Phylogenetic placement of the anamorphic tribe Ustilaginoideae ...

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Mycologia, 96(5), 2004, pp. 1088–1094. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897

Phylogenetic placement of the anamorphic tribe Ustilaginoideae (Hypocreales, Ascomycota) J.F. Bischoff1 R.F. Sullivan

morphic genera or as anamorphic taxa for which no teleomorphic state is known. The Ustilaginoideae is an anamorphic group of Ascomycetes that infects tropical grasses. Diehl (1950) considered Ustilaginoideae to bear affinity to the Clavicipitaceae based on reports of sexual states that resemble those of Balansia Speg., Claviceps Tul., and Mycomalus Mo¨ller. They differ from other major groups of Clavicipitaceae because of their unique conidial structures. They produce smut teliospore-like (e.g., Ustilago spp.) melanized subglobose conidia holoblastically from multiple loci along the length of a hypha-like conidiophore (FIG. 1B, D, G). Most other graminicolous anamorphic clavicipitaleans (e.g., Ephelis, Neotyphodium, Sphacelia spp.) produce hyaline conidia from a conidiogenous cell with a single conidiogenous locus at the apex (Rykard et al 1984). Diehl included all graminicolous clavicipitaleans in subfamily Clavicipitoideae. Diehl (1950) placed several ustilaginoid genera, including Munkia Speg., Neomunkia Petrak and Ustilaginoidea, in tribe Ustilaginoideae. Because of the absence or rarity of sexual states, the teleomorphic affinities of the ustilaginoid fungi never have been resolved clearly. Subfamily Clavicipitoideae included two other tribes (Diehl 1950), both of which possess teleomorphic states. The monotypic tribe Clavicipeae (Claviceps) infects the florets of various grasses, replacing the ovules with sclerotia. Early in sclerotium development, enteroblastic, hyaline and ovoid conidia are produced in a honeydew-like matrix (Sphaceliastate). Tribe Balansieae currently includes the teleomorphic genera Balansia, Atkinsonella Diehl and Myriogenospora Atk., all of which produce holoblastic, acicular conidia referred to the anamorphic genus Ephelis Fr. (Kuldau et al 1997). The primary aim of this study is to determine whether Ustilaginoideae is a monophyletic group and evaluate its placement through phylogenetic analyses of large subunit (LSU) of the ribosomal DNA (rDNA) data. Observations regarding morphology were made to assess the relative value of structural features for classification to genera.

Department of Plant Biology and Pathology, Cook College, Rutgers University, New Brunswick, New Jersey 08901

K.M. Kjer Department of Ecology, Evolution and Natural Resources, Cook College, Rutgers University, New Brunswick, New Jersey 08901

J.F. White Jr. Department of Plant Biology and Pathology, Cook College, Rutgers University, New Brunswick, New Jersey 08901

Abstract: Tribe Ustilaginoideae (Hypocreales, Ascomycetes) is made up of three anamorph genera, Munkia, Neomunkia and Ustilaginoidea. Species of Munkia and Neomunkia develop on the culms of bamboo (Chusquea spp.) and have a neotropical distribution while species of Ustilaginoidea infect the florets of various grasses and are pantropical in distribution. In this study we evaluated the phylogeny of the tribe and assessed hypotheses regarding its affinity to clavicipitalean teleomorphic groups. To support phylogenetic analyses, morphology of representatives of several key species of Ustilaginoideae was examined also. Phylogenetic analyses using sequences of the large subunit of the ribosomal RNA gene suggest that members of Ustilaginoideae are distinct from teleomorphic genera of Clavicipitaceae and that Ustilaginoideae should be recognized as a monophyletic group within Hypocreales. However, phylogenetic analyses did not resolve the placement of Ustilaginoideae in Clavicipitaceae or Hypocreaceae, suggesting that it might be a distinct lineage within Hypocreales. This evaluation supported the monophyly of tribes Balansieae and Clavicipeae in the family Clavicipitaceae. Key words: Ascomycetes, Clavicipitaceae, Hypocreales, Munkia, Neomunkia, Oryza, rice, Ustilaginoidea

INTRODUCTION

Within Clavicipitaceae a number of anamorphic fungi are recognized either as alternate states of teleo-

