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Departamento de Botánica y Zoología, Universidad de Guadalajara, Apartado postal 1-139, Zapopan, Jalisco, 45101, México
Gregory M. Mueller
Department of Botany, The Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois 60605-2496
Joaquín Cifuentes
Facultad de Ciencias, UNAM, Circuito Exterior, Ciudad Universitaria, México, D.F., 04510, México
Andrew N. Miller
Department of Botany, The Field Museum of Natural History, 1400 S. Lake Shore Drive, Chicago, Illinois 60605-2496
Anne Santerre
Departamento de Biología Celular y Molecular, Universidad de Guadalajara, Zapopan, Jalisco, México
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The traditional classification of Gymnopilus (Agaricales) recognizes two primary groups, Annulati and Gymnopilus, based on the presence or absence of a membranous partial veil. While our analyses of DNA sequence data from the nuclear ribosomal ITS1–5.8S-ITS2 (ITS) gene supports the monophyly of the genus, these traditional subgroups were not recovered. Five well-supported clades within the genus were identified through these analyses: 1) the spectabilis-imperialis group; 2) nevadensis-penetrans group; 3) a clade formed by G. underwoodii, G. validipes and G. cf. flavidellus; 4) aeruginosus-luteofolius group; and 5) lepidotus-subearlei group. Relationships among these subgroups were not resolved.
Key words: Agaricales, Annulati, Cortinariaceae, ITS, phylogeny, Strophariaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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or Strophariaceae sensu Kühner (1980) in the Agaricales. The genus is well characterized macromorphologically (Hesler 1969, Singer 1986). Hesler (1969) monographed the North American species of the genus. Gymnopilus also has been studied in México by Guzmán-Dávalos and Guzmán (1986, 1991, 1995) and Guzmán-Dávalos (1994, 1995, 1996a), in Europe by Høiland (1990), Orton (1993) and Bon and Roux (2002), in Central America by Guzmán-Dávalos (1996b) and Guzmán-Dávalos and Ovrebo (2001), in Zimbabwe by Høiland (1998), and in Australia by Rees and Ye (1999) and Rees et al (1999, 2002).
Gymnopilus, as genus Fulvidula Romagn., first was divided into two groups, Annulatae Romagn. and Cortinatae Romagn., by Romagnesi (1942). Annulatae contained species with a persistent, membranous annulus or "cortina abundantly developed so as to form a distinct annular zone" (Singer 1986). Cortinatae contained species with an arachnoid veil (cortina) or no veil. Singer (1986) accepted the two sections of Romagnesi, naming the latter one Gymnopilus. Hesler (1969) also accepted these groups but under the taxonomic rank of subgenera. He divided subgenus Gymnopilus into two sections: Microspori Hesler and Gymnopilus. Later, Guzmán-Dávalos (1995) proposed another section in subgenus Gymnopilus, Macrospori Guzm.-Dáv. to accommodate large-spored species lacking an annulus.
Most authors have accepted the classification of the genus into two groups (subgenera or sections). However, it often is difficult to assign some species to one or the other group because the annulus or membranous veil is sometimes ephemeral or very easily lost if the specimen is not handled with care. Also, either an annulus or a cortinate veil has been observed on the stipe in different specimens from the same species.
