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Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 16802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A phylogenetic analysis was performed on 51 isolates of the commercially valuable basidiomycete, Grifola frondosa (maitake), using sequences from the Internal Transcribed Spacers and 5.8S region of the nuclear ribosomal DNA (rDNA) and a portion of the ß-tubulin gene. The ß-tubulin gene provided more than twice as many variable characters as the ITS/5.8S regions. The isolates analyzed comprised 21 from eastern North America, 27 from Asia, one from Europe, and two of unknown geographic origin, one of which was the major US commercial production strain in use. Grifola sordulenta was used as an outgroup. Combined and separate analysis of both genes showed a partition separating Asian versus eastern North American isolates. Bootstrap analysis showed strong support for these clades in the ß-tubulin data alone and in the combined data. The major commercial isolate of unknown geographic origin is apparently of Asian descent based on its grouping within the Asian clade. The single European isolate analyzed was distinct from both the eastern North American and Asian clades. These results indicate strong support for a species partition separating eastern North American and Asian isolates of G. frondosa, despite previous studies indicating no morphological distinction between them.
Key words: ß-tubulin, biogeography, genetics, ITS, mushroom cultivation, nucleotide variation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), North America (Gilbertson and Ryvarden 1986
) and Europe (Breitenbach and Kräntzlin 1986
). In North America, it is found throughout regions of the east, Midwest, southeast and the Pacific Northwest. It occurs on a variety of hardwoods, particularly oak and chestnut (Breitenbach and Kräntzlin 1986
). Commonly called maitake or hen-of-the-woods, it is considered a choice edible mushroom with unique culinary and medicinal qualities. Maitake is marketed throughout Asia and, because of increased consumer demand, its commercial production has grown dramatically in Asia and the United States (Chang 1999
, Royse 1997
).
Traditional classification of G. frondosa was based solely on morphological characters. Grifola frondosa was first named Boletus frondosus by Dickson (1785)
and later, Fries (1821)
changed the name to Polyporus frondosus Dicks. : Fr. Even today, P. frondosus, the synonym of G. frondosa, is widely used. The genus Grifola S.F.Gray was first applied by Gray (1821)
and described as a polypore with large compound basidiomes. Previous taxonomic investigations by Gilbertson and Ryvarden (1986)
and Zhao and Zhang (1992)
described similar morphological characters shared between North American and Asian isolates, although these studies separately analyzed North American and mainland China isolates, respectively. Both studies recognized G. frondosa (Dicks. : Fr.) S.F.Gray as the only species in the genus Grifola. However, another Grifola species, G. sordulenta, was identified by Singer in 1969. Recent studies by Hibbett et al (2000)
based on mitochondrial and nuclear small and large subunit rRNA gene sequences showed that G. frondosa is related to other polypores including Laetiporus and Ganoderma.
Biogeographic phylogenetic structure is known to exist in various polypores and agarics, such as Pleurotus (Vilgalys and Sun 1994
), Lentinula (Hibbett et al 1998
, Thon and Royse 1999
), Panellus stypticus (Jin et al 2001
) and Flammulina (Methven et al 2000
). Genetic selection and improvement of cultivated isolates in commercial mushroom production may be facilitated by a better understanding of the biogeographic phylogenetic structure of G. frondosa germplasm. Molecular approaches have been used extensively for examining phylogenetic relationships in other edible fungi (for example see Hibbett et al 1995
, Thon and Royse 1999
, and Vilgalys and Sun 1994
).
To analyze biogeographic structure within G. frondosa, partial regions of rDNA and ß-tubulin genes were analyzed and phylogenetic relationships were inferred among isolates from North America and Asia. Results showed a strongly supported partition separating isolates from North America and Asia, suggesting that these represent separate phylogenetic species.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Cultures were grown in 50 mL of potato dextrose yeast broth (PDYB) for 20 to 30 d at room temperature. Mycelium was harvested by vacuum filtration on Whatman (grade #1) filter paper, and washed once with distilled water. One-hundred milligrams of fresh mycelium was homogenized with a micropestle in a 1.5-mL microfuge tube containing 400 µL of LETS buffer [20 mM Tris-HCL (pH 7.8), 100 mM LiCl, 10 mM EDTA, and 0.5% SDS, pH 7.8]. Five-hundred mL of a 25:24:1 (vol/vol/vol) mixture of phenol-chloroform-pentanol were added, and the tube was vortexed for 30 s and centrifuged at 14 000 x g for 10 min. The upper portion of the aqueous phase (
250 µL) was recovered, and DNA was precipitated by the addition of 0.5 mL of cold absolute ethanol with incubation at -20 C for 10 min. DNA was collected by centrifugation at 14 000 x g for 10 min, dried, and resuspended in 20 µL of sterile water. DNA preparations were diluted with sterile water and used as template for PCR amplification.
