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Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences

     Korean Collection for Type Cultures, Korean Research, Institute of Bioscience and Biotechnology, P.O., Box, 115, Yusong, Taejon 305-600, Korea

Hack Sung Jung ²

     School of Biological Sciences, Seoul National University, NS70, San 56-1 Shinrim-9-dong, Kwanak-ku, Seoul 151-747, Korea

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Characteristics and structures of mt SSU rDNA were investigated for the phylogenetic study of Ganoderma. Phylogenetic information was concentrated mostly in the V1, V4, V5, V6 and V9 variable domains, but informative sites in conserved domains also significantly contributed in resolving phylogenetic relationships between Ganoderma groups. Secondary structure information of variable domains was found to be a useful marker in delineation of phylogenetic groups. Strains of Ganoderma species used in this study were divided into six monophyletic groups. Ganoderma colossus made a distinct basal lineage from other Ganoderma species and Tomophagus, created for G. colosuss, appeared to be a valid genus. Ganoderma applanatum and G. lobatum classified in subgenus Elfvingia made a monophyletic group. Ganoderma tsugae from North America and G. valesiacum from Europe, both living on conifers, were closely related. Ganoderma oregonense and strains labeled G. lucidum from Europe and Canada were grouped with G. tsugae and G. valesiacum. Strains labeled G. lucidum living on hardwoods from the United States and Taiwan were grouped with G. resinaceum, G. pfeifferi and G. subamboinense var. laevisporum, and they all produced chlamydospores. Two strains labeled G. lucidum and three strains labeled G. resinaceum from America were concluded to be conspecific. Strains labeled G. lucidum from Korea and Japan were monophyletic and were distinguished from strains labeled G. lucidum from Europe and North America. Host relationships and the presence of chlamydospores in culture proved to be important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.

Key words: chlamydospores, host relationships, secondary structures, variable domains

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Some species of Ganoderma P. Karst. have been used as traditional medicines in Asia. In recent years, there has been a renewed interest in the medicinal use of these fungi. Intensive studies showed that components of Ganoderma have several medicinal effects, such as inhibition of Ras-dependent cell transformation, antifibrotic activity, immunomodulating activity and free-radical scavenging (Lee et al 1998, Lin et al 1995, Park et al 1997, van der Hem et al 1995). Species of Ganoderma also are important wood-decaying fungi, occurring on conifers and hardwoods throughout the world. They are white-rot fungi with the ability to decay lignin as well as cellulose (Adaskaveg and Gilbertson 1994). Root and stem rot caused by Ganoderma species result in worldwide losses of crops and trees (Martinez et al 1994, Miller et al 1994). Selective delignification by fungi might have diverse industrial applications, including biopulping of wood in the paper industry and degradation of pollutants such as PCB and chlorinated phenols (Adaskaveg and Gilbertson 1994, Elissetche et al 2001).

Members of the family Ganodermataceae are characterized by unique double-walled basidiospores. The shape and size of basidiospores and the texture of pileus surfaces are important characteristics that distinguish members of the Ganodermataceae. The macromorphological and micromorphological characters of Ganoderma are extensively variable, and more than 250 species have been described (Ryvarden 1991). Therefore, it is the most difficult genus among the polypores to classify (Moncalvo and Ryvarden 1997, Ryvarden 1985). Variability of morphological characteristics of Ganoderma led many taxonomists to explore chemical and molecular methods to distinguish between species of Ganoderma.

In phylogenetic studies based on the sequences of ITS and 26S rDNA, it was proved that extensive convergence or parallelism of morphological characters has occurred during the evolution of Ganoderma (Moncalvo et al 1995a). It also was found that monophyletic groups correlate fairly well with the geographical origins of taxa and host relationships. It is evident that traditional classification systems of Ganoderma based on morphological characteristics should be reviewed in light of molecular data. Although phylogenetic studies with ITS sequences gave insights in relationships among the species of Ganoderma, relationships of the genus with other genera of the Polyporaceae still are unclear in many respects after earlier studies.