MATERIALS AND METHODS

Morphological observations.—Observations were made on several species belonging to the Ustilaginoideae, including

Accepted for publication April 26, 2004. 1 Corresponding author. E-mail: [email protected]

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FIG. 1. A, B. Ustilaginoidea virens. A. Stroma developing between rice glumes. Bar 5 6 mm. B. Pleurogenously developing conidia (arrowhead) from slightly raised pores (arrow) on conidiophore. Bar 5 7 mm. C, D. Munkia martyris. C. Stroma. Bar 5 2 mm. D. Pleurogenously developing conidia (arrow). Bar 5 8 mm. E, F. Neomunkia sydowii. E. Stroma (arrow) developing from bamboo culm. Bar 5 60 mm. F. Stromal surface. Bar 5 200 mm. G. Pleurogenously developing conidia (arrows). Bar 5 15 mm. Munkia martyris, M. strumosa, Neomunkia sydowii, Ustilaginoidea dichromenae and U. virens. For this examination, specimens from herbaria (NY, BPI, RUTPP) and newly collected material were obtained (TABLE I). Relevant species were assessed for several features including location of stroma on host, shape of conidioma, conidial size and ornamentation and stromal color (TABLE III). Microscopic observations were made from squash mounts of stromal tissue placed in Permountt. Macroscopic evaluations were made with a Zeiss Stemi SV8 dissecting scope and microscopic observations and measurements were made with a Zeiss Axioskop microscope. Conidial measurements were taken at a magnification of 10003. Sequence data.—Collection and voucher information regarding the isolates from this study are listed in TABLE I. Fresh mycelia of Ustilaginoidea dichromenae and U. virens were grown on cellulose acetate sheets overlaid on PDA. Mycelia then were lifted off and ground in liquid nitrogen. Fungal tissue of Neomunkia sydowii (RUTPP) was taken from the center of an air-dried stroma and ground in liquid nitrogen. DNA extraction, amplification and sequencing of the LSU rDNA were done as described previously by Sullivan et al (2001). Sequences obtained from GenBank as well as sequences obtained for this study are listed in TABLE II. Base frequencies were determined using PAUP version 4b10 Alvitec (Swofford 2002). Only taxa that were found to be

homogeneous, according to the Chi squared analysis as implemented by PAUP, with the other taxa in the matrix were included in phylogenetic analyses. Members of Hypocreaceae (Fr.) Tul. were chosen as outgroup taxa due to their placement as the sister group of Clavicipitaceae (Spatafora and Blackwell 1993). Sequence alignment.—Sequences were aligned manually in Microsoft Word using the ribosomal secondary structure of Saccharomyces cerevisiae as depicted on http://www.rna. icmb.utexas.edu as a guide (Cannone et al 2002). The method of alignment and matrix annotation was done as described by Kjer (1995). Ambiguous regions were delimited and placed in brackets to prevent their use in analyses (Nexus format used in PAUP does not read information that is placed within brackets). The alignment is available at http://www.rci.rutgers.edu/;struwe/students/joe/. Sequence analysis.—Modeltest 3.06 (Posada and Crandall 1998) under the Akaike criterion (Akaike 1974) was used to select the model of evolution that best fit the data. This model was input into MrBayes 3.0, a Bayesian phylogenetic inference program (Huelsenbeck and Ronquist 2001), to determine the proportion of invariable sites (I), gamma distribution (G), base frequencies, more specifically define the reversibility of bases in the model and determine branch support (posterior probabilities). Bayesian analysis was run

Collection no.

s.n. s.n. s.n. s.n. s.n. 3114 s.n. s.n. s.n. s.n. 62370 s.n. 4103 IFO 5923

Collected by

H. Schmidt G.M. W.R. Maxon W.R. Maxon J.F. Bischoff Not noted Whetzel, Kern, & Toro A.S. Muller G.F. Bills J.F. Bischoff F.H.B. Dean H.R. Fulton F.J. Santos ? ?/?/1928 Not noted 6/22/1949 6/22/1949 6/26/2001 ?/?/1901 6/12/1924 10/10/1938 12/12/1996 7/12/2001 5/21/1926 9/29/1906 10/6/1966 ?

