Mycologia, 99(6), 2007, pp. 833–841. # 2007 by The Mycological Society of America, Lawrence, KS 66044-8897

Fomitopsis incarnatus sp. nov. based on generic evaluation of and Rhodofomes

Kyung Mo Kim among mycologists (Carranza-Morse and Gilbertson Jin Sung Lee1 1986, Kotlaba and Pouzar 1990, Ryvarden 1991, Hack Sung Jung2 Ryvarden and Gilbertson 1993, Kotlaba and Pouzar Department of Biological Sciences, College of Natural 1998). Kotlaba and Pouzar (1990, 1998) suggested Sciences, Seoul National University, San 56-1 Shillim- a narrow generic concept for Fomitopsis, emphasizing 9-dong, Kwanak-gu, Seoul 151-747, Korea the wall thickness of basidiospores. Fomitopsis pinicola (Sw.)P.Karst.(thetypespeciesofFomitopsis) has thick- walled basidiospores and a resinous substance on the Abstract: A new polypore in the genus Fomitopsis was upper surface of basidiocarps, while F. rosea is charac- discovered in Kangwon Province, Korea. The species terized by thin-walled spores, the rose context and the was morphologically similar to Fomitopsis rosea and F. absence of a resinous crust on the pileal surface cajanderi, but the pinkish white pore surface, the size (Kotlaba and Pouzar 1998). Based on such morpholog- and shape of the pores and the number of sterigmata ical differences, a monotypic genus, Rhodofomes Kotl. & were different enough for it to be distinguished from Pouzar typified by R. roseus (Alb. & Schwein.) Vlasak (5 the recorded species of Fomitopsis. Based on the results F. rosea), was segregated from Fomitopsis into a new of morphological and phylogenetic analyses, this new genus (Kotlaba and Pouzar 1990). However Ryvarden polypore is proposed as Fomitopsis incarnatus sp. nov. (1991) and Ryvarden and Gilbertson (1993) argued that Key words: , Fomitopsis rosea, the rose-colored context is not of sufficient taxonomic internal transcribed spacer, mitochondrial small importance to warrant segregation into a new genus. subunit rDNA, phylogeny, second largest subunit Many genetic markers recently have been applied of RNA polymerase II to resolve fungal phylogenetic relationships. Internal transcribed spacers of nuclear rDNA (nuclear ITS), INTRODUCTION the small subunit mitochondrial rDNA (mt-SSU) and the genes encoding the second largest subunit of Fomitopsis P. Karst. is a cosmopolitan genus (Polypor- RNA polymerase II (RPB2) have become especially aceae, Aphyllophorales, Hymenomycetes, Basidiomy- useful to classify fungal taxa at the species and genus cota), and most of its species occur in boreal and levels (Ko and Jung 1999, 2002, Lim and Jung 2003, temperate zones (Ryvarden 1991, Ryvarden and Gil- Desjardin et al 2004, Hong and Jung 2004, Matheny bertson 1993). The genus is placed in the Daedalea 2005). When the sequences of nuclear ITS, mt-SSU group among 12 groups of the Polyporaceae (Ryvarden and RPB2 were used for molecular phylogenetic 1991) and is included phylogenetically in the strongly analyses of the collected the thinness of the clustered Fomitopsis-Daedalea-Piptoporus group charac- wall of the basidiospores that typifies the genus terized by brown rot and a bipolar mating system Rhodofomes proved to be of no significant generic (Hibbett and Thorn 2001). The genus is characterized value and our new polypore occupied a unique by sessile to effused-reflexed basidiocarps with white to lineage separated from F. cajanderi and F. rosea. rose-colored pore surfaces; di- to trimitic hyphal systems The fungus was related closely to F. rosea (5 R. with clamps in the generative hyphae; clavate basidia roseus) in our study, but the phylogenetic results with four sterigmata; hyaline, cylindrical or allantoid to rejected the genetic concept of Rhodofomes and subglobose, and smooth-walled basidiospores; and suggested that the fungus should be placed in brown rot activity on living or dead wood (Donk Fomitopsis. In this study we propose a new member 1974, Carranza-Morse and Gilbertson 1986, Gilbertson of Fomitopsis, based on an evaluation of the generic and Ryvarden 1986, Ryvarden and Gilbertson 1993). concepts of Fomitopsis and Rhodofomes through Fomitopsis rosea (Alb. & Schwein.) P. Karst. and its morphological observation of basidiocarps and phy- taxonomic position in the genus has been discussed logenetic analyses of molecular sequences.