Analysis of the sequence and secondary structures of the small-subunit ribosomal RNA (SSU rRNA) of Agrocybe aegerita (Briganti) Singer revealed that there are three variable domains (V4, V6 and V9) with unusually long nucleotide extensions (Gonzalez et al 1997). Gonzalez and Labarère (1998, 2000) found that sequences of those three domains are species-specific and could establish a new approach providing good markers in the taxonomy and phylogeny of basidiomycetes. However, Hong et al (2002) recently found that large sequence extensions are not restricted to above three domains and, in this study, information of variable domains was found to be useful again in defining phylogenetic groups of Ganoderma and the evolutionary pattern of mitochondrial small-subunit ribosomal DNA (mt SSU rDNA) was discussed based on the secondary structure model of Neefs et al (1993).

Phylogenetic studies using partial sequences of mt SSU rDNA showed that Ganoderma lucidum (W. Curt. : Fr.) P. Karst. was classified in a group of trimitic or dimitic species in the polypore core group, including Trametes Fr. and Polyporus P. Karst. (Hibbett and Donoghue 1995, Hibbett et al 1997). However, two strains of G. lucidum did not form a monophyletic group in those studies. One strain of G. lucidum was grouped with Daedaleopsis Schroet. and Lenzites Fr. and the other with Cryptoporus (Peck.) Shear. In recent years, primers for PCR amplification and sequence determination of nearly full mt SSU rDNA were developed (Hong et al 2002), which demonstrated that mitochondrial rDNA sequences had 3.3 times more information than ITS sequences among the species of Ganoderma. In this study, we present the phylogenetic results and relationships of Ganoderma species based on nearly full sequences of mt SSU rDNAs.

	MATERIALS AND METHODS

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Strains, DNA extraction, PCR amplification and sequencing – Thirty-eight strains of Ganoderma used in this study are listed in TABLE I with the catalog numbers of culture stock, geographical origins, host relationships and sequence accession numbers. Fungi were cultured on MEA (2% malt extract, 0.5% peptone, 2% agar) covered with cellophane. After growing at 25 C for 1 wk, mycelia were harvested by scraping the colony off the cellophane with a sterile spatula and transferred to 1.5 mL microfuge tubes. The mycelia then were resuspended in 600 µL of lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], 100 mM NaCl, 2% SDS). After repeating freezing in liquid nitrogen and thawing three times at 70 C, DNA was extracted successively with phenol, phenol : chloroform and chloroform and recovered by iso-propanol precipitation (Lecellier and Silar 1994). DNA for sequencing was amplified enzymatically using the polymerase chain reaction. Double-stranded DNA was amplified from genomic DNA with BMS05 and BMS173 primers (FIG. 1 and TABLE II) (Hong et al 2002). PCR amplification was conducted as follows: initial denaturation at 94 C for 3 min, 30 amplification cycles consisted of 30 s at 94 C, 30 s at 50 C and 2 min at 72 C, and terminal extension at 72 C for 10 min. PCR products were purified with a Wizard PCR prep kit (Promega, Madison, Wisconsin) and directly cycle-sequenced with a Top DNA sequencing kit (Bioneer, Taejon, Korea), [³⁵S]dATPS (10 mCi/mL, Amersham, Buckinghamshire, England) and primers listed in TABLE II. All sequences were read bidirectionally.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Location of primers used in PCR amplification and sequencing of mt SSU rDNA. Locations of nine variable domains (V1–V9) are shown as filled boxes in the diagram of the mt SSU rDNA gene of G. lucidum ATCC 64251. The position of the group II intron found in 11 strains of TABLE I was indicated between V4 and V5 domains.

