mycoscience 56 (2015) 481e485

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Short communication A new violet brown Aureoboletus () from Guangdong of China

* Ming Zhang a,b, Tai-Hui Li a,b, , Jiang Xu 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 Institute of Microbiology, Guangzhou 510070, China article info abstract

Article history: Aureoboletus marroninus sp. nov. is described, illustrated and compared with phenetically Received 6 June 2014 similar and phylogenetically related species. Morphologically, it is characterized by its Received in revised form small basidioma with a pileus of 13e22 mm broad, reddish brown to brownish violet or 11 February 2015 violet brown, glutinous and wrinkled pileus fringed with gelatinized veil remnants at the Accepted 12 February 2015 margin, and basidiospores of (8e)8.5e10 4e4.5(e5) mm in size. Phylogenetic analysis of Available online 18 March 2015 the new species and related taxa based on large subunit of nuclear ribosomal RNA gene (LSU) sequences is provided. Morphological and molecular data show that the new species Keywords: should be placed in genus Aureoboletus, and it is clearly different from any known taxon. © 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.

The genus Aureoboletus was circumscribed by Pouzar (1957) collection is actually a species of Boletus (Zang et al. 1993; with A. gentilis (Quel.) Pouzar as the type species, renowned Klofac 2010); and the species A. reticuloceps has been trans- for its viscid pileus and attractive bright yellow and un- ferred into the genus Boletus based on morphological and changing hymenophore. It is a small genus in the family molecular evidences (Zang et al. 1993; Wang and Yao 2005; Boletaceae and comprises 14 known species and varieties Dentinger et al. 2010; Feng et al. 2012). Recently a new spe- (Klofac 2010, http://www.Indexfungorum.org). Until now, 5 cies belonging to Aureoboletus was discovered from southern species, i.e. A. gentilis (Quel.) Pouzar, A. reticuloceps M. Zang, China, and its formal description, photographs of basidiomata M.S. Yuan & M.Q. Gong, A. tenuis T.H. Li & M. Zhang, A. thibe- and the phylogenetic analysis on the basis of LSU sequences tanus (Pat.) Hongo & Nagas. (ss. Patouillard 1895, non Hongo are provided in this article. and Nagasawa 1980) and A. zangii X.F. Shi & P.G. Liu, have Photographs of basidiomata were taken in the type locality been reported from China (Patouillard 1895; Zang et al. 1993; when collected. Specimens were dried and deposited in the Ying and Zang 1994; Yang et al. 2003; Zang 2006; Shi and Liu Fungal Herbarium of Guangdong Institute of Microbiology 2013; Zhang et al. 2014b). However, the Chinese record of A. (GDGM). Descriptions of macromorphology and the habitat gentilis should be excluded because the misidentified were based upon field notes and photos. Colors were recorded

* Corresponding author. School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China. Tel./ fax: þ86 20 87137619. E-mail address: [email protected] (T.-H. Li). http://dx.doi.org/10.1016/j.myc.2015.02.002 1340-3540/© 2015 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved. 482 mycoscience 56 (2015) 481e485

