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Department of Biology, Clark University, 950 Main Street, Worcester, Massachusetts 01610
Yu-Cheng Dai
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
David S. Hibbett
Department of Biology, Clark University, 950 Main Street, Worcester, Massachusetts 01610
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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Sparassis species show extensive morphological variation, especially when materials from eastern Asia and Australia are compared with collections from North America and Europe. We have been studying the taxonomy of Sparassis from eastern Asia, North America, Australia and Europe, using both morphological and molecular data. DNA was extracted from 32 recent collections of Sparassis from Australia, Canada, China, Finland, France, Germany, Japan, Switzerland, Thailand, the United Kingdom and the United States. The report of a Sparassis taxon from Australia is the first report of this genus from the Southern Hemisphere. Sequences of nuclear and mitochondrial rDNA and the gene encoding RNA polymerase subunit II (RPB2) were used to examine relationships both within the genus Sparassis and between Sparassis species and other members of the polyporoid clade. Equally weighted parsimony analyses and Bayesian analyses were performed using independent datasets and combined datasets of sequences from different regions. Our results suggest that: (i) Polyporoid fungi producing a brown rot may form a clade; (ii) as suggested in a previous study, Sparassis and Phaeolus form a monophyletic group, which is united by the production of a brown rot, the presence of a bipolar mating system and the frequent habit of growing as a root and butt rot on living trees; (iii) at least seven lineages are within Sparassis, represented by S. spathulata, S. brevipes, S. crispa, S. radicata and three taxa that have not been described, which can be distinguished on the basis of fruiting body structure, presence or absence of clamp connections, presence or absence of cystidia and spore size.
Key words: polyporoid clade, multigene phylogeny, MrBayes, biogeography
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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, b). Twelve species with three varieties have been recorded in this genus, according to the CABI Index of Fungi (http://www.indexfungorum.org/). Three species, Sparassis brevipes Krombh., S. crispa Wulf. : Fr., and S. spathulata. Schw. : Fr., have been accepted in recent studies (Martin and Gilbertson 1976, Kreisel 1983, Burdsall and Miller 1988a, b, van Zanen 1988). Sparassis radicata Weir was described on Douglas-fir from western North America, but Martin and Gilbertson (1976) suggested that it should be considered con-specific with S. crispa based on di-mon mating tests and culture studies.
Sparassiella longistipitata Schwarzman is grouped in the family Sparassidaceae Herter (Kirk et al 2001), but no collections of this species are available (Burdsall and Miller 1988a).
The stalked form of the basidiocarp, highly branched flabellae with hymenium on both sides in initial stages, and the monomitic hyphal system make it hard to position the genus Sparassis. Donk (1964) reviewed earlier studies and, apparently based on basidiocarp morphology, concluded that the Sparassidaceae could be a tribe in the Clavariaceae or a distinct family close to the Clavariaceae. Later, the Sparassidaceae was put in the Cantharellales without explanation by Jülich (1981), and this was accepted in the Dictionary of Fungi 8th edition by Hawksworth et al (1995). Hibbett et al (1997) included Sparassis spathulata in their molecular study on major groups of gilled mushrooms, polypores and puffballs, which was based on phylogenetic analyses of nuc-ssu and mt-ssu rDNA. Sparassis was resolved as the sister group of a clade including two polypores Laetiporus sulphureus (Bull. : Fr.) Murr. and Phaeolus schweinitzii (Fr.) Pat. (Hibbett et al 1997). The position of Sparassis in the polyporoid clade was supported in other studies (Hibbett and Donoghue 2001), and that placement is accepted in the Dictionary of Fungi 9th edition (Kirk et al 2001).
Sparassis species are known as brown-rot producers with a bipolar mating system (Weir 1917, Martin and Gilbertson 1976). By analyzing character correlations among wood-decay types, mating systems and host ranges in homobasidiomycetes, Hibbett and Donoghue (2001) did not support Gilbertson’s hypothesis of a correlation between production of a brown rot and possession of a bipolar mating system. They inferred that brown-rot fungi are not monophyletic in the polyporoid clade and that the pattern of transformations between different wood-decay types is complicated. Two strongly supported groups of genera—one of them is Laetiporus-Phaeolus-Sparassis—were united by the production of brown rot on the butt or root of living or dead trees and a bipolar mating system (Hibbett and Donoghue 2001). More genes, including partial nuclear gene sequences coding for the second largest subunit of RNA polymerase II (RPB2), were used in this study to test the relationships among Sparassis and other brown-rot polypores. Genes that encode the subunits of nuclear RNA polymerase, especially RPB1 and RPB2, are promising phylogenetic markers in fungal systematics (Liu et al 1999, Matheny et al 2002).