Accepted for publication 8 August 2007. MATERIALS AND METHODS 1 Present address: Polar Biocenter, Korea Polar Research Institute, KORDI, Songdo Techno Park, 7-50 Songdo-dong, Yeonsu-gu, Specimens and morphological studies.—Specimens examined Incheon 406-840, Korea. in this study are listed (TABLE I). Those for the new 2 Corresponding author. E-mail: [email protected] polypore were numbered SNU (Seoul National University

833 3 M 834

TABLE I. Fungal strains used in this study

Sequencing Strains Sourcesb Substrates Localities PCR primersc primersc GenBank accessionsc

Antrodia albida CBS 308.82 Unknown Wisconsin, USA A, B(C), E H, C, E DQ491414, DQ491441, DQ491387 A. heteromorpha CBS 200.91 Trunk of Abies balsamea Nova Scotia, Canada A, B(C), E H, C, E DQ491415, DQ491442, DQ491388 A. juniperina CBS 117.40 Dead Thuja occidentalis Maryland, USA A, B(C), E H, C, E DQ491416, DQ491443, DQ491389 A. serialis CBS 306.82 Picea abies Reinhausen, Germany A, B(C), D(E) H, C, E DQ491417, DQ491444, DQ491390 A. variiformis CBS 309.82 Gymnosperm log New York, USA A, B(C), E H, C, E DQ491418, DQ491445, DQ491391 A. xantha CBS 155.79 Pinus sp. Teutoburger Wald, Germany A, B(C), E I, C, E DQ491424, DQ491451, DQ491397 Buglossoporus CBS 858.72 Unknown Eberswalde, Germany A, B(C), E G, C, E DQ491419, DQ491446, DQ491392 pulvinus Daedalea quercina DSM 4953 Quercus sp. Germany A, B(C), E H, C, E DQ491425, DQ491452, DQ491398 Fomes meliae CBS 179.34 Unknown Canada A, B(C), E I, C, E DQ491421, DQ491448, DQ491394 Fomitopsis africana MUCL 43284 Dead stump of angiosperm Cameroon A, B(C), E F, C, E DQ491422, DQ491449, DQ491395 F. cajanderi HOU 10773 Picea ajanensis Primorsk, Russia A, B(C), E I, C, E DQ491413, DQ491440, DQ491386 F. cajanderi CBS 127.24 Unknown USA A, B(C), D(E) G, C, E DQ491407, DQ491434, DQ491380 F. cajanderi CBS 195.37 Picea canadensis Quebec, Canada A, B(C), E G C, E DQ491399, DQ491426, DQ491372 F. cupreo-rosea CBS 236.87 Unknown Costa Rica A, B(C), E G, C, E DQ491400, DQ491427, DQ491373 F. dochmia CBS 426.84 Unknown Doi Inthanond, Thailand A, B(C), E G, C, E DQ491401, DQ491428, DQ491374 YCOLOGIA F. feei CBS 424.84 Unknown Michoacan, Mexico A, B(C), E G, C, E DQ491402, DQ491429, DQ491375 F. incarnatus a SNU m-05072501 Base of dead Fraxinus Kangwon province, Korea A, B(C), D(E) G, C, E DQ491409, DQ491436, DQ491382 mandshurica F. incarnatus SNU m- 04010313 Timber of Pinus sp. Kangwon province, Korea A, B(C), D(E) H, C, E DQ491411, DQ491438, DQ491384 F. lilacinogilva CBS 422.84 Unknown New South Wales, Australia A, B(C), D(E) G, C, E DQ491403, DQ491430, DQ491376 F. palustris CBS 283.65 Unknown Georgia, USA A, B(C), E H, C, E DQ491404, DQ491431, DQ491377 F. pinicola SFC 02101132 Base of Pinus sp. Chungcheongnam province, A, B(C), E G, C, E DQ491408, DQ491435, DQ491381 Korea F. pinicola CBS 221.39 Unknown British Columbia, Canada A, B(C), E G, C, E DQ491405, DQ491432, DQ491378 F. rosea HOU 17408 Picea abies Bohim, Slovenia A, B(C), E H, C, E DQ491412, DQ491439, DQ491385 F. rosea ATCC 76767 Picea orientalis Trabzon, Turkey A, B(C), E H, C, E DQ491410, DQ491437, DQ491383 F. spraguei CBS 365.34 Unknown USA A, B(C), D(E) G, C, E DQ491406, DQ491433, DQ491379 Melanoporia nigra CBS 341.63 Unknown Washington DC, USA A, B(C), D(E) H, C, E DQ491420, DQ491447, DQ491393 Piptoporus betulinus CBS 378.51 Unknown Austria A, B(C), E H, C, E DQ491423, DQ491450, DQ491396

