mycoscience 57 (2016) 20e25

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Short communication A new of (, ) with strongly radially arranged pores

* Ming Zhang a,b, Chao-Qun Wang c,d, Tai-Hui Li a,b, , Bin Song b a School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China b State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China c South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China d University of Chinese Academy of Sciences, Beijing 100049, China article info abstract

Article history: Chalciporus radiatus sp. nov., discovered in southern China, is described and illustrated as a Received 13 March 2015 new species based on both morphological and molecular data. Morphologically, it is very Received in revised form distinctive in its brown to reddish brown decurrent hymenophore with pores strongly 18 July 2015 radially arranged and appearing sublamellate at maturity. The arrestive hymenophore, Accepted 20 July 2015 combined with the yellowish white flesh discoloring to light yellow when bruised, the mild Available online 1 September 2015 taste flesh and spore size of 7e8 (3e)3.5e4 mm, can easily distinguish the new species from all the known Chalciporus taxa. Phylogenetic analysis based on the ITS and LSU se- Keywords: quences are provided. © 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. New taxon

Chalciporus Bataille, a small in the family Boletaceae, Betulaceae and Fagaceae) (Singer 1962, 1986; Degreef and contains about 28 known species (Klofac 2006; Degreef and Kesel 2008). Kesel 2008; http://www.indexfungorum.org). The main char- The genus was established in 1908 with C. piperatus (Bull.) acters of the genus include the dull red, ocher-red, reddish Bataille as the type species (Bataille 1908). However, it was not cinnamon, pink to salmon pink instead of yellow nor grey to widely accepted as an independent genus for a long time: it pallid hymenophore, non-reticulated stipe with yellow basal was often treated as a section (Piperati) of the genus (or mycelium, peppery tastes, and short to elongated ellipsoid Ixocomus)(Singer 1938, 1962), or linked to the genus Pulver- smooth spores (Singer 1938, 1962, 1986; Klofac 2006; Degreef oboletus (Singer 1938, 1962; Coker and Beers 1943; Smith and and Kesel 2008; Watling 2008). Chalciporus species are widely Thiers 1971). With further studies, the genus Chalciporus was distributed in the world, and can usually form erratic mycor- finally accepted and the genus Rubinoboletus was merged into rhizal associations with seed plants (mostly conifers, Chalciporus according to their morphological similarities

* Corresponding author. State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Mi- crobial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China. Tel./fax: þ86 20 87137619. E-mail address: [email protected] (T.-H. Li). http://dx.doi.org/10.1016/j.myc.2015.07.004 1340-3540/© 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. mycoscience 57 (2016) 20e25 21

