Soral synapomorphies are significant for the systematics of the Ustilago-Sporisorium-Macalpinomyces complex (Ustilaginaceae).

The genera Ustilago, Sporisorium and Macalpinomyces are a polyphyletic complex of plant pathogenic fungi. The four main morphological characters used to define these genera have been considered homoplasious and not useful for resolving the complex. This study re-evaluates character homology and discusses the use of these characters for defining monophyletic groups recovered from a reconstructed phylogeny using four nuclear loci. Generic delimitation of smut fungi based on their hosts is also discussed as a means for identifying genera within this group. Morphological characters and host specificity can be used to circumscribe genera within the Ustilago-Sporisorium-Macalpinomyces complex.

INTRODUCTION

Three genera of smut fungi (subphylum Ustilaginomycotina), Ustilago, Sporisorium and Macalpinomyces, contain about 530 described species that all infect grasses (Vánky 2012). Several phylogenetic studies have demonstrated that Ustilago and Sporisorium together form a monophyletic group within the Ustilaginomycotina (Swann & Taylor 1995, Bauer et al. 1997, Begerow et al. 1997, 2004b, 2006, Stoll et al. 2003, 2005). Macalpinomyces has an ambiguous position in the Ustilaginales as the type species, M. eriachnes, sits outside the Ustilago-Sporisorium group (Begerow et al. 2006). Morphological characters have proven inadequate for separation of species among the three genera. The three genera are polyphyletic (Stoll et al. 2003, 2005), and collectively form an unresolved complex. Morphological studies (Langdon & Fullerton 1975, Vánky 1991, Piepenbring et al. 1998) and molecular phylogenetic analyses (Stoll et al. 2003, 2005) have not identified characters that define monophyletic groups amongst species within this complex.

Smut fungi in the Ustilago-Sporisorium-Macalpinomyces complex either partially or completely destroy the inflorescence of grasses, forming a sorus that contains fungal spores. Four characteristics of the sorus, namely columellae, sterile cells, spore balls and peridia, have been used traditionally to separate Ustilago, Sporisorium and Macalpinomyces (Vánky 2002). Within the sorus, columellae form a central axis of fungal and host origin (Vánky 2002); sterile cells, either derived from non-sporogenous hyphae or a fungal peridium, are found with the spores (Langdon & Fullerton 1975, 1978); spore balls appear as either an ephemeral or permanent agglomeration of spores (Vánky 2002). A peridium is the outer layer of the sorus and can be composed of host or fungal material (Vánky 2002). Soral characters have had different interpretations by mycologists (Stoll et al. 2005). For example, the columella in Ustilago porosa was considered absent by Langdon (1962) but present by Vánky & Shivas (2001). Similarly, Sporisorium consanguineum was considered to have a columella by Langdon & Fullerton (1975), but not by Vánky & Shivas (2008). Subsequently, soral morphology has been considered too variable to serve as a reliable character that can separate Ustilago, Sporisorium and Macalpinomyces (Piepenbring 2004, Stoll et al. 2005).

The current study discusses morphological characters in the Ustilago-Sporisorium-Macalpinomyces complex. A re-evaluation of their homology is provided in light of the phylogenetic results obtained. The merits of using host specificity and soral synapomorphies are discussed as a basis for delimiting genera.

MATERIALS AND METHODS

Taxon selection

Taxa were selected to represent the main groups recovered in previous studies (Stoll et al. 2003, 2005), with increased sampling of under-represented groups, for example species of Macalpinomyces and smut fungi occurring on Aristida. In total, this study included 136 species (14 species of Macalpinomyces, 81 species of Sporisorium and 38 species of Ustilago), 35 of which had not previously been evaluated in systematic studies (Table 1). Two distinctive members of the complex, Anomalomyces panici and Melanopsichium pennsylvanicum, were also included. Moesziomyces bullatus was included as an outgroup to the complex based on a relationship reported by Stoll et al. (2005).

Table 1

Isolates of Isolates of Ustilaginaceae included in this study.