a Holotype. b Herbarium acronyms: HOU, University of Houston Herbarium; SNU, Seoul National University Herbarium; SFC, Seoul National University Fungus Collection. Culture center acronyms: ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen; MUCL, Mycothe`que de l’Universite´ catholique de Louvain; SFCC, Seoul National University Fungus Culture Collection. c PCR primers, sequencing primers and GenBank accessions are listed in order of nuclear ITS, mt-SSU and RPB2. Letters indicate the primers used in the primary PCR reaction and those within parentheses used in the second PCR reaction as follows. A: ITS5, ITS4; B: bRPB2-6F, bRPB2-7.1R; C: bRPB2-6F, bRPB2-7R; D: BMS05, BMS173; E: MS1, MS2; F: ITS5; G: ITS1; H: ITS4; I: T7 and SP6 primers of T vector. MO KIM ET AL: FOMITOPSIS INCARNATUS SP. NOV. 835

Herbarium) m-04010313 and m-05072501. Basidiocarps 10% glycerol, 0.17 M KH2PO4 and 0.72 M K2HPO4)at were examined in a mounting solution of 3% KOH (w/v) 37 C for 20 h. The plasmids of cultured E. coli were and 1% phloxine (w/v) under a light microscope. miniprepared with the Plasmid Spin Kit (GENENMED, Kornerup and Wanscher (1978) were consulted for color Daejeon, Korea). Miniprepared DNA was incubated with descriptions of specimens. the restriction enzyme EcoRI at 37 C for 4 h on 0.75% agarose gel to check the size of inserts. The inserted region Cultures, DNA extraction, PCR, cloning and sequencing.— was sequenced with T vector primers T7 and SP6 (TABLE I) Strains (TABLE I) were obtained from collection centers using the automated DNA sequencer. (ATCC, CBS, DSMZ and MUCL) and grown at 25 C on potato-dextrose agar (PDA). Total genomic DNA were Sequence alignments, phylogenetic analyses and genetic extracted from specimens and strains with the AccuPrepH distance.—Sequences of nuclear ITS, mt-SSU and RPB2 Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). were aligned with Clustal X v1.83 (Thompson et al 1997) PCR and sequencing primer information for three regions with default penalties for gaps. Ambiguous and uninforma- are listed (TABLE I) in this order: nuclear ITS, mitochon- tive variable characters were removed with BioEdit v5.0.9 drial SSU, and RPB2. Quick PCR Premix (GENENMED, (Hall 1999). The three aligned sequence datasets were Daejeon, Korea) and AccuPowerH PCR Premix (Bioneer, deposited in TreeBase (SN2821) and submitted to phylo- Daejeon, Korea) were used to amplify those regions, and genetic analyses. Parsimony analysis was conducted with a PCR reaction was conducted in a PTC100 Thermal Cycler a heuristic search method with PAUP 4.0b10 (Swofford (MJ Research, Watertown, Massachusetts). The nuclear ITS 2002), with tree bisection reconnection (TBR)branch region was amplified with primer set ITS5 and ITS4 (White swapping and unrestricted MAXTREES. To determine confi- et al 1990). Together with the primers used for PCR, ITS1 dence levels for internal nodes of the most parsimonious (White et al 1990) was used for sequencing. The degenerate trees 1000 nonparametric bootstrap replications (branch primers bRPB2-6F and bRPB2-7.1R (Matheny 2005) were swapping, TBR; MAXTREES, unrestricted) were used (Felsen- used to amplify the region between conserved motifs 6 and stein 1985). All parsimonious trees were rooted by outgroup 7 of RPB2 (Liu et al 1999, Matheny 2005). To obtain taxon, Buglossoporus pulvinus (Pers.) Donk. A partition a sufficient concentration for sequencing, all amplicons for homogeneity test (replicates 