Sparassis crispa has been reported in Asia from Japan to the Tibet highland in western China (Imazeki et al 1988, Teng 1995, Mao et al 1993). In many aspects, Asian "S. crispa" resembles S. radicata Weir from western North America more than "S. crispa" from eastern North America and Europe. The relationship between Asian and North American floras has been discussed for many years among botanists and mycologists (Mueller et al 2001). Early fungal biogeographic studies were based on specimens and descriptions in the literature. These studies were limited by the high degree of morphological variation of macrofungi, the existence of huge unexplored areas in eastern Asia and inconsistent applications of morphological species concepts. Several distribution patterns, such as eastern Asia-eastern North America disjuncts, eastern North America-western North America disjuncts or eastern Asia-western North America disjuncts, had been suggested by studies of vascular plants, bryophytes and lichens (Redhead 1989, Zang 1992, Wu and Mueller 1997, Wen 1999, Wu et al 2000, Mueller et al 2001). Molecular phylogenetic hypotheses coupled with information from morphological studies are becoming key to assessing species concepts and biogeographic relationships in fungi (Hibbett et al 1998, Mueller et al 2001). For example, Wu et al (2000) and Mueller et al (2001) tested the distribution pattern of species of Armillaria, Suillus and Xerula from eastern Asia and North America based on ITS sequences. Given that the ITS sequences can be too variable to be aligned across distantly related taxa, combined data from lsu-rDNA, ITS and partial RPB2 genes were used in this study to resolve the distribution pattern of Sparassis species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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and TABLE II. Morphological descriptions are based on observations of fresh, dried or rehydrated specimens. Microscopic studies used squashed tissues and sections cut with a freezing microtome to a thickness of 15–20 µm. Measurements were made under 1% Congo red in ammonium hydroxide using bright field (Olympus CH-2).
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Sequence data of Sparassis species were generated from six regions (TABLE I
): (1) partial mitochondrial small subunit (mt-ssu) rDNA bounded by primers MS1and MS2 (White et al 1990), representing four species of Sparassis in seven isolates; (2) partial mitochondrial large subunit (mt-lsu) bounded by primers ML5 and ML6 (White et al 1990), representing four species of Sparassis in five isolates; (3) nearly complete nuclear small subunit (nuc-ssu) rDNA bounded by primers PNS1 and NS8 (White et al 1990, Hibbett 1996), representing four species of Sparassis in 10 isolates; (4) partial nuclear large subunit (nuc-lsu) rDNA bounded by primers LR0R and LR5 (Vilgalys and Hester 1990), representing seven species of Sparassis in 29 isolates; (5) complete internal transcribed spacers 1 and 2 and the 5.8S rDNA (nuc-its rDNA) bounded by primers ITS4 and ITS5 (White et al 1990), representing seven species of Sparassis in 29 isolates; (6) partial gene encoding RNA polymerase subunit II (RPB2) bounded by primers RPB2-6F, 5'-TGG GGK WTG GTY TGY CCT GC-3', and RPB2-7R, 5'-CC CAT WGC YTG CTT MCC CAT-3' (http://faculty.washington.edu/benhall/), representing five species of Sparassis in 16 isolates. Additional data of other fungal materials were generated from these six regions or downloaded from GenBank (TABLE II). Strain numbers of some isolates were provided in Binder and Hibbett (2002).
PCR reaction mixes (Promega Corp., Madison, Wisconsin) contained 2.5 µL 10x PCR buffer, 5 µM dNTP, 12.5 pM of each PCR primer and 5 µL DNA in 25 µL. The amplification program included 40 cycles of 94 C for 30s, 45 C for 30s, and 72 C for 1 min. PCR products were purified using GeneClean (Bio 101, Carlsbad, California) and sequenced using the ABI Prism BigDye-terminator cycle sequencing kit (Applied Biosystems, Foster City, California) according to the manufacturer’s protocols. Primers used for sequencing were PNS1, NS19bc, NS19b, NS41, NS51, NS6, NS8, MS1, MS2, ML5, ML6, RPB2-6F, RPB2-6.3F, RPB2-7R, RPB2-7.1R, LR0R, LR3, LR3R, LR5, ITS4, and ITS5. Sequencing reactions were purified using Pellet Paint (Novagen, Madison, Wisconsin) and were run on an Applied Bio-systems 377XL automated DNA sequencer. Sequences were edited with Sequencher version 3.1 (GeneCodes Corp., Ann Arbor, Michigan). Sequences generated in this study were submitted to GenBank (accession numbers AY218373–AY218547).