Few molecular studies have included species of Gymnopilus, and only one has focused exclusively on the genus. Gymnopilus sapineus (Fr.) Maire was used as outgroup in a phylogenetic study of Cortinarius using ITS sequences (Høiland and Holst-Jensen 2000). Moser et al (2001) used ITS sequences to establish the phylogenetic relationships of a new species of Gymnopilus, G. turficola M. M. Moser & H. Ladurner, including in their analysis G. decipiens (W. G. Smith) P. D. Orton, G. luteofolius (Peck) Singer, G. odini (Fr.) Kühner & Romagn., G. penetrans (Fr. : Fr.) Murrill, G. picreus (Pers. : Fr.) P. Karst., G. sapineus and G. spectabilis (Fr.) A. H. Sm. Peintner et al (2001) used Gymnopilus (G. penetrans, G. sapineus and G. spectabilis) as outgroup in a paper on the multiple origins of sequestrate fungi related to Cortinarius. Moncalvo et al (2002), in their molecular study on the relationships among euagarics, found a gymnopiloid clade that included Gymnopilus and Galerina paludosa (Fr.) Kühner. A gymnopilus clade represented by G. aeruginosus (Peck) Singer, G. spectabilis, G. junonius (Fr.) P. D. Orton, G. penetrans and Hebelomina neerlandica Huijsman was nested within this larger clade. Thomas et al (2002) also included Gymnopilus (G. aeruginosus, G. penetrans, G. picreus, G. sapineus and G. spectabilis) to establish the relationships of a new genus, Anamika K. A. Thomas, Peintner, M. M. Moser & Manim., which is closely related to Hebeloma. The only work focused exclusively on Gymnopilus is by Rees et al (2002), in which they report on the relationships among Australian and Northern Hemisphere Gymnopilus species using sequences of the ITS. Høiland (1990) undertook the only cladistic analysis based on morphological data using the known species of Gymnopilus from Norway.
In this paper we use sequence data from the nuclear ribosomal ITS1–5.8S-ITS2 (hereafter referred to as ITS) to test the monophyly of the genus and whether the traditional classification of the genus into two groups based on the presence or absence of an annulus is natural.
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MATERIAL AND METHODS |
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and of Kühner (1980) were employed because representatives from the families Cortinariaceae (Cortinarius, Dermocybe, Galerina, Pyrrhoglossum) and Strophariaceae (Pholiota, Psilocybe) were included as outgroups. Most of the material used in this study was from old (more than 10 yr) or very old (more than 50 yr) herbarium specimens because few recently collected specimens (i.e., 1 year old or less) were available. DNA extraction was not successful with many of the old and very old herbarium specimens. Only one sample (G. cf. subearlei 172) was from mycelium grown on MEA (malt-extract agar, Difco). Specimens included in this study are listed in Table I. Herbarium acronyms are cited as from Holmgren et al (1990).
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, CTAB method of Gardes and Bruns (1993) or employing an extraction kit (Nucleon PhytoPure, Amersham). DNA extraction tests were made to determine the best extraction protocol for obtaining DNA from the minimum amount of material (Santerre et al in preparation). A small part (ca 4 mg) of the pileus, including cutis, context and lamellae, was taken. When possible (e.g., in the case of large basidioma) the cutis was excluded to avoid contamination from exotic spores or material deposited on the surface of the pileus. In very few cases, when the pileus was in poor condition, the sample was taken from the stipe. DNA concentration was determined by spectrophotometry. The DNA extracts were diluted 1:2, 1:5 and 1:10 or were used undiluted in PCR reactions.
PCR amplification –
Polymerase chain reaction (PCR) was performed to amplify the internal transcribed spacer 1 (ITS1), the 5.8S rRNA gene and the internal transcribed spacer 2 (ITS2) following the protocols of Miadlikowska and Lutzoni (2000), with some modifications. Each 50 µL PCR reaction contained 32.7 µL of sterile double-distilled water, 5 µL of 10x reaction buffer (100 µm Tris, 500 µm KCl) with MgCl2 (Behringer-Mannheim), 5 µL of 8 mM dNTPs, 2 units of Taq DNA polymerase (Behringer-Mannheim or Roche), 2.5 µL of each 10 µM primer, 1 µL of BSA (bovine serum albumine) (New England Bio Labs), and 1 µL of DNA template. Ready-To-GoTM PCR Beads (Amersham Pharmacia Biotech) also were used, following the manufacturers' protocols to amplify some taxa. Negative controls, without DNA template, were included to detect 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 amplify the ITS1; and ITS3-ITS4, ITS3-ITS4S or ITS5.8SR-ITS4S to amplify the ITS2 (Vilgalys and Hester 1990, White et al 1990, Gardes and Bruns 1993, Kretzer et al 1996). Two primers specific for Gymnopilus were designed, ITS1G (5'-CGTAACAAGGTTTCCGTAGG-3') and ITS4G (5'-GATATGCTTAAGTTCAGCGGG-3'), and the primer pairs used were ITS1G-ITS4G, ITS1G-ITS2 or ITS3-ITS4G.