Species	Specimen no.	Host	Country	GenBank no.
				ITS	LSU	EF-1α	GAPDH
Anomalomyces panici	BRIP 46421	Panicum trachyrachis	Australia	DQ4593481	DQ4593471	–	–
Macalpinomyces arundinellae-setosae	BRIP 47958	Arundinella nepalensis	Australia	HQ013086	–	–	–
	BRIP 51868	Arundinella nepalensis	Australia	–	–	–	HQ013055
Macalpinomyces bursus	Ust. Exs. 844	Themeda quadrivalvis	India	AY7401542	–	–	–
Macalpinomyces eragrostiellae	Ust. Exs. 960	Eragrostiella bifaria	India	AY7400362	AY7400892	–	–
Macalpinomyces eriachnes	M 54573	Eriachne aristidea	Australia	AY7400372	AY7400902	–	–
Macalpinomyces ewartii	BRIP 51818	Sarga timorense	Australia	HQ013087	HQ013127	HQ013026	HQ013056
Macalpinomyces loudetiae	M 56576	Loudetia flavida	South Africa	AY7401512	–	–	–
Macalpinomyces mackinlayi	BRIP 52549	Eulalia mackinlayi	Australia	GU014817	HQ013131	HQ013027	HQ013057
Macalpinomyces neglectus	RB 2056	Setaria pumila	Germany	AY7400562	AY7401092	–	–
Macalpinomyces simplex	M 56577	Loudetia simplex	Zimbabwe	AY7401522	–	–	–
Macalpinomyces spermophorus	H.U.V. 13634	Eragrostis ferruginea	Unknown	AY7401712	–	–	–
	BRIP 51858	Sporobolus australasicus	Australia	–	FQ013130	HQ013028	HQ013058
Macalpinomyces trichopterygis	M 56578	Trichopteryx dregeana	South Africa	AY7400392	AY7400922	–	–
Macalpinomyces tristachyae	MP 2630	Loudetiopsis chrysothrix	Bolivia	AY7401642	–	–	–
Macalpinomyces tubiformis	BRIP 51865	Chrysopogon fallax	Australia	HQ013088	–	HQ013029	HQ013059
Macalpinomyces viridans	BRIP 49133	Sporobolus actinocladus	Australia	HQ013089	HQ013125	HQ013030	HQ013060
Melanopsichium pennsylvanicum	H.U.V. 17548	Polygonum glabrum	India	AY7400402	AY7400932	–	–
Moesziomyces bullatus	Ust. Exs. 833	Paspalum distichum	India	AY7401532	AY7401532	–	–
Sporisorium absconditum	BRIP 49648	Schizachyrium fragile	Australia	HQ013090	–	–	–
Sporisorium aegyptiacum	Ust. Exs. 756	Schismus arabicus	Iran	AY3449708	AY7401292	–	–
Sporisorium andropogonis	M 56588	Bothriochloa saccharoides	Ecuador	AY7400422	AY7400952	–	–
Sporisorium anthistiriae	BRIP 49775	Themeda triandra	Australia	–	–	HQ013031	HQ013061
Sporisorium anthracoideisporum	BRIP 39176	Pseudoraphis spinescens	Papua New Guinea	AY7400442	AY7400972	–	–
Sporisorium apludae-aristatae	M 56590	Apluda mutica	India	AY7400452	AY7400982	–	–
Sporisorium aristidicola	BRIP 26930	Aristida jerichoensis	Australia	HQ013091	–	HQ013032	–
	BRIP 51871	Aristida sp.	Australia	–	–	–	HQ013062
Sporisorium arthraxonis	M 56592	Arthraxon lanceolatus	China	AY7400462	AY7400992	–	–
Sporisorium bothriochloae	BRIP 51819	Dichanthium sericeum	Australia	HQ013092	–	–	HQ013063
Sporisorium caledonicum	BRIP 51854/BRIP 28043	Heteropogon contortus	Australia	HQ013093	–	HQ013033	HQ013064
Sporisorium cenchri	MP 1974	Cenchrus pilosus	Nicaragua	AY3449728	AF4539432	–	–
Sporisorium cenchri-elymoidis	BRIP 26491	Cenchrus elymoides	Australia	HQ013094	HQ013122	HQ013034	HQ013065
Sporisorium chrysopogonis	Ust. Exs. 407	Chrysopogon fulvus	Sri Lanka	AY3449738	AY7401312	–	–
Sporisorium confusum	BRIP 42670	Aristida queenslandica	Australia	HQ013095	HQ013132	–	HQ013066
	BRIP 52755	Aristida sp.	Australia	HQ013096	–	–	–
Sporisorium consanguineum	BRIP 51839	Aristida hygrometrica	Australia	HQ013096	–	–	–
	BRIP 27723	Aristida hygrometrica	Australia	HQ013098	–	–	HQ013067
Sporisorium cruentum	Ust. Exs. 687	Sorghum halepense	United States	AY3449748	AF4539392	–	–
Sporisorium culmiperdum	MP 2060	Andropogon gerardii	Honduras	AY3449758	AF1335802	–	–
Sporisorium cymbopogonis-bombycini	BRIP 52511	Cymbopogon bombycinus	Australia	HQ013099	–	HQ013035	–
Sporisorium destruens	Ust. Exs. 472	Panicum miliaceum	Romania	AY3449768	AY7470772	–	–
Sporisorium dietelianum	H.U.V. 20560	Tripsacum sp.	Mexico	AY9981003	–	–	–
Sporisorium dimeriae-ornithopodae	Ust. Exs. 472	Dimeria ornithopoda	India	AY3449778	AY7401322	–	–
Sporisorium doidgeae	BRIP 49669	Bothriochloa ewartiana	Australia	–	HQ013126	HQ013036	HQ013068
	M 56595	Capillipedium spicigerum	Australia	AY7400472	–	–	–
Sporisorium elionuri	MP 2601	Elionurus muticus	Bolivia	AY7401572	–	–	–
Sporisorium enteromorphum	M 55602	Themeda triandra	South Africa	AY7401582	–	–	–
Sporisorium erythraeense	Ust. Exs. 849	Hackelochloa granularis	India	AY7400492	AY7401022	–	–
Sporisorium everhartii	MP 2270	Andropogon virginicus	Cuba	AY7401592	–	–	–
Sporisorium fallax	BRIP 27687	Chrysopogon fallax	Australia	AY3339404	–	–	–
Sporisorium fastigiatum	MP 1976	Andropogon angustatus	Nicaragua	AY3449788	AY7401332	–	–
Sporisorium formosanum	Ust. Exs. 688	Panicum repens	Taiwan	AY3449798	AY7401342	–	–
Sporisorium foveolati	MP 2365	Eremopogon foveolatus	Canary Islands	AY7400502	AY7401032	–	–
Sporisorium fraserianum	BRIP 49668	Aristida nitidula	Australia	HQ013100	–	–	–
Sporisorium heteropogonicola	BRIP 51822	Heteropogon contortus	Australia	HQ013101	HQ013135	HQ013037	HQ013069
Sporisorium holwayi	MP 1271	Andropogon bicornis	Panama	AY3449808	AF4539412	–	–
Sporisorium hwangense	M 56607	Sporobolus panicoides	Zimbabwe	AY7400512	AY7401042	–	–
Sporisorium iseilematis-ciliati	BRIP 51870	Iseilema sp.	Australia	HQ013102	–	HQ013039	HQ013070
	BRIP 52517	Iseilema sp.	Australia	–	–	HQ013040	–
Sporisorium lacrymae-jobi	M 56611	Coix lacryma-jobi	India	AY7400522	AY7401052	–	–
Sporisorium lanigeri	BRIP 46819	Cymbopogon ambiguus	Australia	HQ013103		–	–
Sporisorium lepturi	Ust. Exs. 966	Hemarthria uncinata	Australia	AY3449818	AY7401352	–	–
Sporisorium loudetiae-pedicellatae	M 56615	Loudetia pedicellata	South Africa	AY7400532	AY7401062	–	–
Sporisorium manilense	Ust. Exs. 854	Sacciolepis indica	India	AY7400592	AY7401122	–	–
Sporisorium mexicanum	H.U.V. 20498	Andropogon sp.	Mexico	AY9981013	–	–	–
Sporisorium mishrae	Ust. Exs. 967	Apluda mutica	India	AY3449838	AY7401362	–	–
Sporisorium mitchellii	BRIP 52538	Iseilema sp.	Australia	–	–	HQ013041	–
Sporisorium modestum	M 56617	Enneapogon avenaceus	Australia	AY7400542	AY7401072	–	–
Sporisorium monakai	M 56618	Isachne globosa	India	AY7401612	–	–	–
Sporisorium moniliferum	BRIP 52504/Ust. Exs. 851	Heteropogon contortus	Australia/Indonesia	HQ013104	AF4539402	HQ013042	HQ013071
Sporisorium mutabile	BRIP 44111	Cymbopogon refractus	Australia	HQ013105	–	–	–
Sporisorium nealii	M 56621	Heteropogon melanocarpus	India	AY7400552	AY7401082	–	–
Sporisorium nervosum	BRIP 27019/M 56622	Sehima nervosum	Australia	HQ013106	AY7401102	–	–
Sporisorium occidentale	Ust. Exs. 758	Andropogon gerardii	United States	AY3449858	AY7401372	–	–
