the spores would form balls. It is unknown Bayesian analysis conducted by Stoll et al. (2005) grouped two how spore balls are formed in Sporisorium panici-leucophaei. Triodia taxa with the Ustilago esculenta group within Clade 6. Langdon & Fullerton (1975) observed that non-sporogenous Ustilago altilis and U. inaltilis infect the host plant culms, while hyphae in Sporisorium caledonicum, and three other species that U. lituana and U. triodiae destroy the host inflorescence. Near occurred in Clade 4, disintegrated after the spores had matured. identical ITS sequences for U. altilis and U. inaltilis (99 % identi- Sterile cells are rarely present in species of Clade 4 (Fig. 3). Often cal over 98 % query coverage in a BLAST search), and U. litu- peridial cells derived from the fungal peridium were reported as ana and U. triodiae (98 % identical over 88 % query coverage in sterile cells for species in Clade 4, for example in Sporisorium a BLAST search) demonstrate their very close relationships. A loudetiae-pedicellatae. synapomorphy for these four taxa is that they infect species of Species within Clade 4 possess a peridium made of fungal Triodia. They have similar characters to species in Clade 6, in cells surrounded by a layer of host cells. Langdon & Fullerton that they do not possess soral structures such as spore balls, (1975) discussed the formation of this peridium in Sporisorium columellae or sterile cells. caledonicum and three other smut fungi that occurred in Clade 4. They observed that hyphae adjacent to the peripheral host Clade 6 tissues became enlarged, with vacuolate cells and thickened Stoll et al. (2005) recovered Clade 6 as a weakly supported cell walls. These hyphae were orientated in the direction of clade, which included Melanopsichium pennsylvanicum. They the long axis of the sorus and formed a sheath inside the peri­ designated this clade as Ustilago s.l. and defined three sub- pheral layer of host tissue. This fungal sheath and the host cells groups within the clade: i) Ustilago s.str.; ii) the Ustilago davisii external to it constituted the soral peridium, which surrounded group; and iii) the Ustilago esculenta group. Further loci were the soral contents. only sequenced for six taxa of Clade 6 in this study. Host and Members of Clade 4 mostly occur on grasses in the tribes An- morphological synapomorphies have not been resolved for dropogoneae or Paniceae in the subfamily Panicoideae. One Clade 6 in our analysis. exception is Sporisorium hwangense that infects Sporobolus in the subfamily Chloridoideae. It shares characters with other Ustilago s.str. clade taxa in Clade 4, namely filiform columellae, spore balls with Ustilago species that infect grasses in the tribe Pooideae dimorphic spores, and an absence of sterile cells. Other exam- formed a well-supported group that included the type species,

a b c d Fig. 7 Clade 4 character states. a. Localized spikelets infected in Sporisorium heteropogonicola; b. dimorphic spores of S. heteropogonicola; c. entire inflo- rescence destroyed by S. caledonicum; d. permanent spore balls of S. caledonicum. — Scale bars: a, c = 1 cm; b, d = 10 µm. A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 75

U. hordei. Stoll et al. (2005) also recovered this group with Sterile cells are present in Macalpinomyces neglectus, M. sper­ strong support using Bayesian analysis. The stripe smuts mophorus, M. viridans and Ustilago affinis, but are absent in the U. calamagrostidis and U. striiformis, as well as U. sporoboli- other members of this clade. Several taxa formed galls in the host indici (on Chloridoideae) were sister to the smuts that destroy ovaries, while U. affinis, U. drakensbergiana and U. syntherismae the inflorescence of pooid grasses. Stoll et al. (2005) included destroyed the entire inflorescence similar to taxa in Ustilago a subgroup in Ustilago s.str. that contained Ustilago cynodontis, s.str. Columellae were described in several of the species in U. sparsa and U. xerochloae. These three taxa occur on pani- this clade, including Ustilago drakensbergiana, Macalpinomyces coid and chloridoid