PCR amplifications were performed in a MJ Research PTC 200 thermocycler. The DNA was denatured at 95 C for 3 min, except for the PCR Beads (93 C for 7 min). Twenty-five cycles of denaturation at 95 C for 1 min, annealing at 50 C for 45 s, and extension at 72 C for 2 min were followed by 15 cycles of 95 C for 1 min, 50 C for 45 s and 72 C for 2 min increasing 5 s each cycle with an extension step of 72 C for 10 min and final incubation at 4 C. Amplification products were visualized by electrophoresis in 1.5% TAE agarose gels (NuSieve, FMC Bioproducts), containing ethidium bromide (1 mg/ml). When necessary, reamplifications were conducted using the above protocol except template DNA was added from either 1 µL of the original PCR product or from a melted punch from the agarose gel.
Sequencing – Amplification products were excised from 1% TALE agarose gels (NuSieve, FMC Bioproducts), melted at 70 C, and 1 µL of GELaseTM Agarose Gel-Digesting Preparation (Epicentre Technologies) was added to each product and incubated at 45 C for at least 1 h. Sequencing reactions were performed with BigDyeTM Terminator (ABI Prism, Perkin-Elmer Biosystems) in a 10 µL final volume, following manufacturers protocols and using the same primers as in the PCR reactions. Depending on the automated sequencer used, one of these protocols was used to precipitate the products: 1) 10 µL of deionized sterile water, 2 µL of 3 M NaOAc and 50 µL of 95% EtOH; or 2) 30 µL of de-ionized sterile water and 60 µL of isopropanol. Sequences were obtained either by polyacrylamide gel electrophoresis performed on an ABI 377A automated DNA sequencer (Perkin-Elmer, Applied Biosystems) or by capillary electrophoresis on an ABI-Prism 3100 Genetic Analyzer (Perkin-Elmer, Applied Biosystems).
Assembly of sequence fragments, correction of raw sequences and alignment of consensus sequences were carried out with Sequencher 3.0 (Gene Codes Corp.). Every sequence was subjected to a BLAST search in GenBank and doubtful sequences (the ones that did not blast to Gymnopilus) were removed. Fifty-three new sequences were generated and seven were retrieved from GenBank. Alignments were checked by eye and manually corrected when necessary using MacClade 4.0 (Maddison and Maddison 2000). New sequences have been submitted to GenBank and alignments are deposited in TreeBASE (matrix accession numbers M1433, M1434; study accesion number S883).
Phylogenetic analyses –
A total of 57 sequences from 38 taxa, which included six sequences from GenBank (Gymnopilus luteofolius, G. picreus, G. sapineus, G. spectabilis, Cortinarius atrovirens, Dermocybe phoenicea), were used in the analyses (Table I). Two datasets were examined: one which used the full 57 specimen matrix and another which used all ingroup sequences but restricted the outgroup to the single most closely related taxon, Galerina autumnalis, based on analyses of the first dataset. Maximum-parsimony (MP) analyses were performed on both sets, while maximum-likelihood (ML) and Bayesian methods were implemented only on the set of 51 sequences. The MP and ML analyses were performed with PAUP* 4.0b10 (Swofford 2000), while MrBayes 2.01 (Huelsenbeck and Ronquist 2001) was used to perform the Bayesian analysis.