(Singer 1973, 1986; Pegler and Young 1981; Klofac 2006). LSU sequence matrix were aligned using ClustalX (Thomson Recently, phylogenetical-systematic studies also proved that et al. 1997), and then slightly manually edited in BioEdit (Hall Rubinoboletus should be placed within the genus Chalciporus, 1999). Final alignments of ITS and LSU sequences were and the genus Chalciporus was supported as a monophyletic deposited in TreeBASE with submission ID 17226. Phyloge- group (Nuhn et al. 2013; Wu et al. 2014). In addition, a sub- netic analyses were performed in MEGA 5.2 (Tamura et al. family Chalciporoideae G. Wu & Zhu L. Yang was proposed to 2011) using the maximum likelihood method after testing accommodate the genera Pilat and Chalcipo- the best models. rus (Wu et al. 2014). In the LSU dataset, 44 sequences with 925 positions were During a recent study on the species diversity of boletes in included, and Suillus aff. luteus (L.) Roussel (KF112430) served southern China, three collections of a Chalciporus species were as an outgroup (Zeng et al. 2012). The analysis of ITS dataset gathered and taxonomically proved to be a species new to included 19 nucleotide sequences with a total of 711 posi- science through morphological comparisons and phyloge- tions in the final dataset, and sequences of Buchwaldoboletus netic analyses. lignicola (Kallenb.) Pilat (HM003617, HM003618, HM003619) In this study, photographs and records of basidiomata served as outgroup. According to the AICc (Akaike Informa- were obtained in the field. Specimens were dried in an tion Criterion, corrected) value, a general time reversible electric drier and finally deposited in the Fungal Herbarium with a gamma distributed rate heterogeneity and a propor- of Guangdong Institute of Microbiology (GDGM). De- tion of invariant sites (GTR þ G þ I) was chosen as the scriptions of macro-morphological characters and the optimal substitution model for the LSU analysis; for the ITS habitat were based upon field notes and photos. Colors were analysis, a Kimura 2-parameter model of nucleotide substi- described in general terms following Kornerup and tution with a gamma distributed rate heterogeneity (K2 þ G) Wanscher (1978). Micro-morphological features were was chosen (Tamura et al. 2011). Clade robustness was observed from dried materials after sectioning and assessed using a bootstrap analysis with 1000 replicates. mounting in 5% KOH solution, 1% Congo Red or Melzer's Bootstrap values greater than or equal to 50% were indicated reagent under a light microscope (Olympus BX51, Tokyo, along nodes. Japan). For basidiospore descriptions, an abbreviation [n/m/ p] denotes n spores measured from m basidiomata of p Taxonomy collections. A notation (ae)bec(ed) was used to describe basidiospore dimensions, where the range bec represented Chalciporus radiatus Ming Zhang & T.H. Li sp. nov. Figs. 1, 2. 90% or more of the measured values and ‘a’ and ‘d’ were the MycoBank no.: MB 811750. extreme values. Q referred to the length/width ratio of an Differs from C. chontae by the greyish yellow to brownish individual basidiospore; Qm referred to the average Q of all orange pileus, radially arranged porous hymenophore, basidiospores ± sample standard deviation. All line- yellowish white flesh changing to light yellow when bruised, drawings of microstructures were made from microscopic and smaller basidiospores of 7e8 (3e)3.5e4 mm. images based on rehydrated materials. Type: CHINA, Hunan Province, Chenzhou city, Rucheng Total genomic DNA was extracted from each voucher town, Jiulongjiang National Forest Park, at 25380N, 113780E, specimen using the Sangon Genomic DNA Extraction alt. 230 m, 1 Aug 2013, Ming Zhang (Holotype, GDGM 43285) kit (Sangon Biotech, Shanghai, China) according to the man- (Figs. 1A, 2). ufacturer's instructions. The internal transcribed spacer (ITS) Etymology: The specific epithet indicates the strongly and nuclear large subunit (LSU) regions were amplified by radially arranged hymenophore. Polymerase Chain Reaction, using universal primers ITS1 and Sequences: LSU and ITS sequences generated from the ITS4 (White et al. 1990), and LR0R and LR7 (Vilgalys and Hester holotype are KP871800 and KP871804, respectively. 1990), respectively. PCR was performed in a total volume of Pileus 10e30 mm wide, hemispheric at first, then 25 ml containing 0.5 ml template DNA, 0.5 ml of each primer, becoming convex to nearly plane with age, fleshy, surface 11 ml distilled water, and 12.5 ml PCR mix [DreamTaq™ Green dry, smooth to velvety-tomentose, greyish orange, greyish PCR Master Mix (2), Fermentas, Vilnius, Lithuania]. Amplifi- red to light brown (6B4e6D4, 7B4e7D4) when young, cations were performed in a Tprofessional standard thermo- becoming greyish yellow (4B3e4B6, 4C3e4C4), greyish orange cycler (Biometra, Gottingen,€ Germany) under the following (5B3e6B3), greyish brown (5D4e6D4) to brownish orange conditions: pre-denaturation at 95 C for 4 min, then followed (5C3e6C3) when old, with a thin and incurved margin, by 35 cycles of denaturation at 95 C for 60 s, annealing at 53 C slightly decurved and even with age. Flesh 5e8mmthickat (ITS)/56 C (LSU) for 60 s, extension at 72 C for 80 s and a final stipe, soft, yellowish white to pale yellow (2A2e4A2, extension at 72 C for 8 min. The PCR product was electro- 2A3e4A3), gradually changing to pastel yellow to light yellow phoresed on 1% agarose gels with known standard DNA (1A4e2A4, 1A5e2A5) when exposed to air. Tubes sub- ® marker, and sequenced on an ABI Prism 3730 Genetic decurrent to decurrent, shallow, 3e4 mm deep, light orange Analyzer (PE Applied Biosystems, Foster, CA, USA) at Beijing (5A6e6A6) to pastel red (7A4) initially, becoming yellowish Genomic Institute (BGI) using the same primers as in the PCR brown (5D8), brown (6D8e7D8) to reddish brown (8D8) when amplifications. The newly gained 3 ITS and 4 LSU sequences mature, unchanging when bruised. Pores 1e2 mm in diam, have been deposited in GenBank. circular to angular, radially arranged when young, seemingly Apart from sequences obtained from this study, 16 ITS sublamellate at maturity, pore-surface concolorous with sequences and 40 LSU sequences were chosen from GenBank tubes. Stipe 30e40 3e8 mm, central, solid, subcylindrical or and UNITE databases for phylogenetic analysis. Both ITS and clavate, equal or slightly tapered downwards, distinctly 22 mycoscience 57 (2016) 20e25