grasses. Inclusion of this subgroup and the spermophorus, M. neglectus and M. viridans. stripe smuts in Ustilago s.str. was supported by both maximum The columellae of U. drakensbergiana are formed from the rem- likelihood and Bayesian inference. Taxa within the Ustilago s.str. nants of the destroyed inflorescence and are not homologous clade lacked three characters that were found in other clades. with columellae of Clade 1 and 4. Vánky (2012) observed that 1. Absence of sterile cells in the sorus. the sori of species of Macalpinomyces were deciduous and 2. Absence of spore balls formed by coiled sporogenous hy- separated from the host plant at maturity, whereas species of phae. Sporisorium had sori that remained attached to the inflores- 3. Absence of a columella derived from host and fungal mate- cence because the columella was connected to the host plant. rial. The sori of M. spermophorus and M. viridans were deciduous and easily removed from the host plant. These columellae are Ustilago davisii group not formed from the host meristem and are not homologous to Stoll et al. (2005) recovered a strongly supported but unresolved the columellae of Clade 1 and 4. clade containing seven species, Sporisorium aegypticum, S. mo- A synapomorphic character for Clade 7 was not identified. Sub- destum, Ustilago davisii, U. filiformis, U. schroeteriana, U. tra- division of Clade 7 based on morphology is impractical at this gana and U. trichophora. The same clade was recovered in this stage, because the characters are highly variable in the group. study, but it was not well supported by bootstrap values (< 70 %) in maximum likelihood or posterior probabilities (< 0.95) in Clade 8 Bayesian inference. Sporisorium aegypticum, S. modestum Four taxa that destroy the ovaries of Aristida formed a well- and Ustilago trichophora were described as having columellae. supported monophyletic group. Stoll et al. (2005) included Fullerton & Langdon (1968) examined the soral development Sporisorium consanguineum in their study, but were unable to of Ustilago trichophora and concluded that a columella was determine whether it was sister to, or part of Clade 7. The inclu- present, however columellae are not included in the descrip- sion of three additional smuts that infect Aristida has resulted in tions by Vánky & Shivas (2008) or Vánky (2012). The sori of a separate, monophyletic group. The smuts on Aristida share Ustilago trichophora occur in ovaries or on stems and do not two morphological characters. have columellae that are homologous to the columellae formed 1. Formation of galls in the ovaries of their hosts. They can in- in Clade 1 and 4. fect all of the ovaries in an inflorescence (Sporisorium confu- sum, S. consanguineum) or be localised in the inflorescence­ Ustilago esculenta group (S. aristidicola). Stoll et al. (2005) recovered a weakly supported group that con- 2. The spores are commonly compacted into spore balls formed tained several smut fungi found on chloridoid grasses together by coiled sporogenous hyphae, for instance in Sporisorium with the atypical Ustilago esculenta, which occurs on Zizania consanguineum (Langdon & Fullerton 1975). in the subfamily Ehrhartoideae. Ustilago curta, which infects Tripogon in the subfamily Chloridoideae, either occurred in the Macalpinomyces eriachnes Ustilago esculenta group, or as sister to Clade 6 or 8. Stoll et Macalpinomyces eriachnes is the sister taxon to the Ustilago- al. (2005) recovered Ustilago curta (as U. alcornii) in the Us- Sporisorium-Macalpinomyces complex. Stoll et al. (2005) first tilago esculenta group. No synapomorphies were determined indicated that Macalpinomyces was a monotypic genus, with for this group. M. eriachnes the sole representative. This relationship is sup- Stoll et al. (2005) demonstrated a close relationship between ported in this study. Macalpinomyces eriachnes has giant sterile Melanopsichium­ pennsylvanicum and the Ustilago s.str. group. cells formed from non-sporogenous hyphae (Langdon & Ful- Our maximum likelihood analyses placed Melanopsichium in lerton 1977, Vánky 1996) and a peridium, but lacks a