Mycol. Res. 98 (6):625-634 (1994) Printed in Great Britain 625 Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences STEPHEN A. REHNER AND GARY J. SAMUELS USDA, ARS, Systematic Botany and Mycology Laboratory Room 304, Building OIIA, Beltsville, BARC-W, Maryland 20705, U.SA. The phylogenetic distribution of Gliocladium within the Hypocreales was investigated by parsimony analysis of partial sequences from the nuclear large subunit ribosomal DNA (28s rDNA). Two principal monophyletic groups were resolved that included species with anarnorphs classified in Gliocladium. The first clade includes elements of the genera Hypocrea (H. gelatinosa, H. lutea) plus Trichodema, Hypocrea pallida, Hypomyces and Sphaerostilbella, which are each shown to be monophyletic but whose sister group relationships are unresolved with the present data set. Gliocladium anamorphs in this clade include Gliocladium penicillioides, the type species of Gliocladium and anamorph of Sphaerostilbella aureonitens, and Trichodema virens (= G. virens) which is a member of the clade containing Hypocrea and Trichodema. A second clade, consisting of species with pallid perithecia, is grouped around Nectria ochroleuca, whose anamorph is Gliocladium roseum. Other species in this clade having Gliocladium-like anamorphs are Nectriopsis sporangiicola and Roumeguericlla rufula. The species of Nectria represented in this study are polyphyletic and resolved as four separate groups: (1)N. cinnabarina the type species, (2) three species with Fusarium anamorphs including N. albosuccinea, N. haematococca and Gibberella fujikoroi, (3) N. purtonii a species whose anamorph is classified in Fwarium sect. Eupionnofes, and (4) N. ochroleuca, which is representative of species with pallid perithecia. The results indicate that Gliocladium is polyphyletic and that G. penicillioides, G. roseum, and Trichoderma virens (= G. virens), are generically distinct. Gliocladium Corda is a genus of hyphomycetes that includes (DNA) data. Comparison of shared, unique polymorphisms two fungi commonly used in experimental biological control can help link closely related or conspecific anamorphs and of soil-borne plant diseases, G. virens J. H. Mill., Giddens & A. teleomorphs, but the full potential of this approach is best A. Foster (1957) and G. roseum Bainier (Papavizas, 1985; Chet, realized when coupled with phylogenetic analysis. Gene 1987, 1990). While these two species broadly conform to the phylogenies can be considered to be hypotheses of organismal concept of Gliocladium, the differences that separate them relationships, and provide information on both contemporary from each other and from the type species of the genus, G. and historical anamorph and teleomorph connections. Gene penicillioides Corda, indicates that their interrelationship is not phylogenies may ultimately lead to the integration of as close as their current generic placement implies and that anamorphic and teleomorphic fungi into a unified system of they may not be congeneric. classification (Reynolds & Taylor, 1993). Within the short One way of assessing the taxonomic and natural relation- time that molecular methods have been available, there have ships of anamorphic fungi is to compare them with the proven been many demonstrations of the usefulness of this approach anamorphs of ascomycetes. Although most species of to fungal systematics (reviewed by Bms, White & Taylor, Gliocladium, including G. virens, do not have proven 1991), which include investigations of anamorph and tel- teleomorphs, culture studies have demonstrated that many are eomorph relationships (Berbee & Taylor, 1992a. 19926; similar or identical to the anamorphs produced by species of Gaudet et al., 1989). at least seven hypocrealean genera (sensu Rogerson, 1970) The purpose of this study is to evaluate the phylogenetic including Hypocrea (Webster, 1964; Webster & Rifai, 1968; relationships within Gliocladium, focusing particularly on the Doi, 1972: Doi & Yamatoya, 1989), Hypomyces (Petch, 1938), biocontrol fungi G. virens and G. rosewm, and the type species Nectria (e.g. Booth, 1959; Samuels, 1976), Nectriopsis (Samuels, of Gliocladium, G. penicillioides (Figs 1-3). We have constructed 1973,1988),Roumegueriella (Bainier, 1910),Sarawakus (Samuels a gene phylogeny for the 5' region of the nuclear 28s large & Rossman, 1992), and Sphaerostilbella (Seifert, 1985). The subunit rRNA based on sequences determined from conidial linkage of Gliocladium anamorphs with such a diverse array of and ascospore isolates of hypocrealean anamorphs that can be taxonomically distinct teleomorph genera clearly indicates identified as Gliocladium, and other hypocrealean taxa which that Gliocladium, as currently defined, is polyphyletic. do