Sporisorium ophiuri	HB 20	Rottboellia cochinchinensis	Unknown	AY7400192	AJ2361362	–	–
Sporisorium ovarium	MP 1871	Urochloa fasciculata	Mexico	AY7400202	AJ2361372	–	–
Sporisorium panici	BRIP 43942	Paspalidium caespitosum	Australia	HQ170519	–	–	–
Sporisorium panici-leucophaei	DAR 58832a	Digitaria brownii	Australia	AY7400352	AY7400882	–	–
Sporisorium paspali	MP 2101	Paspalum notatum	Cuba	AY3449828	AF4539442	–	–
Sporisorium penniseti	MP 2367	Pennisetum setaceum	Canary Islands	AY3449718	AY7401302	–	–
Sporisorium polliniae	Ust. Exs. 690	Andropogon distachyos	Greece	AY3449878	AY7401382	–	–
Sporisorium provinciale	Ust. Exs. 759	Andoropogon gerardii	United States	AY3449888	AY7470762	–	–
Sporisorium pseudechinolaenae	Ust. Exs. 853	Pseudechinolaena polystachya	Indonesia	AY3449898	AY7401392	–	–
Sporisorium pulverulentum	M 56627	Saccharum strictum	Yugoslavia	AY7401622	–	–	–
Sporisorium queenslandicum	BRIP 49706	Sehima nervosum	Australia	HQ013107	–	–	–
Sporisorium rarum	BRIP 49134	Eulalia aurea	Australia	HQ013108	–	HQ013043	HQ013072
Sporisorium reilianum	Ust. Exs. 527	Sorghum halepense	Greece	AY7401632	AY7401632	–	–
	Sr326	Sorghum sp.	United States	–	–	DQ3528275	DQ3528155
Sporisorium ryleyi	BRIP 49713	Sarga timorense	Australia	HQ013109	–	HQ013044	HQ013073
Sporisorium scitamineum	MP 541	Saccharum sp.	Costa Rica	AY7400702	AY7401472	–	–
	Br532	Saccharum sp.	Brazil	–	–	DQ3528295	DQ3528175
Sporisorium sehimatis	BRIP 49671	Sehima nervosum	Australia	HQ013110	–	–	–
Sporisorium setariae	BRIP 49636	Setaria surgens	Australia	HQ013111	–	HQ013045	–
	BRIP 26910	Setaria surgens	Australia	–	–	–	HQ013075
Sporisorium sorghi	MP 2036a	Sorgum bicolor	Nicaragua	AY7400212	AF0098726	–	–
	CBS 104.17	Sorghum sp.	Indonesia	–	–	DQ3528285	DQ3528165
Sporisorium spinulosum	HMAS 193085	Capillipedium parviflorum	China	GU1391727	GU1391717	–	–
Sporisorium tenue	BRIP 48629	Bothriochloa decipiens	Australia	HQ013112	–	HQ013046	HQ013076
Sporisorium themedae-arguentis	Ust. Exs. 855	Themeda arguens	Indonesia	AY3449918	AY7401402	–	–
Sporisorium trachypogonicola	MP 2463	Trachypogon plumosus	Cuba	AY3449928	AY7401412	–	–
Sporisorium trachypogonis-plumosi	M 56635	Trachypogon plumosus	Venezuela	AY7400602	AY7401132	–	–
Sporisorium trispicatae	BRIP 47730	Eulalia trispicata	Australia	HQ13113		–	–
Sporisorium tumefaciens	Ust. Exs. 231	Chrysopogon aciculatus	Sri Lanka	AY3449698	AY7401282	–	–
Sporisorium vanderystii	MP 2372	Hyparrhenia hirta	Canary Islands	AY7400582	AY7401112	–	–
Sporisorium veracruzianum	MP 735	Panicum viscidellum	Costa Rica	AY3449938	AY7401142	–	–
Sporisorium vermiculum	BRIP 49748	Sarga plumosum	Australia	HQ013114	HQ013134	HQ013047	HQ013077
Sporisorium whiteochloae	BRIP 51860	Whiteochloa semitonsa	Australia	HQ013115	–	HQ013048	HQ013078
Sporisorium wynaadense	BRIP 27640	Sarga leiocladum	Australia	HQ013116	HQ013124	HQ013049	HQ013079
Sporisorium xerofasciculatum	BRIP 49682	Xerochloa laniflora	Australia	HQ013117	–	–	–
Ustilago affinis	G. Rivera s.n.	Stenotaphrum secundatum	Costa Rica	AY3449958	AF1335812	–	–
Ustilago altilis	Ust. Exs. 418	Triodia pungens	Australia	AY7401662	–	–	–
	BRIP 52543	Triodia sp.	Australia	–	HQ013136	–	–
Ustilago austro-africana	M 56516	Enneapogon cenchroides	Zimbabwe	AY7400612	AY7401152	–	–
Ustilago avenae	DB 559	Avena barbata	Germany	AY3449978	AF4539332	–	–
Ustilago bouriqueti	M 56517	Stenotaphrum dimidiatum	La Réunion	AY7401672	–	–	–
Ustilago bromivora	H.U.V. 19322	Bromus catharticus	Argentina	AY7400642	AY7401182	–	–
Ustilago bullata	MP 2363	Bromus diandrus	Canary Islands	AY3449988	AF4539352	–	–
Ustilago calamagrostidis	M 56518	Calamagrostis epigeios	Bulgaria	AY7400652	AY7401192	–	–
Ustilago crameri	Ust. Exs. 995	Setaria italica	India	AY3449998	AY7401432	–	–
Ustilago curta	M 56514/BRIP 26929	Tripogon loliiformis	Australia	AY7401652	HQ013123	–	HQ013080
Ustilago cynodontis	MP 1838/BRIP 51207	Cynodon dactylon	Mexico	AY3450008	AF0098812	HQ013050	HQ013081
Ustilago davisii	H.U.V. 19252	Glyceria multiflora	Argentina	AY7401692	–	–	–
Ustilago drakensbergiana	M 56523	Digitaria tricholaenoides	South Africa	AY7401702	–	–	–
Ustilago echinata	Ust. Exs. 540	Phalaris arundinacea	Germany	AY3450018	AY7401442	–	–
Ustilago esculenta	Ust. Exs. 590	Zizania latifolia	Taiwan	AY3450028	AF4539372	–	–
Ustilago filiformis	RB 3011	Glyceria fluitans	Germany	AY7400662	AY7401202	–	–
Ustilago hordei	Ust. Exs. 784	Hordeum vulgare	Iran	AY3450038	AF4539432	–	–
	Uh362	Hordeum sp.	Canada	–	–	DQ3528325	DQ3528205
Ustilago inaltilis	BRIP 49123	Triodia longiloba	Australia	HQ013118	–	–	–
Ustilago ixophori	MP 2194	Ixophorus unisetus	Costa Rica	AY7400672	AY7401212	–	–
Ustilago lituana	BRIP 46795	Triodia epactia	Australia	HQ013119	–	–	–
Ustilago maydis	RB 3093	Zea mays	Germany	AY3450048	–	–	–
	MS 115	Zea mays	Germany	–	AF4539389	–	–
	B-Pbi-4-1-4	Zea mays	Brazil	–	–	DQ3528305	DQ3528185
Ustilago nuda	H.U.V. 17782	Hordeum leporinum	Greece	AY7400692	AJ2361392	–	–
Ustilago pamirica	Ust. Exs. 887	Bromus gracillimus	Iran	AY3450058	AY7401452	–	–
Ustilago phrygica	BPI 871725	Elymus trachycaulus	Turkey	DQ13996110	–	–	–
Ustilago porosa	BRIP 51842	Sarga timorense	Australia	HQ13120	HQ013128	HQ013051	HQ013082
Ustilago schmidtiae	BRIP 26906	Enneapogon polyphyllus	Australia	–	–	–	–
	BRIP 51848	Enneapogon sp.	Australia	HQ013121	HQ013129	–	HQ013083
Ustilago schroeteriana	Ust. Exs. 887	Paspalum paniculatum	Costa Rica	AY3450068	AY7401462	–	–
Ustilago sparsa	Ust. Exs. 892	Dactyloctenium radulans	India	AY3450088	–	–	–
Ustilago sporoboli-indici	BRIP 39706	Sporobolous pyramidalis	South Africa	AY77273611	–	–	–
Ustilago striiformis	H.U.V. 18286	Alopecurus pratensis	Germany	AY7401722	DQ87537512	–	–
Ustilago syntherismae	Ust. Exs. 998	Digitaria ternata	India	AY7400712	AY7401232	–	–
Ustilago tragana	M 56562	Tragus berteronianus	Zimbabwe	AY7400722	AY7401242	–	–
Ustilago trichophora	MP 2473	Echinochloa colona	Cuba	AY3450092	AY7401482	–	–
	BRIP 49159	Echinolchloa utilis	Australia	–	–	HQ013052	HQ013084
Ustilago triodiae	H.U.V. 17662/BRIP 49124	Triodia microstachya	Australia	AY7400742	AY7401262	HQ013053	HQ013085
Ustilago tritici	not given	Triticum aestivum	Canada	AF13542413	–	–	–
Ustilago turcomanica	H.U.V. 23	Eremopyrum distans	Iran	AY3450112	AF4539362	–	–
Ustilago vetiveriae	H.U.V. 17954	Vetiveria zizanioides	India	AY3450112	AF4539372	–	–
Ustilago xerochloae	Ust. Exs. 1000	Xerochloa imberbis	Australia	AY3450122	AF4539382	–	–
	BRIP 49820	Xerochloa barbata	Australia	–	AF453939	HQ013054	–