5 1000; branch swapping, TBR; RPB2 were diluted 503 and amplified and sequenced with MAXTREES, unrestricted; Farris et al 1994) was performed to degenerate primers bRPB2-6F and bRPB2-7R (Matheny check the compatibility for the dataset combining the 2005). Partial mt-SSU rDNA were amplified with primers sequences of the three regions (nuclear ITS, RPB2 and mt- MS1 and MS2 (White et al 1990). For the genomic DNA for SSU). Genetic distances between species were calculated which the former primer set functioned with a low affinity, with MEGA3 (Kumar et al 2004) under the Kimura 2- primers BMS05 and BMS173 (Hong et al 2002) were used parameter distance model. for the amplification of almost complete mt-SSU; the PCR product that was diluted 503 was amplified by MS1 and RESULTS MS2. PCR conditions for nuclear ITS and almost complete mt-SSU were accomplished respectively according to the Fomitopsis incarnatus K.M. Kim, J.S. Lee & H.S. Jung, methods of Ko and Jung (2002) and Hong and Jung (2004). sp. nov. FIG.1 PCR conditions for partial RPB2 were initial denaturation at Basidiocarpus perennis, sessilis, effuso-reflexus ad un- 95 C for 10 min, followed by denaturation at 94 C for gulatum, proximo 13 3 6 3 7 cm; superficies cum 1 min, primer annealing at 55 C for 1 min, extension at concentrico protuberatione, brunneolus canus ad cineras- 72 C for 1 min + 3 s/cycle for 39 cycles and a final centem nigrum; pororum subroseus-albidus ad roseus, pori extension at 72 C for 10 min. PCR conditions to amplify rotundi, 6–8 per mm; hymenium tubiformis, proximo 1– partial mt-SSU were primer annealing at 54 C for 30 s and 1.2 cm crassus; contextus brunneolus luteus, proximo 3– extension at 72 C for 30 s; the remaining conditions were 5 mm crassus; systema hypharum trimiticum; hyphae identical to those used for the PCR reaction of RPB2. generatoriae fibulatae, 2.3–3 mm latae; hyphae skeletaleae Amplified PCR products were detected on 0.75% agarose brunneolus luteus in KOH, aseptatae, 2.3–4 mmlatae; gel and purified with an AccuPrepH PCR Purification Kit hyphae ligativae ramificatae, brunneolus luteus in KOH, (Bioneer, Daejeon, Korea). Purified products were se- aseptatae, 1.8–3.4 mm latae; basidia clavata, 2-sterigmata, 15– quenced with the primer combinations (TABLE I) using an 19 3 4–6.3 mm; basidiosporae ellipsoidae, curvae, parietae ABI3700 automated DNA sequencer (Applied Biosystems, tenuae, hyalinae, laeves, 4.5–6.3 3 2.2–2.9 mm. Foster City, California). T vector cloning was conducted for HOLOTYPE: KOREA. Mount Chiak, Kangwon purified PCR products that generated uncertain or mixed Province, ca. 37u249N, 128u39E, ca. 400 m a.s.l., on base-calling in the sequencing reaction. Purified amplicons the base of Fraxinus mandshurica, 25 Jul 2005, J. S. and T vectors were ligased at 4 C for 12 h with pGEM-T Lee and K. M. Kim SNU m-05072501 (culture ex- EasyVector System I (Promega, Madison, Wisconsin). 5 Recombinant plasmids were transformed into Escherichia holotype SNU m-05072501 SFCC m-05072501). coli strain JM109 with MicroPulserTM Electroporator at Basidiocarps perennial, sessile, semicircular and 2.5 kV (BIO-RAD, Hercules, California). The E. coli colony broadly attached, effused-reflexed to ungulate, up to containing the recombinant plasmid was cultivated in 13 3 6 3 7 cm; upper surface with broad concentric terrific broth (tryptone: 12 g/L, yeast extract: 24 g/L, bulges, frequently fissured, brownish gray (10F2) to 836 MYCOLOGIA