Culture descriptions – Culture studies were done to ensure that our data and findings are consistent with those of earlier studies (Adaskaveg and Gilbertson 1986, Bazzalo and Wright 1982, Moncalvo et al 1995a, Staplers 1978). All strains were grown on MEA at 25 C. Three replicates were made for each isolate using inocula taken from the actively growing margin of mycelia. Formation of chlamydospores was examined by bright-field microscopy using Melzer’s reagent (Gilbertson and Ryvarden 1986).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sequence analyses of mt SSU rDNA – Sizes of amplified products with primers, BMS05 and BMS173, were divided into two types. Twenty-six strains produced PCR products of about 1.5 kb, corresponding to almost a full sequence of mt SSU rDNA, and 12 strains produced PCR products of about 3.0 kb in size. In comparison with the sequence alignment of E. coli and with the secondary structure model of introns, it was found that the additional 1.5 kb originated from a group II intron, located between positions 788 and 789, corresponding to those of the 16S rRNA gene of E. coli (Hong et al 2002). For strains with an intron, 3' and 5' partial sequences of exons were determined around the exonintron junction region and both exon sequences were submitted to the GenBank database as indicated in TABLE I. For phylogenetic analyses, the two exon sequences were combined. The sequence of G. oregonense ATCC 46751 was quite different from the others. The result of BLAST search suggested that the sequence was similar to those of Ceriporiopsis rivulosa (Berk. & M.A. Curtis) Gilb. & Ryv. and Trametes consors (Berk.) Mitra with 95% (534/561) and 94% (537/567) identity, respectively. Thus, this doubtful sequence was excluded from further analyses.

Characteristics of Ganoderma mt SSU rDNA. – Thirty-seven mt SSU sequences of Ganoderma were aligned unambiguously except for V4 and V6 variable domains (FIG. 1) that had undergone several insertion/deletion events. The alignment for those two domains could be achieved with the aid of the mfold program, version 3.1. When the levels of base substitution among sites were estimated, the site variation of variable domains was much greater than that of conserved domains (FIG. 2) and showed that phylogenetic information was concentrated in variable domains. The average similarity values were 99.0% for 965 sites of conserved domains and 93.9% for 538 sites of variable domains. Among the nine variable domains, V1 (91.1%), V4 (90.6%), V5 (92.6%), V6 (89.5%) and V9 (93.9%) were more variable than the other four variable domains (V2: 96.5%, V3: 99.9%, V7: 99.5%, V8: 96.0%). On the other hand, the similarity values of V3 and V7 were higher than the average one of conserved domains (99.0%).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Maximum site variation of the mt SSU rDNA sequence of Ganoderma. The lower bar indicates the relative locations of nine variable domains (V1–V9) in the mt SSU rDNA. The variation in base substitution rates among sites across the length of mt SSU rDNAs from 37 strains of Ganoderma were assessed and graphed with MacClade 3.0 using a window size of 10 consecutive bases.

View larger version (50K): [in this window] [in a new window]   FIG. 3. One of nine most-parsimonious trees derived from mt SSU rDNA sequences. Branches collapsed in the strict consensus tree were indicated by broken lines. Sequences of C. salicina, M. nigra, P. cinnabarinus and T. versicolor were included to root the phylogeny of Ganoderma, and M. nigra was used as outgroup. Numbers above and bold ones below the branch indicate steps and bootstrap values, respectively. Branches supported by more than 70% of bootstrap values in both parsimony and distance analyses were presented by bold lines. Ganoderma lucidum from three continents were represented by bold letters to show the polyphyly of the species. After the strain number, geographic origins of the isolates were indicated in the bracket. After the geographic origin, host relationships, staining of chlamydospores in Melzer’s reagent, and groups (abbreviated as Gr) based on ITS and nuclear LSU-D2 regions (Moncalvo et al 1995a) were presented. Legends were inset in the left lower box.

Secondary structures of the V4 domain were conserved within the same group but variable between groups because of multiple insertion/deletion events (FIGS. 4, 5). However, members of the G. applanatum group had secondary structures different from one another according to geographical origins of the United States, United Kingdom and Japan. In general, Ganoderma species had a short V4 domain compared to that of other species examined here. Deletion of eight nucleotides from positions 39–46 in the P23_1' region of the domain provided G. lobatum CBS 222.48 (Group II) with a big loop and a short stem. However, G. colossus CBS 268.88 (Group I) and the G. tsugae group (Group III) had slightly elongated stems. Secondary structures of the V6 domain also were conserved within the same group, except for the G. applanatum group where in-group variation was found according to geographical origins (FIGS. 6, 7). In both variable domains, G. lobatum CBS 222.48 from the United States and G. applanatum ATCC 44053 from Japan were grouped together. Ganoderma colossus CBS 268.88 (Group I) has the longest stem, and G. applanatum CBS 175.30 (Group II) an unusually big bulge between 37 and P37_1 regions. In comparison with the V4 domain, the V6 domain had elongated structures and variable sizes for the stem P37_1 due to multiple insertions while those for the stem P37_2 were well conserved in general.

