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Institute of Microbiology, University of Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria
Christian Sturmbauer
Department of Zoology, Karl-Franzens-University of Graz, Universitätsplatz 2, A-8010 Graz, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The evolutionary history of the genus Omphalotus was inferred from DNA sequences of the ITS1-5.8S-ITS2 rDNA region. We analyzed 32 collections from different geographical areas: O. olearius (Europe), O. illudens (Europe, North America), O. subilludens (North America), O. olivascens var. olivascens (North America) and var. indigo (Mexico), O. mexicanus (Middle America), O. nidiformis (Australia), and O. japonicus (Japan). Phylogenetic analysis was performed declaring Nothopanus eugrammus as outgroup. Our analyses show that the genus Omphalotus is split into two major clades, the first consisting of O. illudens and O. mexicanus and the second comprising O. olearius, O. olivascens, O. japonicus, O. nidiformis and O. subilludens. Moreover, the often discussed synonymy of O. illudens and O. olearius is rejected. Omphalotus japonicus, a species formerly placed in the genus Lampteromyces Sing. for morphological reasons, clustered as the sister group of O. olearius.
Key words: Basidiomycetes, Lampteromyces, O. illudens, O. mexicanus, O. nidiformis, O. japonicus, O. olearius, O. olivascens, O. subilludens, taxonomy, ITS1-5.8S-ITS2 rDNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) because of the "occurrence of the variegatic acid type or derivates (or otherwise related to pigments commonly found in boletes)." Because certain "boletales pigments" were found in Omphalotus and Lampteromyces they were included in the Paxillaceae by Bresinsky and Besl (1979). Focusing on physiological characters, the two genera revealed relevant differences from other members of the Paxillaceae, such as Paxillus spp. or Hygrophoropsis spp. Omphalotus and Lampteromyces form a socalled "white softrot" (catalysis of lignin) in contrast to other "brown rot"causing Paxillaceae (catabolism of cellulose). An additional difference is the occurrence of sesquiterpenes of the illudine type in Omphalotus and Lampteromyces, which lead to the establishment of the new family Omphalotaceae Bresinsky (Kämmerer et al 1985). The molecular approach of Thorn et al (2000) revealed that Omphalotus, Lampteromyces and Nothopanus Sing. form a monophyletic group within the family Marasmiaceae (Agaricales), suggesting a close relationship of the genera Omphalotus and Lampteromyces. Moncalvo et al (2002) more recently reported that these genera are a monophyletic group in the clade Omphalotacae, including several genera that traditionally were classified in various families of Agaricales.
Four Omphalotus species have been described in North and Central America: O. illudens (Schwein.) Bresinsky and Besl, O. subilludens (Murr.) Bigelow, O. olivascens Bigelow, Miller and Thiers and O. mexicanus Guzmán and Mora. Omphalotus nidiformis (Berk.) Miller has been described from Australia. In Europe, the occurrence of two species has been reported: O. olearius (DC. : Fries) Singer from southern Europe and O. illudens from northern Europe (Kuyper 1995, Kirchmair and Pöder 2002). Mating studies by Petersen and Hughes (1998) showed that O. nidiformis and O. illudens were isolated reproductively from all other Omphalotus species, while O. subilludens, O. olearius and O. olivascens were intercompatible. Omphalotus mexicanus was not considered in their studies. Additional studies on restriction sites in the ribosomal ITS1-5, 8S-ITS2 region by Hughes and Petersen (1998) led to similar results: The restriction site patterns of O. subilludens and O. olearius were identical. O. olivascens differed by only one restriction site, whereas O. nidiformis, O. illudens and O. mexicanus were less similar.
This study aims to clarify the phylogenetic relationships within the genus Omphalotus and to evaluate the systematic significance of morphological, chemotaxonomical and ecological data that were employed so far for the characterization of species. Representative collections of almost all known species from around the world were examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Mycelia were grown on malt-extract agar plates (20 g malt extract, 2 g peptone from soy meal, 17 g agar-agar, 1 L aqua dest.) for at least 3 wk at 20 C.
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10 mg) was added. The samples were ground with a sterile plastic pestle in a 1.5 mL Eppendorf tube. Volume was adjusted by adding 200 µL CTAB buffer. After vortexing the samples were incubated at 65 C for 10 min. One volume (500 µL) chloroform was added to each sample and mixed gently by inverting the tubes. The samples were centrifuged at 15 700 x g for 5 min. DNA was precipitated from the aqueous supernatant by adding 800 µL 96% ethanol (–20 C overnight) and pelleted by centrifugation at 15 700 x g for 5 min. After washing the DNA-pellet with ice-cold 70% ethanol and drying at 37 C for 15 min the pellet was redissolved in TE buffer (10 mM Tris,10 mM EDTA, pH 8)+ 4.5 U RNase/mL (Gerrits van den Ende and de Hoog 1999).