Phylogenetic analyses. –
Two datasets were prepared, one for higher-level analyses (HLA) and one for lower-level analyses (LLA). The datasets for the HLA included sequences from five genes: mt-ssu rDNA (68 isolates), mt-lsu rDNA (59 isolates), nuc-ssu rDNA (72 isolates), nuc-lsu rDNA (72 isolates) and RPB2 (71 isolates). HLA were intended to resolve the placement of Sparassis among 61 homobasidiomycete genera (TABLE I and TABLE II). The datasets for the LLA included sequences from three genes nuc-lsu (36 isolates), ITS (37 isolates) and RPB2 (24 isolates). LLA were intended to resolve the relationships among Sparassis species (TABLE I and TABLE II). Sequences of nuc-rDNA and mt-rDNA were aligned by eye in the data editor of PAUP* 4.0b (Swofford 1999). Sequences of RPB2 were translated into amino acid sequences and aligned by eye in Sequencher and then converted back to nucleotides for the analyses. Both data-sets were analyzed in PAUP* 4.0b (Swofford 1999) and MrBayes 2.01 (Huelsenbeck and Ronquist 2001), with gaps treated as missing data and ambiguous or unalignable positions excluded. Ambiguous positions were excluded from the datasets before performing the analyses.
The HLA dataset was rooted with Dacrymyces chrysospermus Berk. & M.A. Curtis (Hibbett and Donoghue 2001). Ssu-rDNA sequences of Bondarzewia montana (Quél.) Singer, Piptoporus betulinus (Bull. : Fr.) P. Karst., Pycnoporus cinnabarinus ( Jacq. : Fr.) P. Karst., Ramaria formosa (Pers. : Fr.) Quél, Trametes versicolor (L. : Fr.) Pilát, Polyporus varius (Pers.) Fr., Polyporus melanopus (Pers.) Fr., Polyporus arcularius (Batsch) Fr., Cryptoporus volvatus (Peck) Shear and Lenzites betulina (Fr.) Fr. represented a shorter region than sequences of other taxa. Mt-ssu sequences of Albatrellus fletti (Morse) Pouzar, Grifola frondosa (Fr.) S.F. Gray, Sparassis brevipes GER24, and S. sp. AUS31, and mt-lsu sequences of S. crispa USA9, S. brevipes GER24, Trametes versicolor, Heliocybe sulcata (Berk.) Redhead & Ginns, Lentinula lateritia (Berk.) Pegler, Lentinus tigrinus (Fr.) Fr., Neolentiporus maculatissimus (Lloyd) Rajchenb., Phaeolus schweinitzii (Fr.) Pat., Piptoporus betulinus, Ramaria formosa, Sparassis sp. AUS31, Pycnoporellus fulgens (Fr.) Donk and one unidentified resupinate homobasidiomycete were not available, and those taxa were excluded from the analysis based on mt-DNA. No sequence of RPB2 of S. sp. AUS31 was generated, and it was excluded in analyses using RPB2 data.
Parsimony analyses were performed using equal weighting of characters and transformations. Heuristic searches were performed with 1000 replicate searches, each with a random taxon addition sequence, MAXtrees set to autoincrease, and TBR branch swapping. A bootstrap analysis was performed with 1000 replicates, each with 10 random taxon addition sequences, MAXtrees set to 1000, and TBR branch swapping. Bootstrap consensus trees generated from analysis of each gene were compared to check whether there is an apparent conflict in tree topologies. Bootstrap values higher than 80% were used as the criterion for "strong" conflict. The combined analyses were performed using nuc-rDNA and RPB2 sequences, which were congruent according to this criterion.
Bayesian posterior probabilities were computed using Metropolis-coupled Markov Chain Monte Carlo (MCMCMC) under the GTR+G model in MrBayes 2.01 (Huelsenbeck and Ronquist 2001) by running four chains with 200 000 generations using the program default priors on model parameters. Trees were sampled every 100 generations, and a total of 2001 trees were saved. Likelihoods converged to a stable value after 5000 generations, so the first 50 trees were discarded as "burn-in" before computing a majority rule consensus tree in PAUP*.
The LLA dataset was rooted using Lentinus tigrinus based on the results of the HLA. The same analytical settings as that for the HLA were applied to the LLA for separate and combined data of nuc-lsu rDNA, ITS and RPB2. Isolates without RPB2, ITS and nuc-lsu rDNA sequences were excluded from different analyses. Alignments are available at TreeBASE (accession number M1814–M1815).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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–5).
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). A clade including S. spathulata, S. brevipes, S. crispa, S. sp. AUS31, several other brown-rot species and Grifola frondosa was weakly supported (bootstrap = 53%) within the polyporoid clade. A clade (bootstrap = 90%) containing S. spathulata and S. brevipes and a clade (bootstrap = 100%) containing two isolates of S. crispa were grouped together with the Phaeolus-Laetiporus clade (bootstrap = 50%) as the sister group (bootstrap = 72%). Sparassis sp. AUS31 was grouped with Grifola frondosa and Pycnoporellus fulgens (bootstrap = 100%). The brown-rot polypores Fomitopsis, Piptoporus and Neolentiporus formed a clade (bootstrap = 67%). Relationships between white-rot and brown-rot species in the polyporoid clade were not well resolved.
