Mycologia, 95(6), 2003, pp. 1204±1214. q 2003 by The Mycological Society of America, Lawrence, KS 66044-8897 Traditional infrageneric classi®cation of Gymnopilus is not supported by ribosomal DNA sequence data Laura GuzmaÂn-DaÂvalos1 taining more than 200 lignicolous species. Gymnopi- Departamento de BotaÂnica y ZoologõÂa, Universidad de lus has been treated as a member of the family Cor- Guadalajara, Apartado postal 1-139, Zapopan, tinariaceae sensu Singer (1986) or Strophariaceae Jalisco, 45101, MeÂxico sensu KuÈhner (1980) in the Agaricales. The genus is Gregory M. Mueller well characterized macromorphologically (Hesler Department of Botany, The Field Museum of Natural 1969, Singer 1986). Hesler (1969) monographed the History, 1400 S. Lake Shore Drive, Chicago, Illinois North American species of the genus. Gymnopilus 60605-2496 also has been studied in MeÂxico by GuzmaÂn-DaÂvalos JoaquõÂn Cifuentes and GuzmaÂn (1986, 1991, 1995) and GuzmaÂn-DaÂva- los (1994, 1995, 1996a), in Europe by Hùiland Facultad de Ciencias, UNAM, Circuito Exterior, Ciudad Universitaria, MeÂxico, D.F., 04510, MeÂxico (1990), Orton (1993) and Bon and Roux (2002), in Central America by GuzmaÂn-DaÂvalos (1996b) and Andrew N. Miller GuzmaÂn-DaÂvalos and Ovrebo (2001), in Zimbabwe by Department of Botany, The Field Museum of Natural Hùiland (1998), and in Australia by Rees and Ye History, 1400 S. Lake Shore Drive, Chicago, Illinois (1999) and Rees et al (1999, 2002). 60605-2496 Gymnopilus, as genus Fulvidula Romagn., ®rst was Anne Santerre divided into two groups, Annulatae Romagn. and Cor- Departamento de BiologõÂa Celular y Molecular, tinatae Romagn., by Romagnesi (1942). Annulatae Universidad de Guadalajara, Zapopan, Jalisco, contained species with a persistent, membranous an- MeÂxico nulus or ``cortina abundantly developed so as to form a distinct annular zone'' (Singer 1986). Cortinatae contained species with an arachnoid veil (cortina) or Abstract: The traditional classi®cation of Gymnopi- no veil. Singer (1986) accepted the two sections of lus (Agaricales) recognizes two primary groups, An- Romagnesi, naming the latter one Gymnopilus. Hes- nulati and Gymnopilus, based on the presence or ab- ler (1969) also accepted these groups but under the sence of a membranous partial veil. While our anal- taxonomic rank of subgenera. He divided subgenus yses of DNA sequence data from the nuclear ribosom- Gymnopilus into two sections: Microspori Hesler and al ITS1±5.8S-ITS2 (ITS) gene supports the Gymnopilus. Later, GuzmaÂn-DaÂvalos (1995) proposed monophyly of the genus, these traditional subgroups another section in subgenus Gymnopilus, Macrospori were not recovered. Five well-supported clades within Guzm.-DaÂv. to accommodate large-spored species the genus were identi®ed through these analyses: 1) lacking an annulus. the spectabilis-imperialis group; 2) nevadensis-pene- trans group; 3) a clade formed by G. underwoodii, G. Most authors have accepted the classi®cation of the validipes and G. cf. ¯avidellus; 4) aeruginosus-luteo- genus into two groups (subgenera or sections). How- folius group; and 5) lepidotus-subearlei group. Re- ever, it often is dif®cult to assign some species to one lationships among these subgroups were not re- or the other group because the annulus or membra- solved. nous veil is sometimes ephemeral or very easily lost Key words: Agaricales, Annulati, Cortinariaceae, if the specimen is not handled with care. Also, either ITS, phylogeny, Strophariaceae an annulus or a cortinate veil has been observed on the stipe in different specimens from the same spe- cies. Few molecular studies have included species of INTRODUCTION Gymnopilus, and only one has focused exclusively on The genus Gymnopilus P. Karst. represents an impor- the genus. Gymnopilus sapineus (Fr.) Maire was used tant component of fungal biodiversity on wood con- as outgroup in a phylogenetic study of Cortinarius using ITS sequences (Hùiland and Holst-Jensen Accepted for publication May 8, 2003. 2000). Moser et al (2001) used ITS sequences to es- 1 Corresponding author. E-mail: [email protected] tablish the phylogenetic relationships of a new spe- 1204 GUZMAÂ N-DAÂ VALOS ET AL:PHYLOGENY OF GYMNOPILUS WITH DNA DATA 1205 cies of Gymnopilus, G. tur®cola M. M. Moser & H. are listed in TABLE I. Herbarium acronyms are cited as from Ladurner, including in their analysis G. decipiens (W. Holmgren et al (1990). G. Smith) P. D. Orton, G. luteofolius (Peck) Singer, DNA extraction. DNA was extracted at the Departamento G. odini (Fr.) KuÈhner & Romagn., G. penetrans de BiologõÂa Celular y Molecular, Universidad de Guadala- (Fr. : Fr.) Murrill, G. picreus (Pers. : Fr.) P. Karst., G. jara, MeÂxico, using one of these procedures: phenol meth- sapineus and G. spectabilis (Fr.) A. H. Sm. Peintner et od of Raeder and Broda (1985), CTAB method of Gardes al (2001) used Gymnopilus (G. penetrans, G. sapineus and Bruns (1993) or employing an extraction kit (Nucleon and G. spectabilis) as outgroup in a paper on the mul- PhytoPure, Amersham). DNA extraction tests were made to tiple origins of sequestrate fungi related to Cortinar- determine the best extraction protocol for obtaining DNA ius. Moncalvo et al (2002), in their molecular study from the minimum amount of material (Santerre et al in on the relationships among euagarics, found a gym- preparation). A small part (ca 4 mg) of the pileus, includ- nopiloid clade that included Gymnopilus and Galeri- ing cutis, context and lamellae, was taken. When possible na paludosa (Fr.) KuÈhner. A gymnopilus clade rep- (e.g., in the case of large basidioma) the cutis was excluded resented by G. aeruginosus (Peck) Singer, G. specta- to avoid contamination from exotic spores or material de- posited on the surface of the pileus. In very few cases, when bilis, G. junonius (Fr.) P. D. Orton, G. penetrans and the pileus was in poor condition, the sample was taken from Hebelomina neerlandica Huijsman was nested within the stipe. DNA concentration was determined by spectro- this larger clade. Thomas et al (2002) also included photometry. The DNA extracts were diluted 1:2, 1:5 and 1: Gymnopilus (G. aeruginosus, G. penetrans, G. picreus, 10 or were used undiluted in PCR reactions. G. sapineus and G. spectabilis) to establish the rela- tionships of a new genus, Anamika K. A. Thomas, PCR ampli®cation. Polymerase chain reaction (PCR) was Peintner, M. M. Moser & Manim., which is closely performed to amplify the internal transcribed spacer 1 related to Hebeloma. The only work focused exclu- (ITS1), the 5.8S rRNA gene and the internal transcribed sively on Gymnopilus is by Rees et al (2002), in which spacer 2 (ITS2) following the protocols of Miadlikowska and Lutzoni (2000), with some modi®cations. Each 50 mL they report on the relationships among Australian PCR reaction contained 32.7 mL of sterile double-distilled and Northern Hemisphere Gymnopilus species using water, 5 mLof103 reaction buffer (100 mm Tris, 500 mm sequences of the ITS. Hùiland (1990) undertook the KCl) with MgCl2 (Behringer-Mannheim), 5 mLof8mM only cladistic analysis based on morphological data dNTPs, 2 units of Taq DNA polymerase (Behringer-Mann- using the known species of Gymnopilus from Norway. heim or Roche), 2.5 mL of each 10 mM primer, 1 mL of BSA In this paper we use sequence data from the nu- (bovine serum albumine) (New England Bio Labs), and 1 clear ribosomal ITS1±5.8S-ITS2 (hereafter referred mL of DNA template. Ready-To-Goy PCR Beads (Amer- to as ITS) to test the monophyly of the genus and sham Pharmacia Biotech) also were used, following the whether the traditional classi®cation of the genus manufacturers' protocols to amplify some taxa. Negative into two groups based on the presence or absence of controls, without DNA template, were included to detect an annulus is natural. contamination in the reagents. Primer pairs ITS1F-ITS4, ITS1-ITS4, ITS1-ITS4S or ITS5-ITS4 were used to amplify the entire ITS; ITS1F-ITS2, ITS1-ITS2 or ITS5-ITS5.8S to MATERIAL AND METHODS amplify the ITS1; and ITS3-ITS4, ITS3-ITS4S or ITS5.8SR- ITS4S to amplify the ITS2 (Vilgalys and Hester 1990, White The species included in this study encompass the morpho- et al 1990, Gardes and Bruns 1993, Kretzer et al 1996). Two logical variation of the genus, with special focus on the sub- primers speci®c for Gymnopilus were designed, ITS1G (59- genus Annulati. Specimens were selected to provide broad CGTAACAAGGTTTCCGTAGG-39) and ITS4G (59-GA- taxonomic sampling based on the availability of well-pre- TATGCTTAAGTTCAGCGGG-39), and the primer pairs served material. Some species are known only from the type used were ITS1G-ITS4G, ITS1G-ITS2 or ITS3-ITS4G. specimens, so in these cases permits were obtained from PCR ampli®cations were performed in a MJ Research curators to use small portions of these specimens for DNA PTC 200 thermocycler. The DNA was denatured at 95 C for extraction. The familial concepts of both Singer (1986) and 3 min, except for the PCR Beads (93 C for 7 min). Twenty- of KuÈhner (1980) were employed because representatives ®ve cycles of denaturation at 95 C for 1 min, annealing at from the families Cortinariaceae (Cortinarius, Dermocybe, 50 C for 45 s, and extension at 72 C for 2 min were followed Galerina, Pyrrhoglossum) and Strophariaceae (Pholiota, Psil- by 15 cycles of 95 C for 1 min, 50 C for 45 s and 72 C for ocybe) were included as outgroups. Most of the material 2 min increasing 5 s each cycle with an extension step of used in this study was from old (more than 10 yr) or very 72 C for 10 min and ®nal incubation at 4 C. Ampli®cation old (more than 50 yr) herbarium specimens because few products were visualized by electrophoresis in 1.5% TAE recently collected specimens (i.e., 1 year old or less) were agarose gels (NuSieve, FMC Bioproducts), containing ethi- available.