Equally weighted and unequally weighted MP analyses were performed. In the equally weighted MP analyses, ambiguous regions due to gaps were excluded. In the unequally weighted MP analyses, changes among transitions/transversions were subjected to a specific symmetric step matrix with costs among changes calculated as the negative natural logarithm of their relative frequencies using StepMatrix 2.1 (François Lutzoni and Stefan Zoller, Department of Biology, Duke University). The ambiguously aligned regions also were included as unequivocally coded characters using the program INAASE 2.3b (Lutzoni et al 2000) to recode these regions. Each coded character was subjected to a specific symmetric step matrix derived from pairwise comparisons of sequences, accounting for the optimal number of changes between all possible combinations of any two sequences. Gaps were treated as missing characters in all MP analyses.
Heuristic searches were conducted under these conditions: starting trees obtained by stepwise addition, random addition sequence with 1000 replicates, tree-bisection-reconnection (TBR) as the branch swapping algorithm, branches collapsed if maximum branch length is zero, and MulTrees option in effect. In some analyses the option to limit branch swapping to 5 or 10 million rearrangements per replicate was implemented. This procedure was necessary due to the large number of equally most-parsimonious trees generated from the inclusion of identical sequences that could not be resolved. Support for nodes recovered from MP analyses was obtained from 1000 bootstrap replications (Felsenstein 1985). The conditions were the same as above, except that the number of random-addition replicates was set to 10 and in some cases branch swapping was limited to 1 million rearrangements per replicate.
For ML and Bayesian analyses, the most likely model of evolution was determined through nested likelihood ratio tests as implemented in Modeltest 3.06 (Posada and Crandall 1998). In both analyses, the ambiguously aligned characters were excluded and the constant characters were included. The ML analysis was performed with 100 random-addition replicates, while the Bayesian analysis was run for 10 million generations with trees sampled every 1000th generation. The first 2000 trees representing the burn-in phase of the analysis were discarded, and posterior probabilities were calculated from a consensus of the remaining 8000 trees. This analysis was repeated five times, starting from random trees to ensure the same set of trees were being sampled during each analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The equally weighted parsimony analyses, excluding the ambiguously aligned regions, were possible to run in a branch-and-bound search due to the small number of informative characters. For the rest of the parsimony analyses, the only option available due to the size of the data matrix was heuristic searches. Because the results of the first analyses with the 57 taxa dataset documented that Galerina autumnalis was the closest outgroup to Gymnopilus, subsequent analyses were undertaken with only this taxon as outgroup. Excluding the more-distant outgroups reduced the number of gaps and correspondingly, the size and number of ambiguously aligned regions, which improved the alignment and increased the number of parsimony-informative characters.
Equally weighted parsimony analysis of the 57 sequences with a branch-and-bound search resulted in a tree with low bootstrap support for many of the branches (not shown). The heuristic search with transitions/transversions unequally weighted, plus the ambiguous regions unequivocally coded, resulted in 76 840 trees, with a tree-length of 649 steps. The consistency index (CI) excluding uninformative characters was 0.72, homoplasy index (HI) = 0.28, retention index (RI) = 0.86, and rescaled consistency index (RC) = 0.62. The strict-consensus tree (Fig. 1) has the same general topology as the one that resulted from the branch-and-bound search but with higher bootstrap values. The Gymnopilus clade is present (Fig. 1), although with only 60% bootstrap support. Excluding G. picreus, the remaining Gymnopilus species form a well-supported clade, with a bootstrap of 91%.