Fig. 1 e Basidiomata of Chalciporus radiatus. A: GDGM 43285 (holotype). B: GDGM 50080 (paratype). Bars: 10 mm.

punctate with brown to reddish brown (6D8e7D8, 8D8) scales smooth, yellowish white to hyaline, and usually containing on a yellow to reddish yellow (2A6e4A6) ground color, often some yellowish brown oleiferous scattered substance in 5% reddish brown (8D8) at maturity, sometimes concolorous KOH. Cheilocystidia 48e70 6e15 mm, abundant and con- with pores, basal mycelium vivid yellow (2A8e3A8). Stipe spicuous, fusoid to subclavate, smooth, thin-walled, hyaline context yellowish white to pale yellow (2A2e3A2, 2A3e3A3), or more frequently with scattered dark yellowish brown changing to light yellow (2A5e3A5) when exposed. Odor content. Hymenophoral trama subparallel, yellowish white to none and taste mild. hyaline in 5% KOH, with hyphae 8e12 mm broad. Pileipellis Basidiospores [50/4/2] 7e8 (3e)3.5e4 mm, Q ¼ (1.75e) trichodermium of erect hyphae 5e7 mm in diam, branched, 2e2.2(e2.5), Qm ¼ 2.12 ± 0.18, fusiform, cylindrical to some- yellowish white to hyaline in 5% KOH, dark brown to rusty what oblong, barely inequilateral in profile, smooth, thin- brown in Melzer's reagent; terminal cells 15e40 5e6 mm, walled, yellowish to yellowish brown in 5% KOH and yellow cylindrical, clavate or nearly fusoid. Stipitipellis a tangled brown to dark brown in Melzer's reagent. Basidia layer of repent to suberect hyphae 5e7 mm in diam, forming 18e23 6e8 mm, 4-sterigmate, clavate, yellowish white to the dotted ornamentation over surface of stipe, yellowish hyaline in 5% KOH. Pleurocystidia 50e70 7e13 mm, scattered, white to hyaline in 5% KOH, end cells 19e25 5e8 mm. Clamp projecting and conspicuous, narrowly fusiform, thin-walled, connections absent in all tissues.

Fig. 2 e Microstructures of Chalciporus radiatus (drawing from the holotype, GDGM 43285). A: Basidiospores. B: Basidia. C: Pileipellis. D: Cheilocystidia. E: Pleurocystidia. F: Stipitipellis. Bars:A,B,D,E10mm; C, F 20 mm. mycoscience 57 (2016) 20e25 23

Additional specimens examined: CHINA, Hunan, Chenz- Molecular results hou city, Rucheng town, Jiulongjiang National Forest Park, at 25380N, 113770E, alt. 300 m, 1 Aug 2013, Ming Zhang (GDGM A total of 44 fungal samples representing 34 taxa were used in 50080). CHINA, Guangdong Province, Chebaling National Na- the LSU analysis, and the ML tree generated in this study ture Reserve, at 24430N, 114150E, alt. 500 m, 11 Oct 2013, (Fig. 3) showed a similar topology in main branches as those in Chao-Qun Wang (GDGM 43305). the previous studies (Nuhn et al. 2013; Wu et al. 2014), and Habitat and distribution: Solitary or scattered on the Chalciporus was supported as a monophyletic group. The new ground under Cunninghamia lanceolata (Lamb.) Hook, Cyclo- species nests into the well supported Chalciporus Clade, but balanopsis spp. and Castanopsis spp., at 230e500 m alt. obviously differs from the other chosen morphologically Currently known from Guangdong and Hunan provinces, similar species with known sequences, i.e., C. amarellus (Quel.) China. Bataille, C. pseudorubinellus (A.H. Sm. & Thiers) L.D. Gomez,