columella. the Ustilago esculenta group rather than sister to the Ustilago The spore balls of Macalpinomyces eriachnes were not formed s.str. group. Only the two nuclear rDNA loci obtained by Stoll et from coiled sporogenous hyphae (Langdon & Fullerton 1977). al. (2005) were included for Melanopsichium in the combined analysis of molecular loci. Begerow et al. (2004a) discussed the Taxa of uncertain placement complicated coevolution between smut fungi and their hosts. A few taxa moved between clades in trees reconstructed us- Melanopsichium pennsylvanicum may represent a jump from ing different datasets and different phylogenetic assessment to the distantly related Polygonaceae. criteria. These taxa were not supported in any group, although previous analyses have grouped most of these taxa in Clade Clade 7 6 (Stoll et al. 2005). Sporisorium aegypticum, S. modestum This clade was recovered in studies by Stoll et al. (2003, 2005) Ustilago schmidtiae and U. tragana often grouped together and was strongly supported by both maximum likelihood and after maximum likelihood analysis, although they were only Bayesian inference in this study. Stoll et al. (2005) noted that represented by data from two molecular loci in most cases. taxa in this clade had a combination of characters observed These taxa, except for Ustilago schmidtiae, were included with in Sporisorium and Ustilago. Taxa in this group have often taxa now assigned to Clade 6 by Stoll et al. (2005). been placed in Macalpinomyces because of the mixed soral Maximum likelihood analyses placed Ustilago curta in a number characteristics associated with both Sporisorium and Ustilago. of clades. Stoll et al. (2005) recovered U. curta (as U. alcornii) They occur on grasses in the tribe Paniceae and the subfamily in the Ustilago esculenta group of Ustilago s.l. after Bayesian Chloridoideae. analysis of data from two nuclear rDNA loci. With the addition of 76 Persoonia – Volume 29, 2012 nuclear loci, U. curta was often placed as sister to the Aristida protein coding loci and a thorough study of morphological di- group or as sister to the Triodia group. It is not known to which versity in Australian taxa. The determination of monophyletic group Ustilago curta belongs. groups and synapomorphic characters within the complex necessitates taxonomic reassessment of some genera and the Can host classification delimit smut genera? creation or resurrection of others in future studies. The major Taxa within the Ustilago-Sporisorium-Macalpinomyces complex outcomes of resolved character homology in the Ustilago- infect hosts in the Poaceae, with the exception of Melanopsi- Sporisorium-Macalpinomyces complex are: chium, which occurs on Polygonaceae. The systematics of 1. Sporisorium can be subdivided by soral characteristics. Poaceae has been well resolved and the relationships of the Sporisorium s.str. must be described explicitly to prevent subfamilies and tribes are well understood (Hsiao et al. 1999, ambiguity for future taxonomic placement of new species. Kellogg 2000, Stevens 2001, Bouchenak-Khelladi et al. 2008). 2. New genera are required for the placement of taxa that Host classification has often been used in the classification of form monophyletic groups and no longer fit the definition of smut fungi. Within Ustilago, Sporisorium and Macalpinomyces, Sporisorium s.str. putative host specificity is used to differentiate morphologically 3. Ustilago maydis and other taxa with localized tubular sori indistinguishable species (Bauer et al. 2001). Many of the keys and usually with sterile cells form a monophyletic group with to these genera are based on host . Higher-level host the morphologically similar systemic group, which usually taxonomy has been used to delimit smut genera, for example destroy the entire inflorescence. A taxonomic resolution for Ustilago is restricted to members of Poaceae (Bauer et al. these taxa cannot be proposed at this stage, however, if the 2001). method of soral infection is synapomorphic within the groups, Begerow et al. (2004a) concluded that the phylogenetic rela- the two names, Mycosarcoma and