View larger version (36K): [in this window] [in a new window]   FIG. 4. Secondary structure models of the V4 domain. Models were arranged according to the phylogenetic relationships based on the full sequence of the domain.

View larger version (98K): [in this window] [in a new window]   FIG. 5. Sequence alignment of the V4 domain. Stems were enclosed by "[" and "]" and bulges by "{" and "}". The helix numbering was presented at the bottom line. Three sister taxa and even-numbered Ganoderma groups were shaded for better vision.

View larger version (31K): [in this window] [in a new window]   FIG. 6. Secondary structure models of the V6 domain. Models were arranged according to the phylogenetic relationships based on the full sequence of the domain.

View larger version (89K): [in this window] [in a new window]   FIG. 7. Sequence alignment of the V6 domain. Stems were enclosed by "[" and "]" and bulges by "{" and "}". The symbol "^" equals "][". The helix numbering was presented at the bottom line. Three sister taxa and even-numbered Ganoderma groups were shaded for better vision.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Structures of mt SSU rDNA. – It was reported that the mt SSU rDNA of Agrocybe aegerita had large extensions in the variable domains V4, V6 and V9 (Gonzalez et al 1997) and secondary structures of these domains in Agrocybe Fayod and Pleurotus (Fr.) P. Kumm. were variable (Gonzalez and Labarère 1998, 2000). However, large sequence extensions are not restricted to the above three variable domains in basidiomycetes. For example, Phlebiopsis gigantea (Fr.) Jülich had large extensions in the V2, V6, V8 and V9 domains and Punctularia atropurpurascens (Berk. & Broome) Petch in the V2, V4, V5, V6 and V9 domains (Hong et al 2002). In case of G. lucidum, most of the variable domains were short compared to those of other basidiomycetous fungi (Hong et al 2002). Lengths of mt SSU rDNAs from 37 Ganoderma strains included in this study were relatively uniform, except in the V4 and V6 domains. Those two domains have shown length variations between groups due to multiple insertion/deletion events (FIGS. 4–7). Although most of the phylogenetic information was concentrated in variable domains (81 sites out of 111 informative sites for the dataset of Ganoderma strains, excluding sister group and outgroup taxa), 30 informative sites found in conserved regions significantly contributed to resolving phylogenetic relationships between Ganoderma groups.

Sequences of conserved domains are very helpful in resolving phylogenetic relationships, especially when sequences of variable domains have alignment problems because of multiple insertion/deletion events, as in the variable domains V4, V6 and V9 of mt SSU rDNAs from Agrocybe and Pleurotus (Gonzalez and Labarère 1998, 2000). In contrast to the other variable domains, Hong et al (2002) found that V3 and V7 domains were fairly constant in length in various hymenomycetous fungi. In Ganoderma, lengths and sequences of V3 and V7 variable domains were highly conserved and these domains appear to be taxon-specific in conservation but variable in sequence between taxa. Although secondary structure and evolutionary pattern of mitochondrial and prokaryotic SSU rDNAs are conserved well, it is believed that some regions, such as V3 and V7 domains as in Ganoderma and stem 15 of secondary structure model (Neefs et al 1993), are different from general features of those SSU rDNAs.