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One of the two trees obtained in the maximum-likelihood analysis is shown in Fig. 2 (-ln L = 1622). These trees differed only in that G. spectabilis and G. junonius grouped together in one of them. The most likely model of evolution determined through nested likelihood ratio tests was HKY+G (Hasegawa et al 1985, Posada and Crandall 1998). The number of substitution types was two, the transition/transversion ratio was 3.8307, the proportion of invariable sites was zero and the gamma shape parameter was 0.1848. Node support is indicated by posterior probabilities determined through Bayesian analysis. The same major clades were recovered by ML analysis as were identified in the MP trees; however, relationships among the clades differ (Figs. 1 and 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In other cases, ITS sequences show low variation, being very similar or identical among investigated taxa (e.g., Johannesson et al 2000). Both of these conditions can result in the ITS providing few parsimony-informative characters, leading to low resolution among the investigated taxa. The number of informative characters found among the Gymnopilus taxa in this study (51/75 for 38 taxa in 57 samples, or 73/95 for 32 taxa in 51 samples, ambiguous regions excluded/unequivocally coded) is relatively low compared to the numbers reported for some groups of fungi, such as Ceratobasidium and Thanatephorus where González et al (2001) found 162 phylogenetic informative characters for 28 anastomosis groups in 122 accessions. However, the same relative number of informative characters, or fewer, that were uncovered in our analyses has been reported in studies of other genera. For example, in Antrodiella, Johannesson et al (2000) reported 34 informative characters for 12 taxa among 30 sequences, Wu et al (2000) reported 70 informative characters for 18 taxa of Suillus among 40 samples, while Moser and Peintner (2002) found 58 informative characters for 11 taxa of Cortinarius among 23 sequences.
The genus Gymnopilus –
The ITS sequence data provided sufficient resolution to consider the genus monophyletic, although there is no bootstrap or posterior probability support for including G. picreus in the genus. The limits of the genus are difficult to circumscribe based solely on morphology. The differences among some species of Gymnopilus and Galerina are obscure (Singer 1986, Horak 1989, Rees et al 1999), with the presence of styrylpyrone pigments in Gymnopilus (e.g., Hatfield and Brady 1968, Dangy-Caye and Arpin 1974, Gill and Steglich 1987, Høiland 1990) but lacking in Galerina, the only reliable feature that separates them, according to Rees et al (1999). Gymnopilus picreus always has been considered a Gymnopilus, although the color ("red-brown to almost chestnut-brown", Høiland 1990) and consistency of the basidioma (somewhat cartilaginous, especially the stipe), suggest a relationship with Galerina or Phaeocollybia. The presence of styrylpyrones in G. picreus was demonstrated by Høiland (1990), and he selected it as the lectotype of the genus. Rees et al (1999) and Bon and Roux (2002) did not accept Høiland's lectotypification and recognize G. liquiritiae (Pers. : Fr.) P. Karst. the type species. Moncalvo et al (2002) identify a gymnopiloid clade consisting of four species of Gymnopilus (G. picreus was not included in the analyses), Hebelomina and Galerina paludosa among the 117 clades of euagarics uncovered in their analysis of nuclear ribosomal large-subunit (nrLSU) data. However, Galerina marginata (Batsch) Kühner and Galerina nana (Petri) Kühner were resolved close to the panaeolideae clade, indicating that the genus Galerina is polyphyletic. Galerina marginata is very closely related to G. autumnalis (Smith and Singer 1964), which was one of our outgroup species, or is an earlier synonym of that name (Gulden et al 2001). Moser et al (2001) and Thomas et al (2002) resolved G. picreus basal to the other species of Gymnopilus yet still inside Gymnopilus, but they did not include species of Galerina in these analyses. Rees et al (2002) concluded that Gymnopilus is monophyletic, but with very low bootstrap support of 57% and a decay value of 2, when it includes Galerina eucalyptorum E. Horak and Pyrrhoglossum pyrrhum. However, we found P. pyrrhum outside the Gymnopilus clade and more closely related to Dermocybe phoenicea (Fig. 1).