Endosporisorium, will be tionships between smut fungi and their hosts were not straight- available for the placement of these taxa. forward. While species of Ustilago and Sporisorium showed 4. Macalpinomyces bursus and M. ewartii belong to a monophy- evidence for co-speciation, it was considered more likely that letic group that can be differentiated by soral characteristics smut fungi evolved after their hosts had speciated (Begerow et and host tribe. al. 2004a). Host jumps are evident in Clade 4, which contains 5. The monophyletic group of smut fungi that infect Aristida can taxa that infect grasses in two subfamilies, the Paniceae and be delimited by soral characteristics and host subfamily. the Chloridoideae. 6. Four smut fungi on Triodia form a monophyletic group. 7. Macalpinomyces is a monotypic genus, sister to all other The phylogenetic analyses of the Ustilago-Sporisorium-Macal­ taxa in the Ustilago-Sporisorium-Macalpinomyces complex pinomyces complex recovered several monophyletic groups (Stoll et al. 2005). that shared similar morphological characters and are restricted 8. Until Clade 2 and 7 are resolved, Macalpinomyces will remain to hosts in a specific genus, tribe or subfamily. Four smuts a polyphyletic genus. that occur on Aristida in the subfamily Aristidoideae (Stevens 2001) form a monophyletic group in Clade 8. They have similar morphological characters but there are no unique synapomor- Acknowledgements We thank Nate Hardy for advice on phylogenetic techniques, and Anthony Young and Paul Campbell for advice and instruction phies that separate them unambiguously from other species in molecular techniques. ARM would like to acknowledge the support of the in the complex. Their pathogenicity on hosts in the subfamily Australian Government’s Cooperative Research Centre Program. Aristidoideae is a synapomorphy that distinguishes this clade from other clades in the complex. References Macalpinomyces bursus and M. ewartii, which are members of Clade 3, infect hosts in the tribe Andropogoneae. They possess Anisimova M, Gil M, Dufayard J-F, Dessimoz C, Gascuel O. 2011. Survey of morphological characteristics that are similar to some species branch support methods demonstrates accuracy, power, and robustness of Clade 7 that infect hosts in the Chloridoideae or Paniceae. of fast likelihood-based approximation schemes. Systematic Biology 60: The occurrence of members of Clade 3 on hosts in the tribe An- 685–699. Bakkeren G, Kronstad JW, Levesque CA. 2000. Comparison of AFLP finger- dropogoneae is a synapomorphy that can be used to distinguish prints and ITS sequences as phylogenetic markers in . Macalpinomyces bursus and M. ewartii from taxa in Clade 7. Mycologia 92: 510–521. In many cases morphological characteristics are inadequate Bauer R, Begerow D, Oberwinkler F, Piepenbring M, Berbee ML. 2001. Usti- for recognizing smut taxa. It is proposed that delimitation of laginomycetes. In: McLaughlin DJ, McLaughlin EG, Lemke PA (eds), Mycota VII Part B Systematics and evolution: 57–83. Springer-Verlag, Berlin. smut genera be based on host range, provided monophyletic Bauer R, Oberwinkler F, Vánky K. 1997. Ultrastructural markers and syste­ groups are resolved after molecular phylogenetic analyses. In matics in smut fungi and allied taxa. Canadian Journal of Botany 75: 1273– the absence of contradictory evidence, host subfamily or tribe 1314. is a legitimate criterion for generic delimitation in the Ustilago- Begerow D, Bauer R, Oberwinkler F. 1997. Phylogenetic studies on nuclear Sporisorium-Macalpinomyces complex. large subunit ribosomal DNA sequences of smut fungi and related taxa. Canadian Journal of Botany 75: 2045–2056. Begerow D, Göker M, Lutz M, Stoll M. 2004a. On the evolution of smut fungi on Conclusion their hosts. In: Agerer R, Piepenbring M, Blanz P (eds), Fronteirs in Basidio­ mycote mycology: 81–98. IHW-Verlag, Eching, Germany. A detailed examination of morphology is required to determine Begerow D, John B, Oberwinkler F. 2004b. Evolutionary relationships among homology and to improve classification (Mooi & Gill 2010), al­ beta-tubulin gene