1 Vánky et al. (2006)

2 Stoll et al. (2005)

3 Cunnington et al. (2005)

4 Shivas et al. (2004)

5 Munkacsi et al. (2007)

6 Begerow et al. (1997)

7 Vanky & Lutz unpubl. data

8 Stoll et al. (2003)

9 Piepenbring et al. (2002)

10 Berner et al. (2007)

11 Cunnington & Shivas (2006)

12 Begerow et al. (2006)

13 Bakkeren et al. (2000)

Morphological data

Character and character state selection were based on taxonomic descriptions in monographs of the Ustilaginomycotina (Vánky 1994, 2012, Vánky & Shivas 2008) and from direct observation of 61 Australian species. Columellae were scored as either absent, stout or filiform. Spore states were classified as single spores, permanent spore balls, ephemeral spore balls or dimorphic spores. Sterile cells were scored as present or absent. The peridium was classified as either host derived, hypertrophied-host derived or fungal derived. These characters were mapped onto the final tree topology using MacClade v. 4.08 (Maddison & Maddison 2001).

DNA extraction

DNA was extracted from 120 smut specimens representing 92 taxa, by a combination of enzymatic and mechanical lysis. Smut sori or spores were mechanically lysed using a QIAGEN TissueLyser with 0.5 mm stainless steel beads, then shaken at 55 °C overnight in SNES buffer (0.01 M sodium phosphate pH 7.6, 0.15 M sodium chloride, 0.005 M EDTA, 1 % SDS) containing proteinase K at a final concentration of 0.8 μg/mL. The purification was then completed using the QIAGEN Gentra Puregene kit according to the manufacturer’s instructions.

PCR and sequencing

Genomic DNA was amplified by PCR with high fidelity Phusion® DNA Polymerase (Finnzymes) using the manufacturer-specified cycling and reaction conditions. The ITS region was amplified with primers M-ITS1 (Stoll et al. 2003) and ITS4 (White et al. 1990) at 58 °C; the LSU region was amplified with primers LROR and LR5 (Vilgalys & Hester 1990) at 58 °C; the GAPDH locus was amplified with GAPDH-F (CGGTCGTATCGGMCGTATC) and GAPDH-R (GTARCCCCACTCGTTGTCGTA) at 65 °C; the EF-1α locus was amplified with primers EF-1αF (GCCCTMTGGAAGTTCGAGACYCCCA) and EF-1αR (GAYACCGACAGCRACGGTCTG) at 62 °C. PCR products were purified by ethanol precipitation using standard methods (Maniatis et al. 1982). Purified PCR product was sent to Macrogen Korea or the Australian Genome Research Facility, Queensland for sequencing using the forward and reverse primers from amplification. ABI sequence trace files were assembled using ContigExpress® (Invitrogen™). The 165 novel sequences have been deposited in GenBank (Table 1).

Alignment of sequences

Sequences were aligned using the Muscle algorithm (Edgar 2004) included in the MEGA5 software package (Kumar et al. 2008). Alignments of protein-coding loci (GAPDH and EF-1α) were converted to amino acid sequences in MEGA. The original and curated nucleotide alignments have been deposited as Nexus files in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S11013). The super-matrix consisted of ITS sequences for 134 taxa, LSU sequences for 91 taxa, EF-1α sequences for 32 taxa and GAPDH sequences for 35 taxa.

Curation of alignments

Alignments were uploaded to Phylogeny.fr (available at http://www.phylogeny.fr/) (Dereeper et al. 2008) and curated in Gblocks to remove poorly aligned positions and divergent regions (Talavera & Castresana 2007). Alignments were trimmed as follows: ITS from 1 140 nucleotides, including gaps, to 448 nucleotides with no gaps; LSU from 609 to 593 nucleotides; EF-1α from 935 to 926 nucleotides; GAPDH from 1 158 to 769 nucleotides. The final curated super-matrix consisted of 2 736 nucleotides, which was composed of approximately 47 % missing data.

Phylogenetic analyses

Two phylogenetic assessment criteria were implemented: Bayesian inference using MrBayes (Huelsenbeck & Ronquist 2001, Ronquist & Huelsenbeck 2003) and maximum likelihood using RAxML (Stamatakis 2006) and PhyML 3.0 (Guindon et al. 2010). Resulting trees were observed with FigTree (available at http://www.tree.bio.ed.ac.uk/software/figtree/). Data and command files for both Bayesian and RAxML analyses and the resulting trees are available at TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S11013). The four loci were included as separate partitions in the maximum likelihood and Bayesian analyses so that each locus could be run under different optimal model parameters.

Maximum likelihood analysis — Maximum likelihood was implemented as a search criterion in RAxML (Stamatakis 2006) and PhyML 3.0 (Guindon et al. 2010). GTRGAMMA was specified as the model of evolution in both programs. The RAxML analyses were run with a rapid Bootstrap analysis (command -f a) using a random starting tree and 1 000 maximum likelihood bootstrap replicates. The PhyML analyses were implemented using the ATGC bioinformatics platform (available at: http://www.atgc-montpellier.fr/phyml/), with SPR and NNI tree improvement, and support obtained from an approximate likelihood ratio test (Anisimova et al. 2011).

Bayesian analysis — MrBayes was used to conduct a Markov Chain Monte Carlo (MCMC) search in a Bayesian analysis. Four runs, each consisting of four chains, were implemented until the standard deviation of split frequencies were 0.02. The cold chain was heated at a temperature of 0.25. Substitution model parameters were sampled every 50 generations and trees were saved every 5 000 generations. Convergence of the Bayesian analysis was confirmed using AWTY (Nylander et al. 2008) (available at: ceb.csit.fsu.edu/awty/). Convergence was not reached even after 40 million generations with all datasets. A user-defined tree obtained from the maximum likelihood analyses was used as a starting point for all of the Bayesian analyses, which helped to improve convergence of the four runs. A burn-in was not used to summarize the values that were created with a user-defined tree.