PCR amplification and sequencing –
PCR was performed in 25 µL reactions with a 96-well PCR cycler (PTC-100 Programmable Thermal Controller, MJ Research, Inc.), using 10 ng DNA template, one U of Taq DNA polymerase (Promega, Madison, Wisconsin), 0.2 mM of each dNTP, 2 mM MgCl2, 0.1% Triton, and 0.5 µM of each primer. Amplification of ITS-1, ITS-2, and 5.8 S rDNA was performed by utilizing primers ITS1 (White et al 1990
) and ALR0 (5'-CATATGCTTAAGTTCAGCGGG-3') (R. Vilgalys pers comm). PCR reactions for ITS regions were performed using the following parameters: 94°C/1 min; 35 cycles of 94 C/15 s, 60 C/30 s, 72 C/1 min; and 72 C/5 min. PCR Reactions for ß-tubulin regions were performed with primers (designed by authors) BTG5F (5'-CGTTGTGCCCAGTCCTAAGGTG-3') and BTG8R (5'-GTTCTTGCTCTGCACGTTCTG-3') (Fig. 1
) with the following parameters: 94 C/2 min; 35 cycles of 94 C/15 s, 57 C/30 s, 72 C/1 min; and 72 C/7 min. Amplification products were electrophoresed on a 1.0% agarose gel and checked to ensure that a single DNA band was produced of the expected size (600bp for ITS PCR products and 680bp for ß-tubulin PCR products). For sequencing, the PCR products were purified directly from reactions using the Wizard PCR Preps System (Promega Corp., Madison, Wisconsin) and the concentration adjusted to 20 ng/µL. Sequencing reactions were performed using an ABI dye-terminator kit (ABI/Perkin-Elmer) and analyzed using an ABI Prism® Model 377 automated sequencing system (Applied Biosystems, Foster City, California). Sequences have been deposited in GenBank (accession numbers AY049091–AY049142 for rDNA ITS sequences, AY049143–AY049194 for ß-tubulin sequences).
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) in the Lasergene package (DNAStar, Inc. Madison, Wisconsin). Multiple alignment parameters used were gap penalty = 10 and gap length penalty = 10. Final alignments then were optimized visually. Intron/exon junctions in the ß-tubulin sequences were inferred based on comparisons with the known Schizophyllum commune sequence (Russo et al 1992
). Levels of molecular sequence divergence in each of the data sets were compared by calculating pairwise estimates of nucleotide substitution rates by the two-parameter method of Kimura. Phylogenetic analyses were completed using PAUP Version 4.0b4a (Swofford 2000
). A neighbor-joining (NJ) tree was constructed using the Kimura 2-parameter model. The stability of clades was evaluated by bootstrap tests with 1000 replications (Felsenstein 1985
, Hills and Bull 1993
). A maximum parsimony (MP) analysis was performed using 1000 heuristic searches with random taxon addition searches and TBR branch swapping with MAXTREES unrestricted. Other indices for the generated topology, including tree length, consistency index (CI), and retention index (RI) were calculated. A strict consensus of the minimum length MP trees was calculated. Gaps were considered missing data for all analyses. The sequence alignment has been deposited in TreeBASE (accession number M1020). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Zhao and Zhang 1992
). These studies included descriptions of basidiomes, basidiospores, habitats and hyphal context systems. However, neither study compared North American and Asian isolates side-by-side. No mating tests between the North American and Asian isolates have been conducted to determine if they could be different biological species.
The name Grifola frondosa (Dicks.) Gray was applied to the basionym Boletus frondosus J. Dicks., which was described based on a specimen collected in Yorkshire, England (Dickson 1785
, Gray 1821
). Although only a single, non-type European isolate was analyzed in the current study, the phylogenetic data suggest the possibility that a distinct European lineage may exist in G. frondosa. If this is the case, then phylogenetic taxonomic revisions would require that the name G. frondosa be applied to the European lineage, with different names for the eastern North American and Asian clades. Taxonomic changes must await study of the type material and an expanded sampling of isolates from Europe and the British Isles. Grifola frondosa is common in Europe (Breitenbach and Kränzlin 1986
), but WC493 was the only culture available for this study. The ß-tubulin analysis and combined analysis both showed WC493 to form a distinct basal branch related to the Asian clade. However, because most of the phylogenetically informative sites came from the ß-tubulin sequence, this inferred relationship between WC493 and the Asian clade cannot be considered strong. Isolates from the Pacific Northwest of North America and the Southern Hemisphere were not sampled in this study. Further study of Grifola spp. and relatives from these areas may uncover additional biogeographic patterns.