not have Gliocladium-like anamorphs. The gene phylogeny, Another means of establishing connections between interpreted as a species phylogeny, enables us to make anamorphs and teleomorphs is through analysis of genotypic inferences about the phylogenetic position of G. penicillioides, Molecular systematics of Gliocladium 626 Figs 1-3. For captions see facing page. S. A. Rehner and G. J. Samuels 62 7 Table 1. Source of isolates Taxona Strain Source GenBank accession no. 1 Hypocrea cf. gelatinosa GJS 89-114 Maryland, U.S.A. 2 Hypocrea gelatinosa GJS 88-80 Yunnan Prov., China 3 Trichoderma virens GL18 California, U.S.A. 4 Trichoderma virens GL2O Maryland, U.S.A. 5 Hypocrea lutea GJS 89-129 North Carolina, U.S.A. 6 Hypocrea pallida GJS 89-105 French Guiana 7 Hypocrea pallida GJS 89-83 New Hampshire, U.S.A. 8 Hypomyces berkeleyanw GJS 82-274 New Zealand 9 Sphaerostilbella sp. GJS 82-40 New Zealand 10 Sphaerostilbella aureonit~!ns GJS 83-286 New Zealand 11 Sphaerostilbella lutea GJS 83-277 Gabon 12 Hypomyces aurantiw GJS 74-86 New Zealand 13 Hypomyces awtralis CTR 76-163 Venezuela 14 Hypomyces rosellus CTR 78-121 New York, U.S.A. 15 Roumegueriella rufula GJS 91-169 CBS 346-85 16 Nectriopsis sporangiicola CTR 67-135 New Jersey, U.S.A. 17 Nectria ochroleuca GJS 90.167 Venezuela 18 Nectria ochroleuca GJS 90-179 Venezuela 19 Gliocladium roseum Ns-30 New York, USA 20 Nectria ochroleuca GJS 90-227 Venezuela 21 Nectria aureofulva GJS 91-52 Brazil 22 Nectria aureofulva CTR 71-328 Venezuela 23 Mycoarachis inversa ATCC 22107 Uganda 24 Nectria haematococcac NRRL 13425 N A 25 Nectria albosuccineaC NRRL 20459 Venezuela 26 Gibberella fujikuroc NRRL 13563 North Carolina, U.S.A. 27 Nectria purtoniic NRRL 13625 N A 28 Nectria cinnabarina GJS 89-107 New York, U.S.A. 29 Nectria cinnabarina GJS 89-108 New York, U.S.A. " Strains listed as teleomorphs were isolated from single ascospores, anamorph strains were obtained from conidial soil isolates. Collectors/culture collections: ATCC, American Type Culture Collection; GJS. Gary Samuels; CTR, Clark Rogerson; GL, Robert Lumsden; NS, Systematic Mycology Laboratory, U.S. Dept. Agriculture. Beltsville, MD; NRRL, ARS Culture Collection, National Center for Agricultural Utilization Research, U.S. Dept. Agriculture, Peoria, IL. " Sequences for these isolates were obtained from Keny O'Domell, NCAUR, ARS, USDA, Peoria, IL. NA, locality information not available. G. roseum, and G. virens within the Hypocreales, which and blotted dry, then frozen at -80° and lyophilized. The supports the hypothesis that Gliocladium is polyphyletic. lyophilized mycelia were ground in a 1.5 ml microcentrifuge tube with a sterile applicator stick under liquid nitrogen, then suspended in 500 y1 lysis buffer (200 mM Tris/HCl (pH 8.3), MATERIALS AND METHODS 250 mM NaC1, 50 mM Na,EDTA, 1% SDS) and extracted Strains and culture methods successively with equal volumes of pheno1:chloroform and chloroform. DNA was precipitated with 0-54 volumes of Strains used in this study were obtained either from single isopropanol and pelleted by spinning 10-30 s at 12000 g in ascospores or conidia and are listed in Table I. Cultures, which a microcentrifuge. The supernatant was discarded and the are maintained at BPI, were stored on Difco cornmeal agar tubes were drained of residual supernatant by inverting on a slants at 4OC. Mycelia for DNA extractions were grown for paper towel. The pellets were resuspended in 100 yl TE 2-5 d in 15 ml liquid CYM (Raper & Raper, 1972) in darkness (10 mM Tris/HCl (pH 8-O), 1 mM Na,EDTA) containing at room temperature. 100 mM NaCl and incubated at 55' for 1 h. RNAase A was added to the samples to 50 yg ml-' and incubated at 37' for 30 min. Samples were extracted with an equal volume of DNA extracfion chloroform, adjusted to 2.5 M ammonium acetate and DNA was extracted by a method modified from Raeder & precipitated by the addition of 2.5 volumes of 95 % ethanol Broda (1985). Cultured mycelia were transferred to filter paper and pelleted by spinning for 30 s in a microcentrifuge. The Figs 1-3. Fig. 1. Gliocladium virens with chlamydospores, conidiophores from Trichoderma-like aggregates (left) and from aerial hyphae (right),and conidia. Fig. 2. Nectria ochroleuca (anam. = Gliocladium roseum) showing conidia in imbricate chains from a penidllately branched conidiophore (left), and two verticillately branched conidiophores (right), and conidia. Fig. 3. Sphaerostilbella aureonitens (anam. Gliocladium penicillioides) showing two conidiophores and conidia. Fig. I drawn from the 'type' isolate (ATCC 13213), Fig. 2 drawn from GJS 86-236, Fig. 3 drawn from GJS 83-286. Scale bars: 10 1Am. Molecular systematics of Gliocladium 628 LROR LRlR P2 cation the mineral oil overlay was removed by a single 2-