Fig. 3 e Phylogenetic tree of Chalciporus radiatus and related species based on LSU sequences generated by maximum likelihood method in MEGA 5.2. Suillus aff. luteus was chosen as an outgroup. Bootstrap values (>50%) are shown above or beneath individual branches. The scale represents the number of nucleotide substitutions per site. 24 mycoscience 57 (2016) 20e25

C. piperatus and C. rubinellus (Peck) Singer. An analysis of ITS 2006). Chalciporus pseudorubinellus, originally reported from (Fig. 4) sequences also showed that the new species forms an Colombia, is different in having pinkish red to cinnamon independent branch, suggesting it is a very distinct taxon. pileus, smooth stipe, and also large spores (9e13 3e4 mm) (Smith and Thiers 1971; Gomez 1996; Klofac 2006). Chalciporus Remarks rubinellus, originally described from North America, is distin- guished by the reddish to yellowish pileus, smooth stipe, and Chalciporus radiatus is distinguished by its brown to reddish large spores (up to 15 5 mm) (Smith and Thiers 1971; Singer brown hymenophore with pores strongly radially arranged 1973; Klofac 2006). and appearing sublamellate at maturity, yellowish white flesh It is noteworthy that although Chalciporus species are changing to light yellow when bruised, mild taste, smaller widely distributed in the world, only C. piperatus has been spores of 7e8 (3e)3.5e4 mm, and abundant cystidia con- reported in China and adjacent countries. In the geographic taining yellowish brown substance. Such a combination of distribution, species of the genus Chalciporus are mainly characters is quite unique and makes this fungus easily distributed in temperate and tropical areas, and in the sub- separate from the known species under Chalciporus. tropical area, only C. radiatus is found by now except the The most phenotypically similar relative is C. chontae Hal- world-wide distributed species, C. piperatus (Klofac 2006; ling & M. Mata, which shares similar color and hymenophore Degreef and Kesel 2008). The new species C. radiatus is the shape with the new species. However, C. chontae, originally first taxon of Chalciporus with type locality in southern China; reported from Costa Rica, obviously differs in having a tacky to it should be of mycofloral importance and phylogeographical subviscid and lemon yellow to pale brown pileus, pale significance. According to previous records of the known taxa brownish pink flesh without discoloration, and thick-walled and their distributions, most species within Chalciporus are and larger spores (9e10.5 3.5e4.2 mm) (Halling et al. 2004; found to be endemic to certain continents, and have relatively Klofac 2006). narrow distribution ranges; and taxa with intercontinental The following species are also similar to C. radiatus in some distributions were uncommon. The discovery of the new respects, but they can be distinguished morphologically. The species enriches the knowledge about Chalciporus diversity European species C. amarellus differs in having larger spores and distribution, which could be an important evidence for (12.5e14 5e6 mm) (Klofac 2006). The widely distributed the phylogeographical analysis of the genus. In the phyloge- species C. piperatus differs in having dull reddish brown to netic analysis, C. radiatus obviously represents an indepen- pinkish tan colored pileus, brownish-reddish hymenophore dent geographical branch in the trees although only a few changing to darker brown when injured, yellowish flesh with sequences of Chalciporus are available, which implies that it a very peppery taste, and larger spores (up to 12 5 mm) (Coker could be a good representative of subtropical Asian taxa. To and Beers 1943; Smith and Thiers 1971; Both 1993; Klofac further study the evolution and biogeography of Chalciporus

Fig. 4 e Phylogenetic tree of Chalciporus radiatus and related species based on ITS sequences generated by maximum likelihood method in MEGA 5.2. Buchwaldoboletus lignicola was chosen as outgroup. Bootstrap values (>50%) are shown above or beneath individual branches. The scale represents the number of nucleotide substitutions per site. mycoscience 57 (2016) 20e25 25

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