Continental phylogeographic structure is common in basidiomycete macrofungi. Intersterility groups in the Pleurotus ostreatus species complex correlate well with continental biogeography, and also with an ITS rDNA phylogeny (Vilgalys and Sun 1994
). Phylogenetic partitions may exist that do not correlate with intersterility barriers. In Lentinula (shiitake), strong continental phylogeographic structure is evidenced based on rDNA phylogenies, including fairly distinct Asian and Eastern North American clades (Hibbett et al 1995
, Hibbett et al 1998
). Our results are consistent with the proposal that a biogeographic connection exists between basidiomycete macrofungi from eastern Asia and temperate North America (Wu and Mueller 1997
). In the genus Suillus, this observation is supported by the inference that North American species tend to have closely related Asian sister taxa, based on ITS rDNA data (Wu et al 2000
). The split-gill fungus Schizophyllum commune shows worldwide interfertility and a limited degree of phylogeographic structure in its cosmopolitan range (Raper et al 1958
, James et al 1999
, James et al 2001
).
Multilocus phylogenetics provides a powerful tool for the recognition of species, as phylogenetic partitions shared among different loci indicate a historical reproductive barrier between clades (= genealogical concordance phylogenetic species recognition or GCPSR, Taylor et al 2000
). The shared partition between Asian and Eastern North American isolates indicated by both genes suggests that there is a reproductive barrier between these groups, meeting the criteria of GCPSR. However, we cannot say whether the reproductive barrier is intrinsic (i.e., reflecting intersterility) or extrinsic (i.e., reflecting geographic separation). Because phylogenetic partitioning can precede the evolution of intersterility, it cannot be assumed that Asian and Eastern North American isolates of G. frondosa are intersterile. Indeed, strong geographic and phylogenetic partitions, albeit inferred from single genes, can be observed within intersterility groups in the genus Pleurotus (Isikhuemhen et al 2000
, R. Vilgalys, pers comm), and despite worldwide interfertility (Raper et al 1958
), some degree of continental biogeographic structure can be found in the split-gill fungus Schizophyllum commune (James et al 1999
, James et al 2001
). Recombination may occur among isolates within the two geographic lineages of G. frondosa, but evidence for that cannot be extrapolated from these data. Neither the ß-tubulin nor the ITS rDNA trees show much strongly supported structure within lineages, and the partition homogeneity test (PHT) or incongruence length difference (ILD) test cannot be applied to these datasets to test for historical recombination because of relatively high levels of homoplasy (Farris et al 1994
, Koufopanou et al 1997
, Barker and Lutzoni 2000
).
Both neighbor joining (NJ) and most parsimonious (MP) trees derived from all DNA datasets revealed a consistent grouping of U.S. commercial cultivar WC828 in the Asian clade. This suggested that WC828 has an Asian origin and is closely related to Asian commercial cultivars. It is known that molecular data can be effectively used to select unique shiitake genotypes for evaluation of biological efficiency, quality and average weight of mushrooms (Diehle and Royse 1986
, Levanon et al 1993
). So a better understanding of the phylogenetic relationships of Grifola frondosa may help the selection and breeding of commercial lines and help to improve commercial cultivation of these mushrooms.
The ß-tubulin gene region provided more than twice as many phylogenetically informative nucleotide sites as did the ITS rDNA region, in a similar-sized amplicon. This result is consistent with findings in other fungi (e.g., O'Donnell et al 2000
) and shows that ß-tubulin may be a more powerful tool for analyzing the intraspecific phylogeography of basidiomycete macrofungi than ITS rDNA. Other partial protein-coding genes such as translation elongation factor 1-
may also be extremely useful.