RESULTS AND DISCUSSION

Eight clades were consistently recovered in a phylogenetic analysis of four molecular loci (Fig. 1). The major clades recovered in this study were similar to those obtained in previous molecular phylogenetic analyses using different assessment criteria. For example, several phylogenetic studies have reconstructed two monophyletic groups in Sporisorium (Stoll et al. 2003, 2005, Cunnington et al. 2005, Vánky et al. 2006, Vánky & Lutz 2011), but these studies were not able to separate the two groups using morphological characters. The structure of columellae (Fig. 2), the presence or absence of sterile cells (Fig. 3) and the presence or absence of spore balls (Fig. 4) were traced onto the topology. A discussion of the homology of these characters and their use in identifying the clades of the Ustilago-Sporisorium-Macalpinomyces complex follows.

Fig. 1

Phylogram obtained from a maximum likelihood analysis in RAxML. Maximum likelihood support values (> 70 %) from RAxML 1000 bootstrap replicates and PhyML aRLT values shown above the nodes. Posterior probabilities (> 0.95) from Bayesian inference shown below the nodes.

Fig. 2

Structure of the columella mapped onto five clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Macalpinomyces.

Fig. 3

Distribution of sterile cells within clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Macalpinomyces.

Fig. 4

Presence of spore balls within clades of the Ustilago, Sporisorium and Macalpinomyces complex. S. = Sporisorium, U. = Ustilago, M. = Macalpinomyces.

Clade 1

Clade 1 includes S. sorghi, the type of Sporisorium. The members of this clade share a number of characters.

A hardened or stout columella that either replaces the entire inflorescence, for example in Sporisorium andropogonis, S. doidgeae and S. scitamineum (Fig. 5b), or that occurs in all of the ovaries or spikelets of an inflorescence, for example in S. ryleyi, S. sorghi (Fig. 5d) and S. rarum (Fig. 5e).

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.

Sterile cells formed from non-sporogenous hyphae that are intermixed with spores in the sorus (Fig. 5c, f), except in Ustilago porosa and Sporisorium culmiperdum.

A peridium derived mainly from host tissue, either from leaf sheaths or the ovary wall.

Taxa in Clade 1 mainly infect grasses belonging to the subfamily Panicoideae, in one of two tribes, Paniceae or Andropogoneae. The infection is usually systemic and destroys either the entire inflorescence or all of the ovaries or spikelets.

Langdon & Fullerton (1978) examined the soral ontogeny of several species included in Clade 1, namely Sporisorium andropogonis, S. sorghi and S. vanderystii. They observed that the columella began to form after intercellular hyphae became confluent and caused the host cells to proliferate. Hyphae at the periphery of the columella formed a sheath of elongated, thick-walled, vacuolate cells. Other hyphae were present inter- and intracellularly in the tissue of the columella.

Columellae of species in Clade 1 are stout and woody due to the peripheral formation of thick-walled, vacuolate cells (Fig. 2). These columellae are cylindrical and grow vertically. Occasionally, more than one columella is present in a sorus, for example in S. reilianum (Fig. 5a). Sometimes columellae are branched, for example in S. doidgeae (Fig. 5b). Stout columellae are a synapomorphy for species in Clade 1 (Fig. 2)

Langdon & Fullerton (1978) observed that non-sporogenous hyphae partitioned the sporogenous hyphae in sori of Sporisorium sorghi. The partitioning hyphae formed groups of hyaline cells that mixed with the spores as the sorus matured. This pattern of development accounts for the chains of sterile cells found in many species of Sporisorium (Fig. 3), for example S. rarum (Fig. 5f), S. ophiuri, S. themedae and S. vermiculum. Langdon & Fullerton (1978) termed these ‘partitioning cells’, though subsequent descriptions of smut fungi referred to them as sterile cells. The term sterile cells is maintained to differentiate between the cells formed by non-sporogenous, partitioning hyphae, and the peridial cells formed from the peridium.

Clade 2

Species within Clade 2 have been described in Ustilago, Sporisorium and Macalpinomyces. They share two common morphological characters.

The sori are relatively long, twisted and cylindrical, and are derived from hypertrophied host material, as in Macalpinomyces tubiformis (Fig 6a), M. mackinlayi and Sporisorium dietelianum.

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.

Sterile cells are usually found within the sori.

There are two types of infection in Clade 2: a localized infection seen in most of the species, or a systemic infection seen in a monophyletic group of taxa that destroy the entire inflorescence or infect the culms of the host. The position of the systemic group was ambiguous and only had data from the ITS and LSU regions. It either formed a well-supported monophyletic group within Clade 6, which was also recovered by Stoll et al. (2005) using nuclear rDNA loci; or it occurred sister to Clade 2 when using nuclear rDNA and protein-coding loci, as was also recovered by Vánky & Lutz (2011). The systemic monophyletic group will be discussed separately from Clade 2 because of its uncertain taxonomic position and distinct appearance on the host.

The systemic group of Clade 2 contained four species, Macalpinomyces loudetiae (not included in Fig. 1), M. simplex, M. trichopterygis and M. tristachyae. These smuts infect grasses in the subfamily Arundinoideae, a character first observed by Stoll et al. (2005). The entire inflorescence or every spikelet in the inflorescence is destroyed by tubular sori. Vánky (1995a) described Endosporisorium, based on Sorosporium capillipedii (syn. M. chrysopogonicola), to accommodate smuts with long, tubular, host derived sori that contained sterile cells and lacked columellae (Vánky 1995a, 2002). Endosporisorium was later synoymised with Macalpinomyces, as Vánky (1997) preferred to have few larger, well-known genera rather than many smaller, unresolved genera. Three other taxa not included in the phylogenetic analysis have a similar appearance to members of the systemic group, namely M. effusus, M. magicus and M. ugandensis. These taxa should be included in future studies to determine if this method of infection is synapomorphic and whether the separation of Endosporisorium from Macalpinomyces is warranted.

The remaining taxa in Clade 2 form tubular sori derived from hypertrophied host material in some ovaries of the inflorescence and have sterile cells in the sori, with the exception of U. maydis. The model organism U. maydis occurred in Clade 2 and was considered more closely related to Sporisorium than Ustilago by Piepenbring et al. (2002) and Stoll et al. (2005).

Brefeld (1912) established Mycosarcoma for Ustilago maydis, which he diagnosed as different to Sporisorium sorghi (as Ustilago sorghi) for three reasons: i) the incubation time in the host; ii) the development of the sorus at the site of penetration in the host plant; and iii) the development of aerial conidia. The peridial structure of Ustilago maydis was another character that Brefeld (1912) considered different to other species of Ustilago. Two of the characters that Brefeld (1912) described are unique characters to Clade 2, excluding the systemic group. The hypertrophied, host derived peridium and the localized infection sites on the host inflorescence are morphological synapomorphies of these taxa. Furthermore, the localized, hypertrophied, often tubular sori mostly contain sterile cells. Piepenbring et al. (2002) concluded from a molecular phylogenetic analysis that Ustilago maydis was separate to other Ustilago taxa, and that it may warrant placement in the genus originally assigned to it by Brefeld (1912). Other taxa that may belong to Clade 2, based on soral characters, are Macalpinomyces elionuri-tripsacoidis, M. flaccidus, M. nodiglumis, M. siamensis and M. zonotriches.