The mt-rDNA had an aligned length of 684 bp with 117 uninformative variable positions and 290 parsimony-informative positions. Parsimony analysis based on mt-rDNA (FIG. 2) generated 733 equally parsimonious trees of 1810 steps with CI = 0.364. A clade containing Sparassis spathulata and S. crispa was weakly supported (bootstrap = 63%) and was grouped with white-rot species including Ganoderma, Pycnoporus and Polyporus (bootstrap = 84%).
The RPB2 sequences had an aligned length of 510 bp with 79 uninformative variable positions and 333 parsimony-informative positions. Parsimony analysis based on RPB2 (FIG. 3) generated three equally parsimonious trees of 5253 steps with CI = 0.172. Brown-rot species of Oligoporus, Antrodia, Auriporia, Phaeolus, Laetiporus, Sparassis, Fomitopsis, Piptoporus, Neolentiporus and the white-rot species Albatrellus syringae (Parm.) Pouz. formed a weakly supported clade, within which there was a clade containing S. crispa, S. spathulata and S. brevipes (bootstrap = 93%).
The combined nuc-rDNA and RPB2 genes had an aligned length of 3149 bp with 449 uninformative variable positions and 954 parsimony-informative positions. Parsimony analysis based on this dataset generated 19 equally parsimonious trees of 9866 steps with CI = 0.240 (FIG. 4). Sparassis species, including S. spathulata, S. brevipes and S. crispa, were monophyletic (bootstrap = 87%) with the Phaeolus-Laetiporus clade (bootstrap = 64%) as its sister group (bootstrap = 68%). A brown-rot polypore clade, the Fomitopsis-Piptoporus-Neolentiporus clade, also was supported (bootstrap = 87%), but the relationships among brown-rot and white-rot species in the polyporoid clade still were not well resolved.
Bayesian analysis based on combined nuc-rDNA and RPB2 sequences (FIG. 5) strongly supported (posterior probability = 93%) a clade containing Sparassis, Phaeolus, Pycnoporellus, and Grifola, in which Sparassis species are monophyletic (posterior probability = 99%). Two more clades of brown rot polypores were supported: the Fomitopsis-Piptoporus-Neolentiporus-Laetiporus clade (posterior probability = 85%) and the Oligoporus-Antrodia clade (posterior probability = 61%). In addition, the clade including white rot species of Cryptoporus, Ganoderma, Lentinus, Polyporus, Dentocorticium, Lenzites, Pycnoporus and Trametes is strongly supported (posterior probability = 96%).
LLA..
The lower-level relationships among Sparassis species were estimated using five different data-sets: nuc-lsu rDNA; ITS; RPB2; nuc-lsu rDNA+ITS; and nuc-lsu rDNA+ITS+RPB2. Trees inferred from single regions showed similar topologies to trees inferred from the combined sequences but with weaker branch support (FIGS. 6–9).
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). A clade containing Sparassis crispa, S. cf. crispa, S. spathulata, S. brevipes and S. sp. THAI was supported weakly (bootstrap = 61%) with S. sp. THAI as a basal branch. One North American isolate and four European isolates of S. crispa and six Chinese isolates of S. cf. crispa formed a clade (bootstrap = 100%). Isolates of S. spathulata formed a clade (bootstrap = 96%) with S. brevipes as its sister group (bootstrap = 100%). The relationship among the Sparassis clade and other brown-rot polypores were not resolved. Unfortunately, no RPB2 sequences were obtained for S. radicata, S. sp. AUS31 and isolates of S. crispa collected from Georgia (AME28), Tennessee (AME32) and western North America (TABLE I).
The combined sequences of nuc-lsu rDNA, ITS and RPB2 had an aligned length of 2080 bp with 286 uninformative variable positions and 505 parsimony-informative positions. Parsimony analysis based on this dataset generated 20 equally parsimonious trees of 1818 steps with CI = 0.633 (FIG. 7). Sparassis species formed a monophyletic group (bootstrap = 79%) with S. sp. THAI as a basal branch. Five clades were found within the Sparassis clade: Asian S. cf. crispa clade (bootstrap = 96%), European-North American S. crispa clade (bootstrap = 64%), S. spathulata clade (bootstrap = 100%), S. brevipes and S. sp. THAI. Laetiporus and Phaeolus formed a clade (bootstrap = 98%), which is the sister group of the Sparassis clade.