Kühner (1980), placing a strong value on chemical characters, grouped Gymnopilus, Galerina, Pholiota and others in the Strophariaceae because of the presumed presence of styrylpyrones. However no styrylpyrones have been found in any of the tested species of Galerina (Rees et al 1999). Høiland and Holst-Jensen (2000) found that the ITS sequence of Gymnopilus sapineus was more similar to that of Hypholoma capnoides (Fr.) P. Kumm. (Strophariaceae) than to Cortinarius. Results from our study support those of Høiland and Holst-Jensen (2000), with Psilocybe cubensis (= Stropharia cubensis), Pholiota, Galerina and Gymnopilus forming a clade with a bootstrap value of 100% (Fig. 1) distinct from Pyrrhoglossum, Dermocybe and Cortinarius.
Infrageneric relationships – Our phylogenetic analyses of ITS sequences did not recover sufficient information to completely resolve infrageneric relationships in Gymnopilus. However, five well-supported clades were identified. Relationships among these clades varied among the trees obtained during our separate analyses. However, because there is no support for the backbone in any of these trees, these differences might not be significant.
Some genera, e.g., Amanita (Drehmel et al 1999), have many morphological characters that have been used to support infrageneric groups. Unfortunately, this is not the case for Gymnopilus, where only the presence or absence of a partial membranous veil and the size of the basidiospores have been used. Both characters have been shown to be highly homoplastic and of little value at this taxonomic level.
The two subgenera or sections considered by Hesler (1969) and Singer (1986) were not supported by our analyses. Singer (1951), discussing the split of the genus into the two groups, noted, "the veil may not in all cases be a character of primary importance." The three sections in subgenus Gymnopilus also were not supported. Section Gymnopilus was represented in this study by G. cf. flavidellus, G. luteofolius, G. nevadensis, G. penetrans (considered as synonym of G. sapineus by some authors, including Høiland [1990]), G. picreus and G. sapineus. Section Macrospori was represented by G. medius, and section Microspori by G. cf. punctifolius and G. underwoodii. The latter species was a questionable member of Microspori because its spores are relatively large (6–7 µm sensu Hesler 1969). Section Microspori was defined with spores 3.5–7 µm long (Hesler 1969). The material used in our study had spores 6.4–8 µm long (Guzmán-Dávalos 1996b). We unsuccessfully attempted to extract and sequence DNA from other species from this section. Many species from section Microspori have small basidiomata, and it seems that there is some relation between the size of the basidiomata and the success in DNA extraction in Gymnopilus, as already noticed by B. Rees (pers comm). Gymnopilus lepidotus was included in section Microspori by Hesler (1969) but was transferred to section Gymnopilus by Guzmán-Dávalos (1995) due to the size of the spores. The taxon is now considered in subgenus Annulati (Guzmán-Dávalos, unpubl data) because of the presence of a thick arachnoid veil that sometimes develops as a submembranous annulus.
Gymnopilus robustus Guzm.-Dáv. is another species that is difficult to place in either subgenus. The type specimen fits the circumscription of subgenus Gymnopilus, section Macrospori because it displays a fibrillose, evanescent partial veil and has large basidiospores (Guzmán-Dávalos 1995). Recently collected specimens, however, showed that the species could have basidiomata with a submembranous veil, placing it in subgenus Annulati (Guzmán-Dávalos and Ovrebo 2001). Our analyses did not completely resolve the affinities of this species, but it is clear that G. robustus is not part of the spectabilis-imperialis clade, as was anticipated due to its large and annulated basidioma. Gymnopilus robustus is resolved distant from the spectabilis-imperialis group in both the MP (Fig. 1) and ML trees (Fig. 2).
The groups identified in this study are described below. Although each of these groups is well supported by the MP, ML and Bayesian analyses (Figs. 1 and 2), some of them have no obvious morphological synapomorphies that clearly define them.
The aeruginosus-luteofolius clade. All species included here have the pileus covered by purplish to reddish erect squamules, at least when young. They are from temperate to tropical regions. In some of them (G. aeruginosus, G. luteofolius, G. cf. punctifolius and G. subpurpuratus), the basidioma stains green when bruised or has a greenish-blue or bluish coloration that suggests the presence of psilocybin (Gartz 1984).