FIG. 1. Basidiocarps (A) and microscopic characters (B) of F. incarnatus (holotype). 1. basidiospores; 2. basidia; 3. generative hyphae; 4. skeletal hyphae; 5. binding hyphae. Bars: A 5 2 cm; B 5 15 mm. grayish black (H1) with age; margin acute, becoming those of F. cajanderi and commonly tended to be light brown (6D5) to grayish black (H1); pore surface curved in contrast to those of F. rosea.WhileF. pinkish white (12A2); pores circular, 6–8 per mm; cajanderi and F. rosea have four sterigmata on hymenophore tubulate, stratified, up to 1–1.2 cm a basidium, F. incarnatus had only two. thick, sometimes separated by a thin layer of contextual tissue; context brownish yellow (5C8), Phylogenetic analyses and genetic distances.—From five azonate, fibrous or woody, up to 3–5 mm thick. specimens of Fomitopsis and 22 strains including Hyphal system trimitic; generative hyphae with Fomitopsis, Antrodia P. Karst., Buglossoporus Kotl. & clamps, 2.3–3 mm wide; skeletal hyphae thick-walled, Pouzar, Daedalea Pers., Fomes (Fr.) Fr., Melanoporia nonseptate, yellow to brown (5B7-5E7) in KOH, Murrill and Piptoporus P. Karst. the regions of nuclear straight, 2.3–4 mm wide; contextual binding hyphae ITS, partial RPB2 and partial mt-SSU were amplified thick-walled, nonseptate, much branched, yellow to and sequenced (TABLE I). All sequences generated in brown (5B7-5E7) in KOH, 1.8–3.4 mm wide; basidia this study were deposited in GenBank (TABLE I). The clavate, 2-sterigmate, 15–19 3 4–6.3 mm, simple region of nuclear ITS had sequences 592–630 bp septate at the base; basidiospores ellipsoid, frequently long. The aligned sequences 666 bp long with 232 curved, thin-walled, hyaline, smooth, 4.5–6.3 3 2.2– parsimony informative characters. Analyzing the 2.9 mm. aligned dataset of nuclear ITS sequences with the Etymology. ‘‘incarnatus’’: paler than pale pure red. parsimony method resulted in five most parsimonious Known distribution. Oak-pine mixed forests of the trees (tree length 5 949, CI 5 0.530, RI 5 0.575), one Taebaek Mountains, Kangwon Province, Korea. of which is shown (FIG. 2). In this phylogenetic tree Other specimen. Mount Taebaek, Kangwon Province, two strains of the new polypore F. incarnatus were 37u06915.70N, 128u55956.20E, ca. 1240 m a.s.l., on timber of strongly clustered together having a bootstrap value Pinus sp., 13 Jan 2001, J. S. Lee (SNU m-04010313, paratype). of 99%. The three species of F. cajanderi, F. rosea and Remarks. Fomitopsis incarnatus has a pinkish white F. incarnatus formed a monophyletic group (clade A) pore surface and effused-reflexed to ungulate basidio- with a 75% bootstrap value. Within this clade F. carps and is morphologically similar to the closely cajanderi was more closely grouped with F. rosea than related species, F. rosea and F. cajanderi. However the with F. incarnatus. Fomitopsis pinicola, together with size of the pores (6–8/mm) was apparently smaller Piptoporus betulinus (Bull.) P. Karst., F. palustris than those of F. rosea (3–5/mm) and F. cajanderi (4– (Berk. & M.A. Curtis) Gilb. & Ryvarden and F. meliae 5/mm). Together with the size of pores the pinkish (Underw.) Murrill, formed a separate clade (B) with white pore surface made it possible to discriminate the a79% bootstrap value. Clades A and B were separated species from the above two species. Microscopically the distinctly from species of Antrodia, Daedalea quercina basidiospores of F. incarnatus were less elongated than (L.) Pers. and F. dochmia (Berk. & Broome) Ryvarden MO KIM ET AL: FOMITOPSIS INCARNATUS SP. NOV. 837