PCR amplification. –
Primers used for PCR amplification and for sequencing of the internal transcribed spacer region were ITS1 and ITS4 (White et al 1990). Amplifications were performed in 0.2 mM of each dNTP, 1 mM of each primer, 10% of dilution buffer and sterile double-distilled water. BiothermTMTaq DNA polymerase (genXpress, Vienna, Austria) was added at 2.5 u/100 µL of reaction mix; 5 µL of genomic DNA template was used in each 50 µL reaction. Amplifications were carried out in a Techne Unit Progenethermocycler in 200 µL reaction tubes (94 C, 1 min; 50 C, 1 min; 72 C, 2 min; 35 cycles). PCR products (8 µL aliquots) were checked by electrophoresis in 1.5% aga-rose gels with 0.003% ethidiumbromide in 0.5 x TBE buffer (0.045 M Tris, 1.1 mM EDTA, 0.044 M boric acid, pH 8).
DNA sequencing. –
PCR products were purified using NU-CLEOTRAP
CR PCR purification kit (Machery-Nagel, Germany). Sequencing was performed using ABI BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, USA). The sequence products were analyzed with an automated 373A DNA Stretch sequencer (Applied Biosystems). Analysis was performed using ABI Prism Sequencing Analyses Software (Version 3.0, Perkin Elmer). The sequences obtained were deposited in the National Center for Biotechnology Information (NCBI) GenBank (TABLE I
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Data analyses. –
Alignment initially was carried out by using the computer program Clustal W 1.81 and subsequently increased by eye. Nothopanus eugrammus was chosen as out-group based on previous studies of pleurotoid-lentinoid fungi (Thorn et al 2000). Their analyses of nuclear 25S rDNA sequences revealed that N. eugrammus is a sister group of Omphalotus. Phylogenetic analyses were performed with PAUP* 4.0b8 (Swofford 1998). The search options MULPARS on, steepest descent not in effect, Max-Trees 10 000, and gaps treated as fifth character state were used for maximum parsimony (MP). Transversion mutations were weighted 2:1 over transition mutations using a step matrix. The most parsimonious trees were searched with tree-bisection addition (TBR) branch swapping. Starting trees were obtained by random addition sequence. One hundred heuristic searches were performed and the shortest trees over all replicates were kept and assumed to be the most parsimonious reconstructions, to increase the chance of finding the best tree(s). In neighbor joining (NJ) analysis, distances between the taxa were measured with Kimura’s two-parameter correction (Kimura 1980). Gaps were treated as missing data, ties were broken systematically. Negative branch lengths were allowed but set to zero for tree score calculation. Maximum likelihood (ML) analysis was performed using parameters derived from Modeltest 3.04 (Posada and Crandall 1998). Bootstrap support for branches in MP and NJ searches were estimated with 1000, for ML with 500 pseudoreplicates using the search modus full heuristic (Felsenstein 1985).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. The tree is divided into two major clades. The first—"O. olearius clade"—with a bootstrap support of 98% included O. olearius, O. japonicus, O. olivascens, O. nidiformis and O. subilludens. The latter forms the most ancestral branch of this group, as the sister group of O. olivascens, O. olearius, O. nidiformis and O. japonicus, supported by a bootstrap value of 97%. Within this subclade not all phylogenetic relationships were strongly supported: Branches of O. nidiformis and O. japonicus were resolved with strong bootstrap supports of 100% and 93%, while O. olivascens var. olivascens and O. olivascens var. indigo were separated with a bootstrap support of 67%. The latter two taxa were related to O. nidiformis with a weak bootstrap support of 56%. All analyzed collections of O. olearius formed a monophyletic group (bootstrap support 73%). The genetic distances within the major groupings were small, especially within O. olearius, in which many sequences differed by one mutation only. The second clade—"O. illudens"—with a bootstrap support of 100% consisted of O. illudens and O. mexicanus; both species were clearly separated (bootstrap support 96%).
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) as found by the MP analysis, but "O. olearius clade" was not supported as strongly (bootstrap 62%). Internal branches leading to O. nidiformis, O. subilludens, O. olivascens and O. japonicus were supported by bootstrap values of 57–65%, while relationships on the species level were strongly supported (bootstrap values 86–100%. "O. illudens clade" consisting of O. illudens and O. mexicanus collections was supported by a bootstrap value of 100%. Both species were separated with a bootstrap support of 99%.