The combined sequences of nuc-lsu rDNA and ITS had an aligned length of 1432 bp with 193 uninformative variable positions and 295 parsimony-informative positions. Parsimony analysis based on this dataset generated 90 equally parsimonious trees of 1061 steps with CI = 0.644 (FIG. 8). Eight clades were recognized among Sparassis species: crispa clade (European isolates of S. crispa, bootstrap = 69%), radicata clade (western North American isolates of S. crispa and S. radicata, bootstrap = 67%), Asian crispa clade (Chinese isolates of S. cf. crispa, bootstrap = 94%), brevipes clade (European isolates of S. brevipes, bootstrap = 100%), spathulata clade (eastern North American isolates of S. spathulata, bootstrap = 100%), and two unidentified Sparassis species, S. sp. THAI and S. sp. AUS31. Two eastern North American isolates of S. crispa (S. crispa AME9 and AME28) formed a branch close to the radicata clade and the Asian crispa clade without bootstrap support. Relationships among those clades and lineages were not completely resolved. The crispa clade, the radicata clade, the Asian crispa clade and two eastern North American S. crispa were supported as monophyletic (bootstrap = 100%), whereas the brevipes clade and the spathulata clade were grouped together (bootstrap = 62%). S. sp. AUS31 was grouped with Grifola and Pycnoporellus (bootstrap value less than 50%).
Bayesian analysis based on the combined sequences of nuc-lsu rDNA and ITS provided higher confidence for all clades and lineages of Sparassis observed in the parsimony analysis (FIG. 9). The crispa clade (posterior probability = 98%), the radicata clade (posterior probability = 74%), the Asian crispa clade (posterior probability = 100%), the brevipes clade (posterior probability = 100%), and the spathulata clade (posterior probability = 100%) were confirmed. Two eastern North American isolates of S. crispa formed a weakly supported clade (posterior probability = 64%). Sparassis species were strongly supported as monophyletic (posterior probability = 86%) with Grifola and Pycnoporellus as the sister group. All species from the Northern Hemisphere formed a clade (posterior probability = 100%). S. brevipes and S. spathulata were grouped together (posterior probability = 99%), and the crispa clade, the radicata clade, the Asian crispa clade and eastern North American S. crispa formed a monophyletic group (posterior probability = 100%).
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MORPHOLOGY AND TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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, Kreisel (1983), and Burdsall and Miller (1988a, b), have produced authoritative taxonomies of Sparassis. This study used recent collections because they provide high quality DNA for molecular studies. For nomenclatural purposes, however, it would be useful to attempt to extract DNA from the older specimens studied by Gilbertson and others. All the collections were studied morphologically following widely accepted species concepts, which are based on Weir (1917), Kreisel (1983) and Burdsall and Miller (1988a, b). Simplified taxonomic descriptions and habit illustrations (FIG. 9) based on our own observations are provided in addition to the major clades found in phylogenetic analyses (as in FIG. 8). Commentary is provided regarding morphological incongruence or conflict among the members of each clade. brevipes clade (represented by Sparassis brevipes Krombh.)
Base of basidiocarps composed of a round mass. Flabellae spathulate, all originating directly from a common base, split into pieces and then fused with others several times on the way to the top, azonate to not distinctively zonate, margin entire. Hyphal system monomitic, simple septate, no clamp connection observed. Basidiospores hyaline, thin-walled, smooth, broadly elliptic to subglobose, (3.9–)4.4–5.2(–5.5) x (6.2–)6.8–7.8(–8.7) µm. Distribution: Europe.
Specimens examined. – GERMANY. BADEN-WÜRTTEMBERG: In mixed forest of Abies, Picea, Pinus and Fagus on limestone, 24-IX-1995, MBUH (ILKKA96-1044); VIII-1978, RB8/78.
Commentary. –
Sparassis brevipes has been documented in Europe without precise typification (Burdsall and Miller 1988a). Morphological studies showed that there are at least two distinct Sparassis species in European samples. We followed Kreisel’s (1983) concept and accepted the widely used name S. brevipes to represent the European species mostly growing on Abies, Fagus and Quercus. S. brevipes is similar to the eastern North American S. spathulata in spore size and possession of simple septate hyphae, but the flabellae of S. brevipes are azonate.
crispa clade (represented by Sparassis crispa Wulf. : Fr.)
Base of basidiocarps branched and elongated to form several flattened branches. Flabellae extend from the branches, broad but short, dissected and contorted, azonate, margin entire. Hyphal system monomitic, clamp connections present, gelatinous hyphae rarely present. Basidiospores hyaline, thin-walled, smooth, subglobose, 4.0–4.9 x 4.9–6.0(–6.9) µm. Distribution: Europe, eastern North America.