FIG. 2. One of the five most parsimonious trees inferred from the nuclear ITS sequences (666 bp; tree length 5 949, CI 5 0.530, RI 5 0.575). Buglossoporus pulvinus was used as outgroup to root the tree. Nonparametric bootstrap values were shown above branches supported by more than 50% from 1000 replications; bold lines were used where branches were supportedby more than 90%. and had a paraphyletic relationship to each other. nious trees, but branch exchanges existed between Fomitopsis cupreorosea (Berk.) J. Carranza & Gilb., F. the groups. lilacinogilva (Berk.) J.E. Wright & J.R. Deschamps, F. The region between conserved motifs 6 and 7 of feei (Fr.) Kreisel, F. spraguei (Berk. & M.A. Curtis) RPB2 was sequenced and aligned to be 648 bp long Gilb. & Ryvarden and F. africana Mossebo & Ryvarden with 256 parsimony informative sites. In five species, were grouped together independently and moderate- F. pinicola, F. palustris, F. meliae, Antrodia xantha ly supported by 60% bootstrap value. In an excep- (Fr.) Ryvarden and F. spraguei, it was found that one tional one of most parsimonious trees this clade amino acid residue was inserted additionally. Parsi- clustered paraphyletically with the group of A. serialis mony analysis based on the nucleotide sequences of (Fr.) Donk and A. variiformis (Peck) Donk, main- RPB2 generated four equally parsimonious trees (tree taining the separation of clades A from B. The group length 5 1109, CI 5 0.445, RI 5 0.599), one of which of A. juniperina (Murrill) Niemela & Ryvarden and F. is shown (FIG. 3). The phylogenetic tree of RPB2 dochmia and the group of A. serialis and A. variiformis showed a somewhat different topology compared to maintained a sister relationship in all most parsimo- that of the nuclear ITS. In clade A (FIG.3)F. rosea was 838 MYCOLOGIA

FIG. 3. One of the four most parsimonious trees inferred from the RPB2 sequences (648 bp; tree length 5 1109, CI 5 0.445, RI 5 0.599). Buglossoporus pulvinus was used as outgroup to root the trees. Nonparametric bootstrap values were shown above branches supported by more than 50% from 1000 replications; bold lines were used where branches were supportedby more than 90%. more closely related to F. incarnatus than to F. clade B and the group of F. cupreorosea, F. feei, F. cajanderi with a bootstrap value of 57%. Melanoporia lilacinogilva, F. africana, F. dochmia and D. quercina. nigra (Berk.) Murrill positioned at the basal line of The partial region of the mt-SSU had sequences the nuclear ITS tree (FIG. 2) was clustered with clade 493–625 bp long. The edited alignment consisted of A as the closest sister taxon. The group that 639 characters among which 211 sites were parsimony comprised F. cupreorosea, F. feei, F. lilacinogilva, F. informative. Parsimony analysis based on the aligned africana, F. dochmia and D. quercina had a paraphy- dataset of mt-SSU yielded one most parsimonious letic relationship with clade A. Among the species tree (tree length 5 619, CI 5 0.637, RI 5 0.770). As outside clades A and B in the nuclear ITS tree (FIG.2) shown in the trees of nuclear ITS (FIG. 2) and RPB2 the group of Antrodia albida (Fr.) Donk, A. hetero- (FIG. 3) clades A and B members also were main- morpha (Fr.) Donk and A. xantha were clustered by tained in the mt-SSU tree (FIG. 4). Within clade A F. 75% bootstrap value and positioned on the basal line cajanderi and F. incarnatus formed a strongly sup- of the RPB2 tree (FIG. 3). The variable topologies ported group (bootstrap 5 92%), which was clustered shown at higher relationships among clade A, clade B to F. rosea with 84% bootstrap support. The group and their sister taxa also were indicated by bootstrap including clade A and its four sister taxa was values of less than 50% at inner nodes (FIGS. 2, 3). separated from the group including clade B and its Four most parsimonious trees generated from RPB2 seven sister taxa by 100% bootstrap support (FIG. 4). sequences showed the same topology for clade A, The group of F. cupreorosea, F. feei, F. lilacinogilva MO KIM ET AL: FOMITOPSIS INCARNATUS SP. NOV. 839