Sporisorium dietelianum and S. trachypogonis, which are mem-bers of Clade 2, were both described as having columellae (Fig. 2). It is unlikely that these structures are homologous to the stout and filiform columellae in Clades 1 and 4, which are synapomorphies for these clades. Vánky (2004) combined Sporisorium dietelianum into Lundquistia because he did not consider the fascicles of host tissue as true columellae. Vánky (2012) later re-considered this view, equating these fascicles with columellae. The columellae of Sporisorium dietelianum are filiform and similar to the columellae of species in Clade 4. Sporisorium dietelianum can be distinguished from species in Clade 4 because it does not form either a fungal peridium or spore balls, and it possesses sterile cells.

The columella of Sporisorium trachypogonis was described by Vánky (1995b) as well-formed and central, which is typical to those formed in the taxa of Clade 1. Sporisorium trachypogonis can be distinguished from other species in Clade 1 by the presence of a localized tubular sorus, rather than a systemic infection.

The recently described, monotypic genus, Tubisorus was not included in the current study. Vánky & Lutz (2011) recovered Tubisorus within a clade congruent to Clade 2. The infection of Tubisorus is consistent with other members of Clade 2 that possess long tubular sori. However, Tubisorus is described as lacking sterile cells and possessing spore balls, which are two characters considered synapomorphies of Clade 4. The establishment of Tubisorus sets a precedent for creation of monotypic genera that have an eclectic mix of characters within Clade 2.

Clade 3

Macalpinomyces bursus and M. ewartii occur in a strongly supported clade separate from other clades recovered in the analysis. Macalpinomyces bursus and M. ewartii are morphologically very similar in appearance and occur on Themeda and Sorghum respectively, which are members of the tribe Andropogoneae. The sori form hypertrophied galls in the host ovaries. Sterile cells formed from partitioning hyphae are present in the sori, which never have a columella. The spores are prominently echinulate. These characters are similar to smuts in Clade 7 that infect grasses in the subfamily Chloridoideae and the tribe Paniceae. Host classification is the simplest character to separate these two clades. Other smut taxa that may occur in this clade are Macalpinomyces bothriochloae, M. ovariicolopsis and M. pseudanthistiriae.

Clade 4

Species in Clade 4 either destroy the entire inflorescence, as in Sporisorium caledonicum (Fig. 7c) and S. tumefaciens; whole racemes, as in S. enteromorphum; or are localized in the inflorescence, as in S. heteropogonicola (Fig. 7a), S. anthistiriae and S. bothriochloae. Species in Clade 4 exhibit a number of common morphological characters.

Fig. 7

Clade 4 character states. a. Localized spikelets infected in Clade 4 character states. a. Localized spikelets infected in Sporisorium heteropogonicola; b. dimorphic spores of S. heteropogonicola; c. entire inflorescence destroyed by S. caledonicum; d. permanent spore balls of S. caledonicum. — Scale bars: a, c = 1 cm; b, d = 10 μm.

Filiform or slender columellae (Fig. 7a, c).

Persistent spore balls (Fig. 7d). Two distinct spore types are usually present within the spore ball, namely inner and outer spores. Outer spores are often ornamented and are darker than the inner spores (Fig. 7b).

A sorus surrounded by a peridium composed mostly of fungal tissue.

Sterile cells derived from non-sporogenous hyphae are rarely present within the sorus.

Langdon & Fullerton (1978) examined the soral ontogeny of two species found in Clade 4, S. anthistiriae and S. caledonicum. They described the columella of Sporisorium caledonicum as a vascular bundle surrounded by host parenchyma, with tissues permeated by inter- and intracellular hyphae. Five to seven columellae were formed by growth of hyphae in the parenchyma between the vascular bundles that separated the central column. Host cells close to intercellular hyphae in some instances were distorted but there was little destruction of host tissue. Langdon & Fullerton (1975) also studied the soral ontogeny of Sporisorium cryptum, which had a single columella made of several vascular bundles of parenchyma and mycelium that did not separate.

Species within Clade 4 have filiform or slender columellae (Fig. 2). These columellae are typically flattened in one plane and are never cylindrical. They are flexuous and do not grow vertically without support from the sorus as there are no thickened cells to sustain vertical growth. Many columellae are present in the sorus, for example in Sporisorium caledonicum, S. enteromorphum and S. fallax. A single, filiform columella comprised of several vascular bundles is sometimes present, for example in Sporisorium bothriochloae and S. cryptum. The columellae formed in this fashion are not hardened or woody, although they are sufficiently robust to persist in the sorus.

The presence of a columella was the defining character of Sporisorium (Link 1825, Langdon & Fullerton 1978, Vánky 2002). Members of Clades 1 and 4 that were examined by Langdon & Fullerton (1975, 1978) possessed two differences in development and structure of columellae. The first difference was that peripheral cells of Clade 4 species were not distorted or hardened in contrast to the thickened, vacuolated peripheral cells in Clade 1 species. The second difference was that the central columns were separated into several columellae in Sporisorium caledonicum or were made of numerous vascular bundles, as in S. cryptum; the columellae of Clade 1 members, S. andropogonis and S. sorghi were not separated into vascular bundles. Filiform columellae composed of vascular bundles constitute a synapomorphy in species of Clade 4 (Fig. 2).

Many species of Sporisorium that possess permanent spore balls were originally described as members of Sorosporium. Most of these species belong to Clade 4 (Fig. 4). Langdon & Fullerton (1975) observed spore balls in several Sporisorium (as Sorosporium) species and described their formation. Coils of sporogenous hyphae were produced among mycelium that grew from the columellae as the sorus elongated. Coils consisted of two or three intertwined hyphae. Non-sporogenous hyphae, present between the spore balls, disintegrated and did not form sterile cells. Spores formed in spore balls were dimorphic. The peripheral spores developed surface ornamentation in the form of warts or spines and the internal spores were smooth.

Sporisorium panici-leucophaei has spore balls and occurs in Clade 4. According to Vánky (2001) the spore balls of Lundquistia fascicularis (syn. S. panici-leucophaei) differentiate from non-concentric, sporogenous hyphae. This differed from the mode of formation described for Sporisorium by Langdon & Fullerton (1975), and was one reason Vánky (2001) established Lundquistia. The mode of spore ball development in Lundquistia fascicularis (syn. S. panici-leucophaei) cannot be determined from the images provided by Vánky (2001). The spore balls are not agglutinated by sterile cells, as in Moesziomyces, and if the sporogenous hyphae are intertwined, as for species in Clade 1, then it is unlikely that the spores would form balls. It is unknown how spore balls are formed in Sporisorium panici-leucophaei.