Specimens examined. – USA. MASSACHUSETTS: Purgatory Chasm State Park, 15-IX-2001, FH (ZW-Clarku003); GEOR-GIA: Fort Top Mountains, 13-IX-1983, TENN44575. FINLAND. North Kotinen Forest: on Pinus, 12-IX-1997, MBUH (YCD2637); PAINO TUOREENAN: 25-IX-1983, MBUH (Savolaninen). FRANCE. LOZERE: in mixed forest of Fagus, Picea and Quercus, 21-X-1994, MBUH (PIR-JO&ILKKA94-1587). SWEDEN. NARKE: on Pinus, 26-X-1988, MBUH (ILKKA88-2036). UK. ESHER COMMON, SURREY: collected by P.M. Kirk, on Pinus sylvestris, 29-X-2000, BMS2857. GERMANY. 6-IX-1987, RB9/6/87; collected by Doris Laber, 27-IX-1996, MBUH.
Commentary. –
Neotypification of Sparassis crispa was based on a European collection (Burdsall and Miller 1988b). S. crispa has been reported from Europe, eastern Asia and North America (Breitenbach and Kränzlin 1986; Burdsall and Miller 1988a, 1988b; Gilbertson 1980; Imazeki et al 1988; Mao et al 1993; Martin and Gilbertson 1976). Asian collections under the name of S. crispa are morphologically different from European S. crispa. European S. crispa collections are characterized by branched basidiocarps, strongly dissected and contorted flabellae and are strictly growing on conifers. Two North American isolates, FH-ZW-Clarku003 (S. crispa AME9) and TENN44575 (S. crispa AME28), were not nested in the crispa clade in two analyses (FIGS. 8–9) and need further study. FH-ZW-Clarku003 collected from Massachusetts is similar to the European S. crispa except for possessing gelatinous hyphae. TENN44575 collected from Georgia is similar to the western North American S. radicata, which has bigger, slightly dissected and contorted flabellae.
Asian crispa clade (represented by Sparassis cf. crispa Wulf. : Fr.)
Base of basidiocarps branched or not. Flabellae mostly extend from a common central mass, broad, dissected and slightly contorted, azonate, margins sometimes tooth-like. Hyphal system monomitic, clamp connections present. Basidiospores hyaline, thin-walled, smooth, subglobose, 4.0–5.0 x 5.0–6.0 (–6.6) µm. Distribution: eastern Asia ( Japan and China).
Specimens examined. – CHINA. JILIN, ANTU: Changbaishan Mountains, on Larix sp., 16-IX-1995, MBUH (YCD2145); on Larix sp., 14-VIII-1997, MBUH(YCD2470); on conifers, 25-VII-1960, HMAS30269; on conifers, 1-VIII-1960, HMAS30270; NEIMENGU: Arxan Mountains, 14-VIII-1991, HMAS60590; SICHUAN, DAOCHENG: on Picea, 11-VIII-1984, HKAS15728; SICHUAN, XIANGCHENG: on Quercus sp., 17-VII-1998, HKAS 32363; YUNNAN, ZHONG-DIAN: on Larix, 25-VII-1986, HKAS17477. JAPAN. NAGA-NO PREF, USUDA: on Larix, VII-1988, FFPRI(Tsengoku); 5-IX-1989, FFPRI(F-15706).
Commentary. –
Sparassis crispa reported and collected from Japan and China are macro-morphologically distinct from European S. crispa and are referred to as S. cf. crispa in this study. Flabellae of S. cf. crispa are similar to those of S. radicata, but they are bigger and not as contorted as in European S. crispa collections. Chinese collections show a host range from conifers such as Larix, Pinus to hardwood plants (dactyls) such as Quercus. S. radicata so far has been reported only on Douglas-fir and Pines (Martin and Gilbertson 1976).
radicata clade (represented by Sparassis radicata Weir)
Basidiocarps expand from a round mass. Flabellae anastomosed, vertically subdivided, broad, dissected and slightly contorted, azonate. Hyphal system monomitic, clamp connections present. Basidiospores hyaline, thin-walled, smooth, broadly elliptic to subglobose, 3.9–5.0 x 6.3–7.0 µm. Distribution: western North America.
Specimens examined. – CANADA. VANCOUVER: Stanley Park, on Tsuga heterophylla, collected by P. Koreger, 6-X-1985, UBC(F12464). USA. WASHINGTON: on the base of Tsuga sp., 4-X-1984, TENN45811; on base of conifer, 3-X-1992, TENN52558; CALIFORNIA: 13-II-1998, TENN56253; TENNESSEE: Great Smoky Mountains National Park, on base of Pinus sp., 28-VII-1991, TENN50232.
Commentary –
S. radicata is well studied and was thought to be conspecific with the European and Asian S. crispa, mainly based on culture studies and dikaryon-monokaryon mating tests (Martin and Gilbertson 1976). Morphologically, S. radicata is similar to Asian S. cf. crispa in having large but not strongly contorted flabellae. The flabellae arise radially from a common center rather than from a branched base as in the European S. crispa.
spathulata clade (represented by Sparassis spathulata Schw. : Fr.)