sequences of basidiomycetous fungi. Mycological Re- though in many groups of fungi this is impossible. The synapo- search 108: 1257–1263. Begerow D, Stoll M, Bauer R. 2006. A phylogenetic hypothesis of Ustila­ morphies outlined here based on gross morphology and host ginomycotina based on multiple gene analyses and morphological data. coevolution allow confident placement of new taxa within the Mycologia 98: 906–916. Ustilago-Sporisorium-Macalpinomyces into well delimited Berner DK, Dubin HJ, Smallwood EL. 2007. Slender wheatgrass is suscepti- clades. Although there are some morphological anomalies, the ble to smut caused by Ustilago phrygica from Turkey. Plant Disease 91: 906. monophyletic groups are robust and well supported. Bouchenak-Khelladi Y, Salamin N, Savolainen V, Forest F, Bank M van der, et al. 2008. Large multi-gene phylogenetic trees of the grasses (Poaceae): Morphological synapomorphies within the Ustilago-Sporisorium- Progress towards complete tribal and generic level sampling. Molecular Macalpinomyces were identified after incorporation of nuclear Phylogenetics and Evolution 47: 488–505. A.R. McTaggart et al.: Soral synapomorhies of the Ustilago-Sporisorium-Macalpinomyces complex 77

Brefeld O. 1912. Die Brandpilze und die Brandkrankheiten. V. Untersuchun- Piepenbring M, Stoll M, Oberwinkler F. 2002. The generic position of Usti- gen aus dem Gesammtgebiete der Mykologie. XV. Commissions-Verlag lago maydis, Ustilago scitaminea, and Ustilago esculenta (Ustilaginales). von Heinrich Schoningh, Munster. Mycological Progress 1: 71–80. Cunnington JH, Shivas RG. 2006. Phylogenetic considerations for predicting Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic infer- the host range of Ustilago sporoboli-indici, a potential biological control ence under mixed models. Bioinformatics 19: 1572–1574. agent for Sporobolus species in Australia. Plant Protection Quarterly 2: Shivas RG, Cunnington JH, Vánky K. 2004. Two new species of Ustilaginomy- 95–99. cetes on Chrysopogon fallax from Australia. Fungal Diversity, 16: 147–156. Cunnington JH, Vánky K, Shivas RG. 2005. Lundquistia is a synonym of Stamatakis A. 2006. RAxML-VI-HPC: Maximum likelihood-based phyloge- Sporisorium (Ustilaginomycetes). Mycologia Balcanica 2: 95–100. netic analyses with thousands of taxa and mixed models. Bioinformatics Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, et al. 2008. Phylogeny.fr: 22: 2688–2690. robust phylogenetic analysis for the non-specialist. Nucleic Acids Research Stevens PF. 2001. “Angiosperm Phylogeny Website. Version 9, June 2008.” 36: W465–W469. Retrieved 18th September 2010, from http://www.mobot.org/MOBOT/ Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy research/APweb/. and high throughput. Nucleic Acids Research 32: 1792–1797. Stoll M, Begerow D, Oberwinkler F. 2005. Molecular phylogeny of Ustilago, Fullerton RA, Langdon RFN. 1968. A study of some smuts of Echinochloa Sporisorium, and related taxa based on combined analyses of rDNA se- spp. Proceedings of the Linnean Society of New South Wales 93: 281–293. quences. Mycological Research 109: 342–356. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. Stoll M, Piepenbring M, Begerow D, Oberwinkler F. 2003. Molecular phylo­ New algorithms and methods to estimate maximum-likelihood phylogenies: geny of Ustilago and Sporisorium species (, Ustilaginales) Assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. based on internal transcribed spacer (ITS) sequences. Canadian Journal Hsiao C, Jacobs SWL, Chatterton NJ, Asay KH. 1999. A molecular phylogeny of Botany 81: 976–984. of the grass family (Poaceae) based on the sequences of nuclear ribosomal Swann EC, Taylor JW. 1995. Phylogenetic perspecitives on Basidiomycete DNA (ITS). Australian Systematic Botany 11: 667–688. systematics – evidence from the 18S ribosomal-RNA gene. Canadian Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian inference of phylo- Journal of Botany 73: S862–S868. genetic trees. Bioinformatics 17: 754–755. Talavera G, Castresana J. 2007. Improvement of phylogenies after removing Kellogg EA. 2000. The grasses: A case study in