From the construction of secondary structure models (FIGS. 4–7), it was found that secondary structures could be used to delineate the phylogenetic groups of Ganoderma. Secondary structures of the variable domain V4 of a same group were similar to one another with minor changes from base substitutions. For example, the base change from A to C at position 16 causes the creation of a bulge at positions 40 and 41 in six strains from the G. resinaceum group (Group VI, FIGS. 4, 5) and the base change from U to C at position 20 reduces one bulge base at position 37 in G. lucidum CBS 270.81 of the G. tsugae group (Group III, FIGS. 4, 5). On the basis of the secondary structure and sequence alignment of G. lobatum CBS 222.48 (Group II, FIGS. 4, 5), it is judged that the multiple deletion occurred only in one strand of the stem P23_1 whereas the sequence of the other strand P23_1' was conserved and the length of the stem has been changed by a sequential mode of insertion or deletion events. The same situation was found in the variable domain V6 based on the secondary structure and sequence alignment for the stem P37-1 of G. applanatum CBS 175.30 (Group II, FIGS. 6, 7). In both V4 and V6 domains, the strains of Ganoderma always grouped together and made a monophyletic clade at the base of which G. colossus (Group I) was placed (FIGS. 4, 6). The G. applanatum group (Group II), with secondary structures different from one another caused by in-group variation according to geographical origins, made one subclade and the remaining groups formed the other subclade, as in the phylogenetic tree of FIG. 3.

The secondar y structures of variable domains showed that the structural information was useful in describing Ganoderma groups. Secondary structures of V4 and V6 domains were conserved within the same group but variable between different groups except for the G. applanatum group (FIGS. 4, 6); the phylogenetic relationships based on full sequences of both domains (FIGS. 4, 6) actually developed the same basic topology as the parsimony tree (FIG. 3) derived from the total sequence of mt SSU rDNA, suggesting that the phylogeny of the variable domain sequence reflects that of the total mt SSU rDNA sequence. Because of group-specific characteristics, such secondary structures of variable domains of the mt SSU rDNAs could be used as a marker in phylogenetic and typing studies of polypores, agarics or other basidiomycetes to discriminate species or strains from one another within a given taxon.

Phylogenies of Ganoderma. – The phylogenetic analysis of polypores based on partial sequences of mt SSU rDNA, Hibbett and Donoghue (1995) included two G. lucidum strains in Group I, which contained Cryptoporus, Daedaleopsis, Datronia Donk, Fomes (Fr.) Fr., Lentinus Fr., Lenzites Fr., Polyporus, Pycnoporus P. Karst. and Trametes. Those two strains of Ganoderma did not make a monophyletic group. One of them showed a close relationship with Cryptoporus volvatus (Peck) Shear and the other with Daedaleopsis confragosa (Bolton) J. Schroet. and Lenzites betulinus (Fr.) Fr. However, from the phylogenetic analysis of Ganoderma (FIG. 3), it was evident that the genus Ganoderma made a monophyletic group. Monophyly of Ganoderma also was supported by the phylogenetic analyses of partial mt SSU rDNA sequences retrieved from Ganoderma species of the Polyporoid clade (Hibbett and Thorn 2001) and Group I (Hibbett and Donoghue 1995) (data not shown).

Ganoderma colossus is the only species described in Tomophagus Murrill. It easily is distinguished from other Ganoderma species by its thick, pale context and soft, light basidiocarps when dry (Moncalvo and Ryvarden 1997). It has a yellow laccate pileal surface, slightly amyloid skeletal hyphae, large basidiospores and prominent chlamydospores. Creation of Tomophagus by Murrill (1905) was based on the light basidiocarp, floccose, soft, spongy and light-colored context and friable tubes. However, Tomophagus has been regarded as a synonym of Ganoderma because major differences between the two genera were restricted to macromorphological characters that have been treated as adaptable at a generic level (Furtado 1965, Ryvarden 1991, Steyaert 1972). Nevertheless, phylogeny based on LSU and ITS rDNA sequences (Moncalvo et al 1994) and phylogeny based on mt SSU rDNA sequences (FIG. 3), together with the secondary structure model of two variable V4 and V6 domains (FIGS. 4, 6), supported Murrill’s opinion that Tomophagus colossus (Fr.) Murrill is a distinct species from Ganoderma and proved that Tomophagus is a taxonomically appropriate genus.

All strains of G. applanatum and G. lobatum were included in the G. applanatum group. The two species have a nonlaccate basidiocarp and have been classified in the Elfvingia group by Imazeki (1939). The present phylogenetic study supported their monophyly. However, G. applanatum isolates were found to be polyphyletic according to geographical origins (FIG. 3) and it was supported by secondary structure models of V4 and V6 domains (FIGS. 4 and 6). Therefore, it is necessary that the classification of G. applanatum and related taxa be revised again when more strains and molecular data are available.