Base of basidiocarps composed of a round mass. Flabellae spathulate, all originating from a common base, anastomosing and vertically subdivided, distinctively zonate, margin entire. Hyphal system monomitic, simply septate, no clamp connections observed. Basidiospores hyaline, thin-walled, smooth, broadly elliptic to subglobose, 4.7–5.8 x (5.9–)6.9–8.0 µm. Distribution: eastern North America and Japan (?).
Specimens examined. – USA, MASSACHUSETTS: on Quercus sp., 13-IX-2001, FH (ZW-Clarku001); WORCESTER: on Quercus sp., 1-X-2000, FH (ZW-Clarku004); NEW HAMPSPHERE: Walpole, 7-IX-2001, FH (ZW-Clarku002); SOUTH CAROLINA: Oconee, 18-VIII-1992, TENN51767. JAPAN. HONSHU, TODAI-JI: collected by Kumiko and Korf, 22-VIII-1959, CUP(JA-1385).
Commentary. –
The large, zonate, vertically oriented flabellae of S. spathulata distinguish this species from other Sparassis species. S. spathulata is similar to the European S. brevipes (synonym: S. laminosa) in some aspects, and therefore validity of S. spathulata as a separate species was doubted by Burdsall and Miller (1988a). Eastern North American S. spathulata have been found on both Pinus and Quercus, whereas S. brevipes has been found mostly on Quercus. One Japanese collection (CUP JA1385) was identified as S. spathulata, which might be introduced from North America and so far is the only report of this species from Asia. Basidiocarp development of S. spathulata begins with central daedaloid ridges on the surface. These ridges expand somewhat parallel to form flabellae with an amphigenous hymenium.
Sparassis sp. AUS
Base of basidiocarps complex, flabellae narrow and highly branched, azonate. Branch tips flattened, often daedalioid arranged, then crested. Lower branches light ochraceous buff, upper branches pinkish buff to light ochraceous salmon. Margin with 2–4 fork-like teeth when dried. Hyphal system monomitic, simply septate, no clamp connections observed. Basidiospores hyaline, thin-walled, smooth, broadly elliptic to subglobose, 4.8–5.9 x 5.8–7.5 µm. Distribution: Australia.
Specimens examined. – Australia. TASMANIA, VIS. GEESTON: Tahune Forest Reserve, 5-VI-1991, by R. Peterson and A. Mills, TENN50289, Alan Mills (3996).
Commentary. – This fungus shows a unique macromorphology and was identified as Sparassis sp. by the collectors. The amphigenous hymenium and the arrangement of the flabellae make this fungus a Sparassis species. There is only one collection of this fungus, and the base was rotten away. More and better materials from the southern hemisphere are necessary for further studies.
Sparassis sp. THAI
Basidiocarps composed of a rosette of flabellae. Flabellae loosely arranged, up to 120 mm broad, margin not dissected, wavy, azonate. Hyphal system monomitic, clamp connections present. Hymenium layer composed of basidia and cystidia. Basidiospores hyaline, thin-walled, smooth, subglobose to broadly ellipsoidovoid, 6–7 x 7–9 µm. Distribution: Thailand.
Specimens examined. – THAILAND, CHIANG MAI PROVINCE: Doi Inthanon National Park, at the base of a living oak tree (Quercus eumorpha), 27-VI-2002, SFSU (D.E. Desjardin 7410).
Commentary. –
Sparassis sp THAI is distinct from other Sparassis species in possessing cystidia and larger basidiospores. Taxonomy of this fungus is discussed elsewhere (Desjardin et al 2004).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSMORPHOLOGY AND TAXONOMYDISCUSSIONLITERATURE CITED |
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Parsimony analyses, except for the one based on the RPB2 gene alone, showed a similar tree topology for the major clades in homobasidiomycetes. The nucleotide sequences of the RPB2 gene, between the primer pairs bRPB2-6f and bRPB2-7r, are highly variable, which suggests that nucleotide sequences in this part of the gene are saturated among taxa sampled here. Analyses based on the amino acid sequences (data not shown) suggest that amino acid sequences in this part of the gene are too conserved to be informative for HLA, however. Combining RPB2 sequences with nuc rDNA sequences increased the number of resolved clades (FIGS. 1, 3, 4) and reduced the number of equally parsimonious trees.