macroevolution. Annual divergent and ambiguously aligned blocks from protein sequence align- Review of Ecology and Systematics 31: 217–238. ments. Systematic Biology 56: 564–577. Kumar S, Dudley J, Nei M, Tamura K. 2008. MEGA: A biologist-centric soft- Vánky K. 1991. Spore morphology in the taxonomy of Ustilaginales. Transac- ware for evolutionary analysis of DNA and protein sequences. Briefings in tions of the Mycological Society of Japan 32: 381–400. Bioinformatics 9: 299–306. Vánky K. 1994. European smut fungi. Fischer Verlag, Stuttgart, Germany. Langdon RFN. 1962. A study of some smuts of Sorghum spp. Proceedings Vánky K. 1995a. Taxonomical studies on Ustilaginales. XII. Mycotaxon 54: of the Linnean Society of New South Wales 87: 45–50. 215–238. Langdon RFN, Fullerton RA. 1975. Sorus ontogeny and sporogenesis in Vánky K. 1995b. Taxonomical studies on Ustilaginales. XIII. Mycotaxon 56: some smut fungi. Australian Journal of Botany 23: 915–930. 197–216. Langdon RFN, Fullerton RA. 1977. Macalpinomyces, a new genus of smut Vánky K. 1996. The genus Macalpinomyces (Ustilaginales). Mycotaxon fungi. Transactions of the British Mycological Society 68: 27–30. 59: 115–126. Langdon RFN, Fullerton RA. 1978. The genus Sphacelotheca (Ustilagina- Vánky K. 1997. Taxonomical studies on Ustilaginales. XVII. Mycotaxon 65: les): criteria for its delimination and the consequences thereof. Mycotaxon 159–182. 6: 421–456. Vánky K. 2001. Lundquistia, a new genus of Ustilaginomycetes. Mycotaxon Link HF. 1825. Cryptogamia. Gymnomycetes. Species Plantarum exhibentes 77: 371–374. Plantas Rite Cognitas ad Genera Relatas. Linne Ca. 2: 1–128. Vánky K. 2002. Illustrated genera of smut fungi. APS Press, St. Paul, Min- Maddison DR, Maddison WP. 2001. MacClade 4: analysis of phylogeny and nesota, USA. character evolution. Sinauer Associates, Sunderland. Vánky K. 2004. New smut fungi (Ustilaginomycetes) from Mexico, and the Maniatis T, Fritsch EF, Sambrook J. 1982. Molecular cloning, a laboratory genus Lundquistia. Fungal Diversity 17: 159–190. manual. Cold Spring Harbor Laboratory, Cold Spring Habor, New York. Vánky K. 2012. Smut fungi of the world. APS Press, St. Paul, Minnesota. Mooi RD, Gill AC. 2010. Phylogenies without synapomorphies – a crisis in Vánky K, Lutz M. 2011. Tubisorus, a new genus of smut fungi (Ustilagino- fish systematics: time to show some character. Zootaxa 2450: 26–40. mycetes) for Sporisorium pachycarpum. Mycologia Balcanica 8: 129–135. Munkacsi AB, Stoxen S, May G. 2007. Domestication of maize, sorghum, Vánky K, Lutz M, Shivas RG. 2006. Anomalomyces panici, new genus and sugarcane did not drive the divergence of their smut pathogens. and species of Ustilaginomycetes from Australia. Mycologia Balcanica 3: Evolution 61: 388–403. 119–126. Nylander JAA, Wilgenbusch JC, Warren DL, Swofford DL. 2008. AWTY (are Vánky K, Shivas RG. 2001. Smut fungi (Ustilaginomycetes) of Sorghum we there yet?): a system for graphical exploration of MCMC convergence (Gramineae) with special regard to Australasia. Mycotaxon 80: 339–353. in Bayesian phylogenetics. Bioinformatics 24: 581–583. Vánky K, Shivas RG. 2008. Fungi of Australia: The smut fungi. ABRS, Can- Piepenbring M. 2004. Comparative morphology of galls formed by smut berra & CSIRO Publishing, Melbourne, Australia. fungi and discussion of generic concepts. In: Agerer R, Piepenbring M, Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of en- Blanz P (eds), Fronteirs in Basidiomycote mycology: 117–164. IHW-Verlag, zymatically amplified ribosomal DNA from several Cryptococcus species. Eching, Germany. Journal of Bacteriology 172: 4238–4246. Piepenbring M, Bauer R, Oberwinkler F. 1998. Teliospores of smut fungi – White TJ, Bruns TD, Lee S, Taylor JW. 1990. Amplification and direct sequenc- Teliospore connections, appendages, and germ pores studied by electron ing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand microscopy; phylogenetic discussion of characteristics of teliospores. DH, Sninsky JJ, White TJ (eds), PCR Protocols: A guide to methods and Protoplasma 204: 202–218. applications: 315–322. Academic Press, Inc., San Diego.