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) with rate heterogeneity, transition/transversion ratio = 1.5088. The model accounted for among-site rate variation with a gamma-shape parameter estimation of 0.3446 and four rate categories represented by mean. The observed nucleotide frequencies were A = 0.2266, C = 0.2110, G = 0.2363, T = 0.3261. Number of substitution types = 2. The overall topology of the ML tree (—ln likelihood = 2118.2827) (FIG. 3) corresponded well with the four trees of the MP analysis: the ML analysis resulted in the same groupings of collections.
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The "O. illudens clade" with O. illudens and O. mexicanus was supported by 54%, separating the two taxa by a bootstrap value of 79%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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: O. olearius and O. subilludens, which could not be separated by them, were close to O. olivascens. In a relatively distant second clade, O. nidiformis and O. illudens were separated by one restriction site. Omphalotus mexicanus seemed to be relatively isolated from all other taxa. The sequence analyses presented here confirm the close relationships of O. olearius, O. olivascens and O. subilludens. Omphalotus nidiformis seems not as isolated as suggested by RFLP data. In contrast to their findings, O. mexicanus forms one clade with O. illudens. Different results of RFLP and sequence analyses of the same DNA region might be explained by a different quality and quantity of characters used: 15 restriction sites were found by Hughes and Petersen (1998); eight of them were variable and, therefore, useful for the calculation of a neighbor joining tree. In this study, a sequence dataset of 632 characters, 140 of which were parsimony informative, was analyzed.
Mating experiments by Petersen and Hughes (1998) revealed high intercompatibility between O. olearius, O. olivascens and O. subilludens. In the case of O. illudens and O. nidiformis they reported a little intercompatibility with every other species. Omphalotus japonicus (Lampteromyces japonicus) and O. mexicanus were not considered in their mating experiments. Morphological and chemotaxonomical data (Kirchmair et al 2002), mating intercompatibility (Petersen and Hughes 1998), RFLP data (Hughes and Petersen 1998) as well as the present results of sequence analyses indicate a close relationship of the three taxa O. olearius, O. olivascens and O. subilludens. This might encourage a hypothesis on the conspecifity of these taxa. But in this case, O. olearius becomes paraphyletic because O. japonicus and O. nidiformis are excluded (FIGS. 1–3). The latter two, however, must be considered as distinct species; prominent phenotypic characters distinguish these two taxa from all others: (i) Their basidiomatal context is unpigmented (white) in contrast to the distinctly colored context of all other Omphaloti (yellow-orange or bluish to black); (ii) O. japonicus, known solely from Japan, is the only species with an annulate zone at the stipe apex and, in relation to all others, its spores are gigantic. Moreover, the mating compatibility of O. nidiformis (it is restricted to Australia) with all other Omphalotus species is weak (Peterson and Hughes 1998). Data on the mating behavior of O. japonicus still are lacking.
Miller (1994) discussed interspecific and infraspecific taxonomical problems concerning O. nidiformis from which two color variants are known: a dark form with a deep brownish black pileus center, margin brown to orange-brown; and a light form with nearly white basidiomes. The latter exhibits a much more prominent luminescence (Miller 1994). He also reported strong intercompatibility between these two phenotypes. In agreement with the aforementioned study, no separation of the two color variants could be observed in the present analysis: The strains VT 1490 ("dark form") and VT 1946 ("light form") used by Miller (1994) have identical ITS1-5.8S-ITS2 rDNA sequences.
Mycologists have been at odds concerning the interpretation of O. illudens: Some authors treat O. illudens and O. olearius as conspecific (Pegler 1977, Watling and Gregory 1989), others separate the two taxa (Kuyper 1995, Kirchmair et al 2002). In addition, the interpretations of O. illudens among taxonomists who favor the existence of separate species are not uniform; Kirchmair et al (2002) said that O. illudens can be distinguished from O. olearius by smaller spores and an umbonate pileus. Lighter forms of O. olearius from southern Europe were misinterpreted as O. illudens or led to constructions such as O. olearius var. illudens (Schwein.) A. Ortega and Esteve-Rav. (Ortega et al 2000). According to Petersen and Hughes (1998), the mating intercompatibility between O. olearius and O. illudens is weak: RFLP analyses (Hughes and Petersen 1998) separated the two taxa as well. Our most recent study (Kirchmair and Pöder 2002) substantiated the separation of these taxa by a comprehensive revision of all available taxonomically relevant data including rDNA-sequence data.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: Martin.Kirchmair{at}uibk.ac.at
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