Langdon & Fullerton (1975) observed that non-sporogenous hyphae in Sporisorium caledonicum, and three other species that occurred in Clade 4, disintegrated after the spores had matured. Sterile cells are rarely present in species of Clade 4 (Fig. 3). Often peridial cells derived from the fungal peridium were reported as sterile cells for species in Clade 4, for example in Sporisorium loudetiae-pedicellatae.

Species within Clade 4 possess a peridium made of fungal cells surrounded by a layer of host cells. Langdon & Fullerton (1975) discussed the formation of this peridium in Sporisorium caledonicum and three other smut fungi that occurred in Clade 4. They observed that hyphae adjacent to the peripheral host tissues became enlarged, with vacuolate cells and thickened cell walls. These hyphae were orientated in the direction of the long axis of the sorus and formed a sheath inside the peripheral layer of host tissue. This fungal sheath and the host cells external to it constituted the soral peridium, which surrounded the soral contents.

Members of Clade 4 mostly occur on grasses in the tribes Andropogoneae or Paniceae in the subfamily Panicoideae. One exception is Sporisorium hwangense that infects Sporobolus in the subfamily Chloridoideae. It shares characters with other taxa in Clade 4, namely filiform columellae, spore balls with dimorphic spores, and an absence of sterile cells. Other examples of smut fungi that share characters in Clade 4 but occur on chloridoid grasses are S. cynodontis, S. normanensis, S. pa-rodii and S. saharianum.

Anomalomyces panici

Anomalomyces panici is sister to Clades 1, 2, 3 and 4. In terms of soral morphology, this species is similar to M. bursus and M. ewartii as it forms globose hypertrophied sori localized in the host ovaries. Anomalomyces infects Panicum trachyrachis in the tribe Paniceae. The sorus is filled with hardened spore balls formed by coiled sporogenous hyphae (Vánky et al. 2006), dimorphic spores and sterile cells. Anomalomyces possessed a unique combination of characters that warrants a monotypic genus within the Ustilago-Sporisorium-Macalpinomyces complex.

Clade 5

Four taxa that occur on the arid grass Triodia form a clade supported in maximum likelihood and Bayesian inference. The Bayesian analysis conducted by Stoll et al. (2005) grouped two Triodia taxa with the Ustilago esculenta group within Clade 6.

Ustilago altilis and U. inaltilis infect the host plant culms, while U. lituana and U. triodiae destroy the host inflorescence. Near identical ITS sequences for U. altilis and U. inaltilis (99 % identical over 98 % query coverage in a BLAST search), and U. lituana and U. triodiae (98 % identical over 88 % query coverage in a BLAST search) demonstrate their very close relationships. A synapomorphy for these four taxa is that they infect species of Triodia. They have similar characters to species in Clade 6, in that they do not possess soral structures such as spore balls, columellae or sterile cells.

Clade 6

Stoll et al. (2005) recovered Clade 6 as a weakly supported clade, which included Melanopsichium pennsylvanicum. They designated this clade as Ustilago s.l. and defined three subgroups within the clade: i) Ustilago s.str.; ii) the Ustilago davisii group; and iii) the Ustilago esculenta group. Further loci were only sequenced for six taxa of Clade 6 in this study. Host and morphological synapomorphies have not been resolved for Clade 6 in our analysis.

Ustilago s.str. clade

Ustilago species that infect grasses in the tribe Pooideae formed a well-supported group that included the type species, U. hordei. Stoll et al. (2005) also recovered this group with strong support using Bayesian analysis. The stripe smuts U. calamagrostidis and U. striiformis, as well as U. sporoboli-indici (on Chloridoideae) were sister to the smuts that destroy the inflorescence of pooid grasses. Stoll et al. (2005) included a subgroup in Ustilago s.str. that contained Ustilago cynodontis, U. sparsa and U. xerochloae. These three taxa occur on panicoid and chloridoid grasses. Inclusion of this subgroup and the stripe smuts in Ustilago s.str. was supported by both maximum likelihood and Bayesian inference. Taxa within the Ustilago s.str. clade lacked three characters that were found in other clades.

Absence of sterile cells in the sorus.

Absence of spore balls formed by coiled sporogenous hyphae.

Absence of a columella derived from host and fungal material.

Ustilago davisii group

Stoll et al. (2005) recovered a strongly supported but unresolved clade containing seven species, Sporisorium aegypticum, S. modestum, Ustilago davisii, U. filiformis, U. schroeteriana, U. tragana and U. trichophora. The same clade was recovered in this study, but it was not well supported by bootstrap values (< 70 %) in maximum likelihood or posterior probabilities (< 0.95) in Bayesian inference. Sporisorium aegypticum, S. modestum and Ustilago trichophora were described as having columellae.

Fullerton & Langdon (1968) examined the soral development of Ustilago trichophora and concluded that a columella was present, however columellae are not included in the descriptions by Vánky & Shivas (2008) or Vánky (2012). The sori of Ustilago trichophora occur in ovaries or on stems and do not have columellae that are homologous to the columellae formed in Clade 1 and 4.

Ustilago esculenta group

Stoll et al. (2005) recovered a weakly supported group that contained several smut fungi found on chloridoid grasses together with the atypical Ustilago esculenta, which occurs on Zizania in the subfamily Ehrhartoideae. Ustilago curta, which infects Tripogon in the subfamily Chloridoideae, either occurred in the Ustilago esculenta group, or as sister to Clade 6 or 8. Stoll et al. (2005) recovered Ustilago curta (as U. alcornii) in the Ustilago esculenta group. No synapomorphies were determined for this group.

Stoll et al. (2005) demonstrated a close relationship between Melanopsichium pennsylvanicum and the Ustilago s.str. group. Our maximum likelihood analyses placed Melanopsichium in the Ustilago esculenta group rather than sister to the Ustilago s.str. group. Only the two nuclear rDNA loci obtained by Stoll et al. (2005) were included for Melanopsichium in the combined analysis of molecular loci. Begerow et al. (2004a) discussed the complicated coevolution between smut fungi and their hosts. Melanopsichium pennsylvanicum may represent a jump from Poaceae to the distantly related Polygonaceae.

Clade 7

This clade was recovered in studies by Stoll et al. (2003, 2005) and was strongly supported by both maximum likelihood and Bayesian inference in this study. Stoll et al. (2005) noted that taxa in this clade had a combination of characters observed in Sporisorium and Ustilago. Taxa in this group have often been placed in Macalpinomyces because of the mixed soral characteristics associated with both Sporisorium and Ustilago. They occur on grasses in the tribe Paniceae and the subfamily Chloridoideae.

Sterile cells are present in Macalpinomyces neglectus, M. spermophorus, M. viridans and Ustilago affinis, but are absent in the other members of this clade. Several taxa formed galls in the host ovaries, while U. affinis, U. drakensbergiana and U. syntherismae destroyed the entire inflorescence similar to taxa in Ustilago s.str. Columellae were described in several of the species in this clade, including Ustilago drakensbergiana, Macalpinomyces spermophorus, M. neglectus and M. viridans.