The lepidotus-subearlei clade. Except for G. cerasinus, which has a fibrillose pileus and a temperate distribution, the species included in this group have erect, reddish squamules in the pileus and are restricted to tropical or subtropical environments. Gymnopilus subearlei stains green when bruised. This clade, together with the previous one, is part of a larger clade (75% bootstrap) in the parsimony trees (Fig. 1) that includes species with reddish scales that contain psilocybin (although the latter character state is lost in some species in the overall clade). These two clades also are part of a large nonsupported clade in the ML tree, but in this case the clade also includes the underwoodii-validipes clade, which contains species that are morphologically distinct (Fig. 2).
The spectabilis-imperialis clade. This group contains all the species with large, robust basidioma that have a thick, membranous annulus and fibrillose to slightly squamose pileus, except G. robustus. The complex formed by G. junonius, G. pampeanus and G. spectabilis, considered as synonyms by some authors, has species with ellipsoid spores that are longer than 8 µm. Further studies are needed to elucidate if they represent a single variable species. Singer (1986) suggested that G. suberis could be a subspecies of G. spectabilis, even though there are obvious differences between them. Gymnopilus suberis (Maire 1928) has smaller spores and erect, reddish squamules in the pileus disk of young basidiomata that never are present in G. spectabilis. In our analyses, it is clear that G. suberis is not nested within the spectabilis-imperialis clade but is related to other species with squamose pilei in the aeruginosus-luteofolius clade, such as G. subpurpuratus. Gymnopilus imperialis is the basal species of this clade. This species and G. cf. rugulosus are distinguished from the other species in the clade by having tuberculate and broadly ellipsoid to subglobose basidiospores, rather than ellipsoid and verrucose basidiospores.
The nevadensis-penetrans clade. The three species forming this complex are macromorphologically similar but they display some differences in micromorphology. For example, the pleurocystidia in G. nevadensis are larger than the cheilocystidia while in the other two species the pleurocystidia are of similar size or more often, smaller than the cheilocystidia. Gymnopilus nevadensis is rare and known only from México, while the other two species are commonly encountered and distributed worldwide. Gymnopilus penetrans and G. sapineus are very similar to each other both macro- and micromorphologically and occur in similar habitats (i.e., on branches and logs in coniferous or temperate forests). The primary differences between these two species are in the ornamentation of the pileus, being fibrillose and lacking pileocystidia in the former and squamulose with poorly differentiated pileocystidia in the latter. They are grouped together with a 100% bootstrap and posterior probability (Figs. 1 and 2). Further studies are needed to confirm if they are conspecific or separate species.
The underwoodii-validipes clade. The three species in this clade are known only from North and Central America. Gymnopilus validipes closely resembles G. spectabilis, as well as the included specimen of G. cf. flavidellus (the herbarium specimen originally was identified as G. spectabilis). Gymnopilus underwoodii is a species with medium-size basidioma and morphologically is different from the other two species in the clade. It is known only from the U.S.A. and Guatemala (Guzmán-Dávalos 1996b). This well-supported clade is basal to the core Gymnopilus in the parsimony tree (Fig. 1) but is deeply nested in the likelihood tree, resolving close to the aeruginosus-luteofolius clade (Fig. 2).
In conclusion, the genus is monophyletic, although bootstrap and posterior probability support is lacking for recognizing G. picreus in Gymnopilus. The genus, excluding G. picreus has strong support. The traditional subgenera Annulati and Gymnopilus, as well as the sections of the later, are not supported. Partial veil characters and basidiospore size are highly homoplastic characters. Five well-supported clades were recognized within the genus, but relationships among these clades and for some other taxa were not robustly resolved.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 8, 2003.
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LITERATURE CITED |
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70%) are indicated by thickened branches