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Date collected

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with four Markov Chain Monte Carlo chains (three cold, one heated) for 1 000 000 generations, sampling every 100 generations, which yielded 10 001 trees. These trees were graphed in Microsoft Excel to determine at which point the likelihood scores of the trees being recovered were asymptotic (point at which the curve flattens out). The trees that were not asymptotic were discarded (burn in; Huelsenbeck 2000). This was done twice, and I, G, base reversibility and base frequencies were averaged over all remaining trees. These trees were imported into PAUP 4.0b10 Alvitec (Swofford 2002) and a majority-rule consensus tree was produced to determine posterior probabilities that were reported in the maximum likelihood tree (FIG. 1). A maximum likelihood analysis was performed with model parameters I, G, base frequencies and the R matrix, as taken from the means estimated in the Bayesian analysis. Taxa were added randomly in 100 replicates with a random starting seed. One tree was held at each step during stepwise addition using the TBR algorithm. Branches were collapsed if branch length was less than or equal to 1210.

San Jose, Costa Rica Monkey Hill, Jamaica Monkey Hill, Jamaica Monkey Hill, Jamaica Cerro de la Muerte, Costa Rica Manaos, Brazil Guaynabo Farm, Puerto Rico Caracas, Venezuela Costa Rica La Selva, Costa Rica India Crowley, Louisiana, USA Laguna, Philippines ? BPI 183581 BPI 183662 BPI 183660 BPI 183661 RUTPP BPI 183602 BPI 183606 BPI 183600 RUTPP RUTPP BPI 180512 BPI 183676 BPI 183674 ATCC 16180 Munkia martyris (as Ustilaginoidea sp.) Munkia strumosa (as Ustilaginoidea strumosa)* Munkia strumosa (as Ustilaginoidea strumosa)* Munkia strumosa (as Ustilaginoidea strumosa)* Neomunkia sydowii† Ustilaginoidea dichromenae* Ustilaginoidea dichromenae* Ustilaginoidea dichromenae* Ustilaginoidea dichromenae† Ustilaginoidea dichromenae† Ustilaginoidea virens Ustilaginoidea virens Ustilaginoidea virens Ustilaginoidea virens†

* Indicates it is a type. † Indicates that DNA was extracted for this study. a s.n. 5 sine numbero, ? 5 unknown.

Collection location Herbarium and accession number Taxon

TABLE I.

Specimens examined for morphological evaluationa

RESULTS

Morphological observations.—Ustilaginoidea dichronemae and U. virens developed in the host florets. The mycelial balls (FIG. 1A) were covered largely by conidia that were powdery and orange-brown to ocherbrown. No evidence of a perithecial state was observed. Microscopic examination of conidia revealed the characteristic ustilaginoid conidial state with porospore-like conidiophores bearing multiple conidiogenous loci at which a single conidium is produced (FIG. 1B). Stromata of Neomunkia sydowii (FIG. 1C) and Munkia strumosa (FIG. 1E) were produced on culms and were gray-green, large (5–25 mm 3 4–20 mm) and subglobose to fusoid in shape. Conidia were produced in cup-shaped sporodochia in the specimen of Munkia martyris, but cupules were not present on specimens of M. strumosa and N. sydowii. The ustilaginoid conidia of these genera were produced under an epidermal layer of the fungus that fractures to expose the underlying conidiophores. As in Ustilaginoidea spp., typical ustilaginoid conidia were observed (TABLE III). Sequence analyses.—The alignment consisted of 927 total characters, of which 90 were ambiguous and removed from the analysis. Modeltest 3.06 selected a general time reversible model 1 I 1 G (6ST-GTR1I1G; Rodrı´guez et al 1990). The trees determined in the first run of MrBayes were asymptotic after the first 70 trees, and the trees in the second run were asymptotic after the first 68 trees. This left a total of 19 864 trees to be evaluated. Likelihood model assumptions were: base frequencies A 5 0.245, C 5 0.229, G 5 0.329, T 5 0.197; number of substitution types 5 6; I 5 0.629,

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Large Subunit Ribosomal DNA sequences included in analyses Taxa