The columellae of U. drakensbergiana are formed from the remnants of the destroyed inflorescence and are not homologous with columellae of Clade 1 and 4. Vánky (2012) observed that the sori of species of Macalpinomyces were deciduous and separated from the host plant at maturity, whereas species of Sporisorium had sori that remained attached to the inflorescence because the columella was connected to the host plant. The sori of M. spermophorus and M. viridans were deciduous and easily removed from the host plant. These columellae are not formed from the host meristem and are not homologous to the columellae of Clade 1 and 4.

A synapomorphic character for Clade 7 was not identified. Subdivision of Clade 7 based on morphology is impractical at this stage, because the characters are highly variable in the group.

Clade 8

Four taxa that destroy the ovaries of Aristida formed a well-supported monophyletic group. Stoll et al. (2005) included Sporisorium consanguineum in their study, but were unable to determine whether it was sister to, or part of Clade 7. The inclusion of three additional smuts that infect Aristida has resulted in a separate, monophyletic group. The smuts on Aristida share two morphological characters.

Formation of galls in the ovaries of their hosts. They can infect all of the ovaries in an inflorescence (Sporisorium confusum, S. consanguineum) or be localised in the inflorescence (S. aristidicola).

The spores are commonly compacted into spore balls formed by coiled sporogenous hyphae, for instance in Sporisorium consanguineum (Langdon & Fullerton 1975).

Macalpinomyces eriachnes

Macalpinomyces eriachnes is the sister taxon to the Ustilago-Sporisorium-Macalpinomyces complex. Stoll et al. (2005) first indicated that Macalpinomyces was a monotypic genus, with M. eriachnes the sole representative. This relationship is supported in this study. Macalpinomyces eriachnes has giant sterile cells formed from non-sporogenous hyphae (Langdon & Fullerton 1977, Vánky 1996) and a peridium, but lacks a columella. The spore balls of Macalpinomyces eriachnes were not formed from coiled sporogenous hyphae (Langdon & Fullerton 1977).

Taxa of uncertain placement

A few taxa moved between clades in trees reconstructed using different datasets and different phylogenetic assessment criteria. These taxa were not supported in any group, although previous analyses have grouped most of these taxa in Clade 6 (Stoll et al. 2005). Sporisorium aegypticum, S. modestum Ustilago schmidtiae and U. tragana often grouped together after maximum likelihood analysis, although they were only represented by data from two molecular loci in most cases. These taxa, except for Ustilago schmidtiae, were included with taxa now assigned to Clade 6 by Stoll et al. (2005).

Maximum likelihood analyses placed Ustilago curta in a number of clades. Stoll et al. (2005) recovered U. curta (as U. alcornii) in the Ustilago esculenta group of Ustilago s.l. after Bayesian analysis of data from two nuclear rDNA loci. With the addition of nuclear loci, U. curta was often placed as sister to the Aristida group or as sister to the Triodia group. It is not known to which group Ustilago curta belongs.

Can host classification delimit smut genera?

Taxa within the Ustilago-Sporisorium-Macalpinomyces complex infect hosts in the Poaceae, with the exception of Melanopsichium, which occurs on Polygonaceae. The systematics of Poaceae has been well resolved and the relationships of the subfamilies and tribes are well understood (Hsiao et al. 1999, Kellogg 2000, Stevens 2001, Bouchenak-Khelladi et al. 2008).

Host classification has often been used in the classification of smut fungi. Within Ustilago, Sporisorium and Macalpinomyces, putative host specificity is used to differentiate morphologically indistinguishable species (Bauer et al. 2001). Many of the keys to these genera are based on host taxonomy. Higher-level host taxonomy has been used to delimit smut genera, for example Ustilago is restricted to members of Poaceae (Bauer et al. 2001).

Begerow et al. (2004a) concluded that the phylogenetic relationships between smut fungi and their hosts were not straightforward. While species of Ustilago and Sporisorium showed evidence for co-speciation, it was considered more likely that smut fungi evolved after their hosts had speciated (Begerow et al. 2004a). Host jumps are evident in Clade 4, which contains taxa that infect grasses in two subfamilies, the Paniceae and the Chloridoideae.

The phylogenetic analyses of the Ustilago-Sporisorium-Macalpinomyces complex recovered several monophyletic groups that shared similar morphological characters and are restricted to hosts in a specific genus, tribe or subfamily. Four smuts 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 synapomorphies that separate them unambiguously from other species in the complex. Their pathogenicity on hosts in the subfamily Aristidoideae is a synapomorphy that distinguishes this clade from other clades in the complex.

Macalpinomyces bursus and M. ewartii, which are members of Clade 3, infect hosts in the tribe Andropogoneae. They possess morphological characteristics that are similar to some species of Clade 7 that infect hosts in the Chloridoideae or Paniceae. The occurrence of members of Clade 3 on hosts in the tribe Andropogoneae is a synapomorphy that can be used to distinguish Macalpinomyces bursus and M. ewartii from taxa in Clade 7.

In many cases morphological characteristics are inadequate for recognizing smut taxa. It is proposed that delimitation of smut genera be based on host range, provided monophyletic groups are resolved after molecular phylogenetic analyses. In the absence of contradictory evidence, host subfamily or tribe is a legitimate criterion for generic delimitation in the Ustilago-Sporisorium-Macalpinomyces complex.

CONCLUSION

A detailed examination of morphology is required to determine homology and to improve classification (Mooi & Gill 2010), although in many groups of fungi this is impossible. The synapomorphies outlined here based on gross morphology and host coevolution allow confident placement of new taxa within the Ustilago-Sporisorium-Macalpinomyces into well delimited clades. Although there are some morphological anomalies, the monophyletic groups are robust and well supported.

Morphological synapomorphies within the Ustilago-Sporisorium-Macalpinomyces were identified after incorporation of nuclear protein coding loci and a thorough study of morphological diversity in Australian taxa. The determination of monophyletic groups and synapomorphic characters within the complex necessitates taxonomic reassessment of some genera and the creation or resurrection of others in future studies. The major outcomes of resolved character homology in the Ustilago-Sporisorium-Macalpinomyces complex are:

Sporisorium can be subdivided by soral characteristics. Sporisorium s.str. must be described explicitly to prevent ambiguity for future taxonomic placement of new species.

New genera are required for the placement of taxa that form monophyletic groups and no longer fit the definition of Sporisorium s.str.

Ustilago maydis and other taxa with localized tubular sori and usually with sterile cells form a monophyletic group with the morphologically similar systemic group, which usually destroy the entire inflorescence. A taxonomic resolution for these taxa cannot be proposed at this stage, however, if the method of soral infection is synapomorphic within the groups, the two names, Mycosarcoma and Endosporisorium, will be available for the placement of these taxa.

Macalpinomyces bursus and M. ewartii belong to a monophyletic group that can be differentiated by soral characteristics and host tribe.

The monophyletic group of smut fungi that infect Aristida can be delimited by soral characteristics and host subfamily.

Four smut fungi on Triodia form a monophyletic group.

Macalpinomyces is a monotypic genus, sister to all other taxa in the Ustilago-Sporisorium-Macalpinomyces complex (Stoll et al. 2005).

Until Clade 2 and 7 are resolved, Macalpinomyces will remain a polyphyletic genus.