FIG. 4. The most parsimonious tree inferred from the mt-SSU rDNA sequences (639 bp; tree length 5 619, CI 5 0.637, RI 5 0.770). Buglossoporus pulvinus was used as outgroup to root the trees. Nonparametric bootstrap values were shown above branches supported by more than 50% from 1000 replications; bold lines were used where branches were supportedbymorethan90%. and F. africana was related more closely to clade B DISCUSSION than to A. Most taxa whose phylogenetic positions were Fomitopsis incarnatus was distinguished macromor- variable on the trees of nuclear ITS and RPB2 were placed as single lineages or clustered groups, which phologically from F. rosea and F. cajanderi by its were supported by bootstrap of 50% or less. The pinkish white pore surface. In addition the number of partition homogeneity test on the combined dataset of pores (6–8/mm) makes it possible to discriminate three regions (nuclear ITS, RPB2 and mt-SSU) this fungus from F. cajanderi (4–5/mm; Gilbertson generated a P value of 0.001. Because this test result and Ryvarden 1986, Ryvarden and Gilbertson 1993) was much lower than the significance level of 0.05, and F. rosea (3–5/mm; Gilbertson and Ryvarden 1986, subsequent phylogenetic analyses were not conducted Ryvarden and Gilbertson 1993). Microscopically the for the combined dataset. Genetic distances of nuclear basidiospores (4.5–6.3 3 2.2–2.9 mm) of F. incarnatus ITS, RPB2 and mt-SSU were estimated (FIG. 5) for the were slightly shorter and less elongated than those specimens and strains of F. cajanderi, F. incarnatus and (5–7 3 1.5–2 mm; Gilbertson and Ryvarden 1986, F. rosea. Fomitopsis incarnatus diverged from F. Ryvarden and Gilbertson 1993) of F. cajanderi cajanderi and F. rosea by sequence differences of (FIG. 1B). While the spores of F. rosea (5.5–7.5 3 2– 0.032–0.073, but the sequence variations within the 2.5 mm; Gilbertson and Ryvarden 1986, Ryvarden and species was 0–0.013. Gilbertson 1993) are straight, those of F. incarnatus 840 MYCOLOGIA