Twelve strains identified as G. lucidum were distributed in three groups—the G. tsugae group, the Asian G. lucidum group and the G. resinaceum group. The three groups were distinguished in terms of host relationships, geographical distribution and chlamydospore formation. The G. tsugae group contained one North American strain (G. lucidum ATCC 46755) isolated from conifer and two European strains (G. lucidum CBS 176.30, CBS 270.81) from unknown hosts. Moncalvo et al (1995a) reported that G. lucidum CBS 270.81 produced rare, small and bullet-shape chlamydospores but, in this study, no chlamydospores were found from isolates included in the G. tsugae group.

Ganoderma tsugae was reported first only on Tsuga canadensis. It later was reported on Abies, Picea, Larix and other conifers (Adaskaveg and Gilbertson 1986, Imazeki 1939, Zhao 1989). It was found even on the hardwood Betula in North America (Adaskaveg and Gilbertson 1988). Ganoderma valesiacum was reported on Larix and other conifers in Europe, China and Japan (Hongo and Izawa 1994, Pegler and Young 1973, Ryvarden and Gilbertson 1993, Zhao 1989). Stalpers (1978) concluded from his culture study that G. tsugae and G. valesiacum are conspecific. Adaskaveg and Gilbertson (1986) also found that G. tsugae from North America and G. valesiacum from Europe were similar in morphology and temperature relationships. In the molecular phylogenetic studies based on ITS (Moncalvo et al 1995a) and mt SSU rDNA (FIG. 3), it was shown that they had a close relationship and were not separated from each other. Based on sequence variation of ITS and nuclear LSU-D2 regions, G. tsugae ATCC 46754 from an unknown locality and G. valesiacum CBS 282.33 from Europe in the G. tsugae group belonged to the same Group 1 of Moncalvo et al (1995a).

Ryvarden and Gilbertson (1993) stated that the microscopic structures of G. valesiacum were similar to those of G. lucidum and the only character that separates two species was the white, punk-like context under a cracking crust. They questioned the distinction between G. oregonense and G. tsugae (Gilbertson and Ryvarden 1986), although G. oregonense has larger basidiocarps, pores and basidiospores than G. tsugae. In the phylogenetic analysis, G. lucidum, G. oregonense, G. tsugae and G. valesiacum in the G. tsugae group were grouped together but weakly supported by bootstrap analysis. However, the two European isolates labeled G. lucidum were separated into an independent lineage from others within the group by the support of a high bootstrap value (FIG. 3). Although distinction between G. oregonense and G. tsugae was not supported by high bootstrap value, they were divided into two separate groups, where two G. oregonense strains were related closely to the strain labeled G. lucidum and four strains labeled G. tsugae were not phylogenetically discriminated from two G. valesiacum strains. This result is consistent with the opinion of Stalpers (1978) that, in the survey of culture characteristics of wood-rotting fungi, G. tsugae was regarded as a synonym of G. valesiacum.

The Asian G. lucidum group contained six Asian G. lucidum isolates and one G. oerstedii isolate. In the case of G. oerstedii, Bazzalo and Wright (1982) once reported that numerous culture differences were found among four G. oerstedii strains. Among them, two strains, including ATCC 52409 of the G. tsugae group, exhibited a tight, felt-like texture, absence of chlamydospores, abundance of fibrous hyphae and low proportion of cuticular cells. The other two strains, including ATCC 52411 of the Asian G. lucidum group, exhibited a farinaceous texture, dextrinoid chlamydospores, smaller proportion of fibrous hyphae and compact pseudoparenchyma of abundant cuticular cells. Later, they re-identified ATCC 52411 as G. resinaceum from culture similarities (Moncalvo et al 1995a). However, ATCC 52411 certainly did not belong to the G. resinaceum group, as Moncalvo et al (1995a) indicated, but was proved to have a relationship with the Asian G. lucidum group. In addition, we observed that ATCC 52411 produced amyloid chlamydospores rather than dextrinoid ones. Ganoderma oerstedii ATCC 52411 thus should be re-examined from the viewpoint of its taxonomic identity in connection with G. lucidum.