Atkinsonella hypoxylon (Pk.) Diehl Atricordyceps harposporifera Samuels Balansia brunnans E.A. Lewis & J.F. White Balansia henningsiana (Mo¨ller) Diehl Balansia obtecta Diehl Balansia strangulans (Mont.) Diehl Claviceps africana Frederickson, Mantle & De Milliano Claviceps fusiformis Loveless Claviceps paspali F. Stevens & J.G. Hall Claviceps purpurea (Fr.) Tul. Claviceps ranunculoides Mo¨ller Cordycepioideus bisporus Stifler Cordyceps capitata (Fr.) Link Cordyceps cochlidiicola Kobayasi & Shimizu Cordyceps inegoensis Kobayasi Cordyceps japonica Lloyd Cordyceps kanzasiana Kobayasi & Shimizu Cordyceps militaris (L.) Link Cordyceps ophioglossoides (Ehrenberg) Link Cordyceps prolifica Kobayasi Cordyceps ramosopulvinata Y. Kobayasi & D. Shimizu Cordyceps subsessilis Petch Cordyceps takaomontana Y. Kobayasi Cordyceps tuberculata (Lebert) Maire Dussiella tuberiformis (Berk. & Rav) Atk. Epichloe¨ amarillans J.F. White Epichloe¨ typhina (Pers. : Fr.) Tul. Hyperdermium bertonii (Speg.) J.F. White et al. Hypocrea lutea (Tode) Petch Hypocrea schweinitzii (Fr.) Sacc. Hypomyces armeniacus Tul. & C. Tul. Hypomyces odoratus G.R.W. Arnold Hypomyces rosellus (Albertini & Schwein.) Tul. Myriogenospora atramentosa (Berk & Curt.) Diehl Neomunkia sydowii Petrak* Torrubiella luteorostrata Zimm. Ustilaginoidea dichromenae P. Henn.* Ustilaginoidea virens (Cooke) Takah.*

Genbank accession numbers U57087 U17400 AY327046 U57678 U17395 U57679 AF245294 U17402 U47826 U57085 AF245295 AF009654 U57086 AB027377 AB027368 AB027367 AB027371 AF043135 U47827 AB027370 AB027372 AF373285 AB044637 AF327384 U57083 U57680 U17396 AF242354 AB027384 U47833 AF160239 AF160240 AF160242 U57084 AY327047 AF327388 AF373280 AF245299

* Indicates new Genbank accessions as a result of this study.

G 5 0.538; number of rate categories 5 4; rate matrix was A↔C 5 0.5198, A↔G 5 3.7377, A↔T 5 1.3683, C↔G 5 2.19332, C↔T 5 20.712, G↔T 5 1. Maximum likelihood analysis found the most likely tree (2ln 3463.96137; FIG. 2) in 19 of 100 random additions. All teleomorphic members of Clavicipitaceae were recovered in the Clavicipitaceae clade (Clades A–E). Clade Clavicipitaceae was weakly supported. All members of tribe Balansieae grouped in Clade A. Clade B was supported weakly and contained species from the genera Atricordyceps, Cordycepioideus, Cordyceps and Hyperdermium. Clade C (76% support) included the three Elaphomyces Nees pathogens included in this analysis (Cordyceps capi-

tata, C. ophioglossoides and C. japonica), C. subsessilis and C. inegoensis. All members of the tribe Clavicipeae grouped in Clade D (100% support). Clade E included Epichloe¨ amarillans, E. typhina, Dussiella tuberiformis and Torrubiella luteorostrata but was supported poorly. Clade F (100% support) supported the monophyly of Ustilaginoideae. Tribe Ustilaginoideae was placed in the ancestral position to the teleomorphic groups of Clavicipitaceae. DISCUSSION

Ustilaginoidea includes 16 species (Wang and Bai 1997), all of which occur on grasses in subtropical

Oryza Dichronema, Paspalum Chusquea Chusquea Chusquea Ustilaginoidea virens Ustilaginoidea dichronemae Munkia martyris Munkia strumosa Neomunkia sydowii

a ‘‘1’’ indicates that the structure is present; ‘‘2’’ indicates that they are absent. In the case of echinulate conidia the ‘‘11’’ indicates that the wart-like appendages are consistently larger than what was found on species with ‘‘1’’.