Sparassis species were grouped with some white-rot polypores in the parsimony analysis based only on mitochondrial rDNA, whereas other analyses suggested a close relationship between Sparassis species and other brown-rot polypores. Much of the mt-rDNA has multiple deletions and insertions and was excluded from the dataset. The mt-lsu rDNA sequences of one-fifth of the taxa sampled, including Phaeolus schweinitzii, were not available, so inferences regarding the higher-level phylogenetic relationships of Sparassis species based mostly on the combined nuc-rDNA and RPB2 sequences.
Sparassis species form a monophyletic group within the polyporoid clade supported by multilocus molecular data (FIGS. 1–5). Sparassis is closely related to Phaeolus, Laetiporus, Pycnoporellus, Auriporia, Oligoporus and other brown-rot polypores. Phaeolus and Laetiporus were confirmed as the sister group of Sparassis using equally weighted parsimony (FIGS. 1, 3, 4) as in previous studies (Hibbett et al 1997). Phaeolus, Laetiporus and Sparassis are united by the production of a brown rot, bipolar mating system and the habit of growth as a root and butt rot on living trees (rarely on dead trees and logs). Bayesian analysis (FIG. 5) suggests a close relationship among Sparassis species, Phaeolus, Grifola and Pycnoporellus. Pycnoporellus was thought to be closely related to Phaeolus (Gilbertson and Ryvarden 1987), and the cystidia similar to those found in Phaeolus and Pycnoporellus were found in Sparassis sp. THAI as well (Desjardin et al 2004). Bayesian analysis supports a clade including Fomitopsis, Piptoporus, Neolentiporus and Laetiporus, which are all brown rot polypores with bipolar mating systems.
Sequences from three loci, nuc-lsu rDNA, ITS and RPB2, have been used to infer the lower-level phylogenetic relationships of Sparassis. ITS sequences of sampled taxa are divergent and difficult to align, and parsimony analysis based on ITS sequences alone generated more than 53 000 equally parsimonious trees with little resolution (data not shown). As expected, RPB2 were easier to align and provided more informative sites than ITS sequences. Combining RPB2 gene with nuc-lsu rDNA and ITS provided higher support to branches and better resolution (FIGS. 6–8). Unfortunately, RPB2 sequences were not obtained for most Sparassis collections from North America.
Except for two unidentified Sparassis species, S. sp. AUS and S. sp. THAI, five well-supported clades were found among Sparassis species. Asian isolates of S. cf. crispa show morphological differences compared with European S. crispa. Western North American collections of Sparassis, including three isolates identified as S. crispa and one as S. radicata, formed a clade, and they morphologically are similar to the Asian S. cf. crispa. Sparassis spathulata, S. brevipes and the European S. crispa are distinct species delimited by both molecular data and morphological characters. The recently collected cystidioid Sparassis sp. THAI was grouped with all Sparassis species and might provide clues to the plesiomorphic form of Sparassis (Desjardin pers comm). Recognizing the Australian collection (S. sp. AUS31) as a species of Sparassis denotes that this genus is distributed worldwide, but more material from the Southern Hemisphere is necessary to describe this species and to resolve the distribution pattern.
The relationships among members of Sparassis crispa sensu lato, which include European and eastern North American S. crispa, western North American S. radicata and Asian S. cf. crispa, were not well resolved in all the analyses. Asian S. cf. crispa and western North American S. radicata are morphologically similar, but they are not strongly supported to form a monophyletic group. The eastern North American S. crispa morphologically is identical to the European S. crispa, but molecular data do not suggest that the collections from both locations are conspecific. This eastern North America-Europe distribution pattern was supported by the close relationship between eastern North American S. spathulata and European S. brevipes. North American S. crispa and S. radicata collections show a morphological transition between European S. crispa and Asian S. cf. crispa (i.e., eastern North American S. crispa is similar to European species whereas western North American S. radicata is similar to Asian S. cf. crispa). However, the lack of the RPB2 sequences from western North American collections make it premature to draw conclusions about biogeographic relationships among S. crispa sensu lato.
Sparassis is characterized morphologically by flabellae with an amphigenous hymenium and a central mass giving rise to the flabellae. The presence of clamp connections in Sparassis crispa, S. radicata, S. cf. crispa and S. sp. THAI suggests that clamp connections might have been lost in S. spathulata and S. brevipes. The spore size range in Sparassis species is overlapping and therefore not always reliable to use for recognizing species. Basidia with 2–4 sterigmata have been observed in all examined collections and basidiospores generally are larger when fewer spores are produced on single basidium.
Host shifts must have occurred in the evolution of Sparassis species. S. sp. THAI was thought to grow strickly on Quercus, and this trait has been kept in the lineages of S. brevipes, S. spathulata and Asian S. cf. crispa, but they all are known to have conifer hosts as well. European S. crispa and western North American S. radicata are found strictly on conifers. Additional ecological and phylogenetic studies could resolve patterns of host shifts in Sparassis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: zwang{at}clarku.edu
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