In the artificial fructification experiment on sawdust medium, it was reported that strains labeled G. lucidum from Korea produced tough fruiting bodies with a context 4.1–5.9 mm thick, compared to 2.3–4.8 mm in Taiwanese and North American isolates (Kim et al 2001). The dikaryon-monokaryon mating experiment also revealed that Korean isolates could not mate with strains labeled G. lucidum from different geographical origins (Kim et al 2002). Therefore, it is evident that G. lucidum from Korea are clearly distinguished from other isolates from North America and Europe in morphological, mating and molecular characteristics.

The conifer-inhabiting strains of the G. meredithiae group did not produce chlamydospores and had slow or moderate growth rates. Ganoderma oregonense ATCC 64487 had an identical mt SSU rDNA sequence as that of G. meredithiae ATCC 64492. The other two strains identified as G. oregonense were included in the G. tsugae group rather than in the G. meredithiae group (FIG. 3). Considering these results, we concluded that G. oregonense ATCC 64487 was misidentified and should be relabeled G. meredithiae.

All strains included in the G. resinaceum group made chlamydospores, although the staining properties associated with Melzer’s reagent were different. Three G. resinaceum strains from North and South America and two strains labeled G. lucidum from North America could not be distinguished phylogenetically. They had 99.72–100% similarity in mt SSU rDNA sequences. We could not find noteworthy differences between the two species in the morphological descriptions, except for the color or texture of basidiocarps that have been regarded as variable characteristics (Gilbertson and Ryvarden 1986). In culture and mating studies, it was shown that G. lucidum from North America and G. resinaceum from Europe had similar culture characteristics and were interfertile (Adaskaveg and Gilbertson 1986). Therefore, we conclude that three strains labeled G. resinaceum and two strains labeled G. lucidum from America are conspecific.

It is known that G. resinaceum from Europe showed similar culture characteristics and mated with G. lucidum from North America (Adaskaveg and Gilbertson 1986). However, the two species formed independent phylogenetic groups and contained considerable sequence differences in ITS rDNA analysis (Moncalvo et al 1995a, b). We also found that G. resinaceum from Europe were distinguished from strains labeled G. lucidum from North America in mt SSU rDNA analysis. However, because of the discrepancy between culture and molecular data, it is too early to consider the two species as conspecific until more morphological, culture and molecular data are available.

In conclusion, mt SSU rDNA sequences of Ganoderma species contained valuable phylogenetic information in conserved domains as well as in variable domains. Secondary structures of variable domains could be a useful marker and were significantly helpful in the description of phylogenetic groups. Thirty-seven Ganoderma strains included in this study were grouped into at least six monophyletic groups. Ganoderma lucidum, the most cosmopolitan member of Ganoderma, was polyphyletic according to geographical origins. Analyses of morphological and culture data proved that host relationships and the presence of chlamydospores in culture were important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.

	ACKNOWLEDGMENTS

 
The authors are sincerely grateful to Dr. Ruey-Shyang Hseu (Applied Microbiology Laboratory, Department of Agriculture Chemistry, National Taiwan University, Taipei, Taiwan) who kindly provided for reference "Ganoderma: systematics, phytopathology and pharmacology" from symposia of the 5th International Mycological Congress. Our appreciation is happily extended to Dr. Jean-Marc Moncalvo (Department of Botany, Duke University, Durham, North Carolina), who proofread the manuscript and made insightful comments. This work was supported by grant No. R01-2002-000-00304-0 from the Basic Research Program of the Korea Science and Engineering Foundation and by the BK21 Research Fellowship from the Ministry of Education and Human Resources Development.

	FOOTNOTES

 
Accepted for publication March 1, 2004.

¹ Present address: Department of Plant Pathology, College of Agriculture and Life Sciences, The University of Arizona, Tucson, AZ 85721-0036.

² Corresponding author. E-mail: minervas{at}snu.ac.kr

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