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FOOTNOTES |
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Accepted for publication October 1, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Breitenbach J, Kräntzlin F., 1986 Fungi of Switzerland, Vol. 2. Non-gilled fungi Lucerne: Verlag Mykologia. 412 p
Chang ST., 1999 World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk.) Sing. in China Int J Med Mushrooms 1:291-300
Chen X, Romaine CP, Ospina-Giraldo MD, Royse DJ., 1999 A polymerase chain reaction-based test for the identification of Trichoderma harzianum biotypes 2 and 4, responsible for the worldwide green mold epidemic in cultivated Agaricus bisporus Appl Microbio Biotech 52:246-250
Dickson J., 1785 Fasciculus Plantarum Crytogamicarum Britanniae Londini: Nicol
Diehle DA, Royse DJ., 1986 Shiitake cultivation on sawdust: evaluation of selected genotypes for biological efficiency and mushroom size Mycologia 78:929-933
Farris JS, Kallersjo M, Kluge AG, Bult C., 1994 Testing significance of congruence Cladistics 10:315-319
Felsenstein J., 1985 Confidence limits on phylogenies: an approach using the bootstrap Evolution 39:783-791
Fries EM., 1821 Systema Mycologicum: Sistens Fungorum Ordines, Genera et Species, Huc U.S.que Cognitas, Quas ad Normam Methodi Naturalis Determinavit, Disposuit Atque Descripsit Lundae: ex officina Berlingiana. 355 p
Gilbertson RL, Ryvarden L., 1986 North American Polypores Norway: Fungiflora, Oslo. p 332–333
Gray SF., 1821 A natural arrangement of British plants, according to their relations to each other as pointed out by Jussieu, De Candolle, Brown, C including those cultivated for use, with an introduction to botany, in which the terms newly introduced are explained London: Baldwin, Cradock and Joy. 643 p
Hibbett DS, Gilbert LB, Donoghue MJ., 2000 Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes Nature 407:506-508[Medline]
Hibbett DS, Hansen K, Donoghue MJ., 1998 Phylogeny and biogeography of Lentinula inferred from an expanded rDNA dataset Mycol Res 102:1041-1049
Hibbett DS, Fukumasa-Nakai Y, Tsuneda A, Donoghue MJ., 1995 Phylogenetic diversity in shiitake inferred from nuclear ribosomal DNA sequences Mycologia 87:618-638
Higgins DG, Bleasby AJ, Fuchs R., 1991 CLUSTAL W: improved software for multiple sequence alignment CABIOS 8:189-191
Hills DM, Bull JJ., 1993 An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis Syst Biol 42:182-192
Isikhuemhen OS, Moncalvo JM, Nerud F, Vilgalys R., 2000 Mating compatibility and phylogeography in Pleurotus tuberregium Mycol Res 104:732-737
James TY, Moncalvo JM, Li S, Vilgalys R., 2001 Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune Genetics 157:149-161
James TY, Porter D, Hamrick JL, Vilgalys R., 1999 Evidence for limited intercontinental gene flow in the cosmopolitan mushroom Schizophyllum commune Evolution 53:1665-1677
Jin J, Hughes KW, Petersen RH., 2001 Biogeographical patterns in Panellus stypticus Mycologia 93:309-316
Koufopanou V, Burt A, Taylor JW., 1997 Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis Proc Natl Acad Sci USA 94:5478-5482
Levanon D, Rothschild N, Danai O, Masaphy S., 1993 Strain selection for cultivation of shiitake mushrooms (Lentinus edodes) on straw Bioresource Tech 45:9-12
Methven A, Hughes KW, Petersen RH., 2000 Flammulina RFLP patterns identify species and show biogeographical patterns within species Mycologia 92:1064-1070
O'Donnell K, Nirenberg HI, Aoki T, Cigelnik E., 2000 A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species Mycoscience 41:61-78
Raper JR, Krongelb JS, Baxter MG., 1958 The number and distribution of incompatibility factors in Schizophyllum commune Am Nat 92:221-232
Royse DJ., 1997 Specialty mushrooms and their cultivation Hort Rev 19:59-97
Russo P, Juuti JT, Raudaskoski M., 1992 Cloning, sequence and expression of a ß-tubulin-encoding gene in the homobasidiomycete Schizophyllum commune Gene 119:175-182[Medline]
Singer R., 1969 Mycoflora Australis Lehre: Cramer. p 382–383
Swofford DL., 2000 PAUP*. Phylogenetic analysis using parsimony (*and other methods), 4.0b4a Sunderland, Massachusetts: Sinauer Associates
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC., 2000 Phylogenetic species recognition and species concepts in fungi Fungal Gen Bio 31:21-32
Thon MR, Royse DJ., 1999 Evidence for two independent lineages of shiitake of the Americas (Lentinula boryana) based on rDNA and ß-tubulin genes sequences Mol Phylogen Evol 13:520-524[Medline]
Vilgalys R, Sun BL., 1994 Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences Proc Natl Acad Sci USA 91:4599-4603
White T, Bruns T, Lee S, Taylor J., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics In Innis et al, eds. PCR protocols: a guide to methods and applications. New York: Academic Press. p 315–322
Wu QX, Mueller GM., 1997 Biogeographic relationships between the macrofungi of temperate eastern Asia and eastern North America Can J Bot 75:2108-2116
Wu QX, Mueller GM, Lutzoni FM, Huang YQ, Guo SY., 2000 Phylogenetic and biogeographic relationships of eastern Asian and eastern North American disjunct Suillus species (Fungi) as inferred from nuclear ribosomal RNA ITS sequences Mol Phyl Evol 17:37-47[Medline]
Zhao JD, Zhang X., 1992 The polypores of china Berlin: Bibliotheca Mycologica. J. Cramer. p 209–210
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