new species it was necessary to evaluate whether the segregation of Rhodofomes from Fomitopsis is proper. Genus Rhodofomes is characterized by thin-walled basidiospores when compared with F. pinicola.In addition the presence of clamps on thin-walled generative hyphae, the rose-colored context and the absence of a resinous crust on the upper surface of the basidiocarps were defined as generic keys for Rhodofomes (Kotlaba and Pouzar 1990, 1998). Genus Pilatoporus Kotl. & Pouzar typified by P. palustris (5 F. palustris) also was described by thin-walled basidio- spores together with the presence of pseudoskeletal hyphae (Kotlaba and Pouzar 1990). In our phyloge- FIG. 5. Genetic distances among F. incarnatus, F. netic trees (FIGS.2–4) F. pinicola was strongly cajanderi and F. rosea. The values in a circle show the clustered with F. palustris, P. betulinus and F. meliae sequence differences within a species. The genetic distances in clade B (bootstrap values: ITS 5 79%, RPB2 5 between two species are indicated on the baseline and 100%, mt-SSU 5 100%) while F. rosea was separated hypotenuses. The numbers within the parentheses were distinctly from F. pinicola. If the spore wall thickness listed in the order of nuclear ITS, PRB2 and mt-SSU markers. is significant enough for the generic delimitation between Rhodofomes and Fomitopsis then the thin- frequently were curved. The widths of generative and walled character of the spores might have been binding hyphae are similar among the three species, derived synapomorphically from a common ancestor but the skeletal hyphae (2.3–4 mmwide)ofF. of the group that comprises F. rosea and its closely incarnatus were thinner than those of the two other related taxa, while such a character has been absent in species (2.5–6 mm for F. cajanderi, 4–6 mm for F. rosea; the species of the group that comprises F. pinicola Gilbertson and Ryvarden 1986, Ryvarden and Gilbert- and its relatives. However F. palustris with thin-walled son 1993). Although the basidia of the three species basidiospores was distinctly separated from the were nearly identical in their dimensions and shapes, lineage of F. rosea and was strongly clustered with F. only two sterigmata were observed in F. incarnatus pinicola with thick-walled basidiospores (Kotlaba and unlike four sterigmata in the other two species Pouzar 1990) as well as P. betulinus and F. meliae (Gilbertson and Ryvarden 1986, Ryvarden and Gil- (FIGS. 2–4). This indicates that the character of thin- bertson 1993). Fomitopsis incarnatus possesses suffi- walled basidiospores has evolved autapomorphically cient morphologically different features to differenti- in the phylogeny of Fomitopsis, Antrodia, Daedalea, ate it from the two closely related taxa, F. cajanderi Fomes, Melanoporia and Piptoporus (FIGS. 2–4). and F. rosea. The rose-colored context along with the wall In the three phylogenetic trees (FIGS. 2–4), F. thickness of the basidiospores also was suggested by cajanderi, F. incarnatus and F. rosea always formed Kotlaba and Pouzar (1990, 1998) to be of importance a monophyletic group (clade A, FIGS. 2–4) moderate- for segregation of Rhodofomes from Fomitopsis. In our ly supported respectively by bootstrap values of 75%, phylogenetic trees (FIGS. 2–4) members of the F. rosea 69% and 84% in ITS, RPB2 and mt-SSU trees. complex consisting of F. cajanderi, F. cupreorosea, F. However the strains of each species were tightly dochmia, F. feei, F. lilacinogilva and F. rosea (Carranza- clustered at the species level by bootstrap values of Morse and Gilbertson 1986) developed polyphyletic 93% to mostly 100% (FIGS. 2–4). The low within- lineages, indicating that the rose-colored context is species divergence (0–1.3%) and the high between- not an absolute key that can define Rhodofomes. Based species divergence (2.7–7.3%)showedthateach on present phylogenetic analyses it is suggested that species has an independent genetic boundary the characters of the spore wall thickness and the (FIG. 5). The bootstrap values and genetic distances context color have no independent generic values to indicate that the new species occupies an indepen- separate Rhodofomes from other Fomitopsis species, dent specific lineage that can be characterized and which coincides with the view of Ryvarden and separated from those of F. cajanderi and F. rosea Gilbertson (1993) that the character of the rose- (FIGS. 2–5); thus it comes under the new phylogenetic colored context is not enough to establish a new species concept defined by Cracraft (1983). genus from Fomitopsis. The phylogenetic character of Fomitopsis incarnatus formerly was related closely to F. incarnatus (FIGS. 2–4) indicates that the species F. rosea (5 R. roseus) in three phylogenetic trees; should be placed in Fomitopsis as a new member and therefore to determine the generic position of the F. rosea (5 R. roseus) needs to be retained in MO KIM ET AL: FOMITOPSIS INCARNATUS SP. NOV. 841

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