4–7 4–8 3–6 3–5 3–5 1 11 1 1 2

Host genus Taxon

Floret Floret Culm Culm Culm

2 2 1 2 2

Conidia diameter (mm) Conidia echinulatea Loc. of stromal Sporodochiumdevelopment like cupulesa Ustilaginoideae morphological data TABLE III.

Orange-brown, black Ocher-brown Gray-green Gray-green Gray-green

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and tropical regions. The genus is typified by U. virens (Cooke) Takah., the causal agent of false smut of rice (Oryza sativa L.). In parts of Asia U. virens has been found to produce sclerotia from its mycelial mass. Hashioka (1951) determined that this growth habit and the production of stipitate perithecial stromata from the sclerotium resembled that of Claviceps spp. and described Claviceps oryzae-sativa Hashioka (5 U. virens) as a new species. von Ho¨hnel (1911) considered Munkia martyris to be linked to a Mycomalus-like teleomorph. Mycomalus Mo¨ller produces a globose stroma that encircles the culm of a bamboo and produces a flattened, light brown Balansia-like perithecial stroma (Mo¨ller 1901). In addition, Diehl (1950) discussed the possibility of Munkia martyris being linked to Balansia chusqueicola P. Henn. based on a Munkia-like stroma that bore a flattened black perithecial stroma. Based on the results of this study, Ustilaginoidea virens does not show affinity to Claviceps. Furthermore, the monotypic tribe Clavicipeae (Claviceps) was found to be monophyletic (Clade D; 100%; FIG. 2) and can be distinguished by the production of enteroblastic hyaline conidia (Sphacelia). It is evident that production of sclerotium-like structures in florets is not evidence of affinity to genus Claviceps. Formation of sclerotia in florets in Ustilaginoidea and Claviceps are independently derived or ancestral characteristics. It also seems likely that formation of a globose stroma and flattened perithecial stromata cannot be taken as evidence that Munkia bears affinity to genus Balansia, where such traits are the norm. Globose stromata and flattened ascostromata might be independently derived characters in Munkia and Balansia. The affinity of Munkia martyris and Mycomalus could not be evaluated in this study. No molecular data or culture of the monotypic genus Mycomalus currently is available. However, descriptions of Mycomalus (Mo¨ller 1901, von Ho¨hnel 1911) indicate the production of the ustilaginoid-like conidial state. Our data suggest that the ustilaginoid anamorph is a synapomorphy linked to Ustilaginoideae and the likely placement of Munkia martyris in Ustilaginoideae. Mycomalus, known only from its type collection in Brazil, represents the only valid teleomorph genus that may be linked to the Ustilaginoideae. Despite comparisons with all of the major teleomorphic groups of Clavicipitaceae, none of these genera were allied with the genera in Ustilaginoideae (FIG. 2). Results of the phylogenetic analyses supported the monophyly of tribe Ustilaginoideae (100%; FIG. 2) but placed it as a sister clade of known clavicipitaleans and distinct from outgroup Hypocreaceae. In a separate phylogenetic analysis (tree

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FIG. 2. The most likely tree (2ln likelihood 5 3463.96137). Numbers on branches are percentages that indicate the posterior probability given the observed data that the group exists. Only percentages above 75% are indicated.

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not shown; matrix available on TreeBase) we included the three ustilaginoid fungi along with the hypocrealean families Bionectriaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae and Niessliaceae. Microascales and Halosphaeriales were used as outgroups based on the results of Spatafora and Blackwell (1993) and Artjariyasripong et al (2001). This analysis did not help resolve the familiar placement of Ustilaginoideae in Hypocreales. The results of the analysis placed Ustilaginoideae as a derived group within Hypocreales and closely related but distinct from Clavicipitaceae and Hypocreaceae clades. ACKNOWLEDGMENTS

The authors would like to thank: Dr Amy Rossman, for her suggestions and review of the manuscript; Dr Marshall Bergen, for his invaluable skills in translation. J.F.B. (OTS 013) would like to thank the Organization for Tropical Studies for its assistance and leadership during his collection and course experience in the summer of 2001.

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