and described in general terms following Kornerup and changing to purplish pink or bluish in places; tubes yellowish Wanscher (1978). Micromorphological features were white initially, later grayish yellow to olive yellow; stipe observed on dried material after sectioning and mounting in glutinous, with white basal mycelium; basidiospores (8e) 5% KOH solution, 1% Congo Red or Melzer's reagent. The no- 8.5e10 4e4.5(e5) mm. Differs from A. thibetanus by having a tation (ae)bec(ed) was used to describe basidiospores di- much smaller, darker (brownish red to violet brown) pileus mensions, where the range bec represents 90% or more of the and shorter basidiospores. measured values and ‘a’ and ‘d’ were the extreme values. Q Type: China, Guangdong Province, Chebaling National referred to the length/width ratio of an individual basidio- Nature Reserve, at 24430N, 114120E, alt. 500 m, 11 July 2013, J. spore; Qm referred to the average Q of all Xu, T. Li, B. Song and W. Wang (holotype, GDGM 43288). basidiospores ± sample standard deviation. Sections were LSU sequence ex-holotype: KJ488958. studied at a magnification of up to 1000 using an Olympus Etymology: The specific epithet marroninus indicates the BX51 microscope. All line-drawings of microstructures were maroon color of the pileus. made from rehydrated material and based on microscopic Basidiomata small (Fig. 1). Pileus 13e22 mm broad, obtuse images. to convex, becoming broadly convex to plane, reddish brown Genomic DNA of the new species was extracted from the (9D8e9E8), brownish red to brownish violet or violet brown herbarium specimen using the Sangon Genomic DNA (maroon) (10C6e10D8, 11D6e11F6, 11D8e11F8 to 13D6e13F6), Extraction kit (Sangon Biotech, Shanghai, China) according to slightly paler towards margin, distinctly wrinkled and often the manufacturer's instructions. The LSU region was ampli- reticulate irregularly with somewhat darker folds at center, fied with primers LR0R and LR5 (Vilgalys and Hester 1990). PCR strongly glutinous when fresh; margin nearly flat and often was performed in a total volume of 20 ml containing 0.5 ml appendiculate with yellowish white to subhyaline, strongly template DNA, 8.5 ml distilled water, 0.5 ml of each primer, and gelatinized veil remnants. Context 2e3 mm thick at the stipe, 10 ml PCR mix [DreamTaq™ Green PCR Master Mix (2), Fer- firm and tough in youth, soft when matured, white on the mentas, Vilnius, Lithuania]. Amplification reactions were whole, purplish red (13A3e13B6, 14A3e14C5) to greyish performed in a Tprofessional standard thermocycler (Bio- magenta (13D6e14D6) beneath the pileipellis, practically un- metra, Gottingen,€ Germany) under the following conditions at changing or slightly changing to purplish pink (14A3) when 95 C for 4 min, then followed by 35 cycles of denaturation at exposed, often slightly changing to blue in places especially in 95 C for 60 s, annealing at 53 C for 60 s, extension at 72 C for the lower portion of the stipe. Tubes 4e5 mm deep, distinctly 80 s and a final extension at 72 C for 8 min. The PCR products depressed around stipe, yellowish white (2A2e4A2) when was electrophoresed on 1% agarose gels with known standard young, becoming pale yellow, greyish yellow, pastel yellow to ® DNA marker, and sequenced was performed on an ABI Prism olive yellow (2A3e4A3, 1B3e2B3, 2A4e3A4, 2C6e3C6) in age, 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, often with an olive tint, practically unchanging or slightly USA) at Beijing Genomic Institute (BGI) using the same bruising blue. Pores 0.5e0.8 mm in diam., mostly subangular, primers as in the PCR amplification. The sequence of the ho- slightly radially elongated around stipe at maturity, smaller lotype was submitted to GenBank. Other LSU sequences of near pileus margin, concolourous with tubes. Stipe related taxa were downloaded from GenBank. 13e20 2e4 mm, central, cylindrical or narrowly clavate, Sequences were aligned with additional sequences down- solid, equal to slightly enlarged downwards, pale red, pink to loaded from GenBank using ClustalX (Thompson et al. 1997). purplish pink (11A3e12A3, 13A3, 14A3), without reticulation, Alignment was manually adjusted to allow maximum align- smooth to faintly longitudinally striate, gelatinous or strongly ment and to minimize gaps using BioEdit (Hall 1999), and the viscid when young and wet, with white basal mycelium. Odor aligned sequence data matrix has been deposited in TreeBASE and Taste mild. (ID: 15947). Maximum likelihood (ML) analysis was applied to Basidiospores (8e)8.5e10 4e4.5(e5) mm, Q ¼ (1.9e) e e ¼ ± the LSU dataset. In phylogenetic construction, sequence of 2.0 2.4( 2.5), Qm 2.2 0.1, ellipsoid, smooth, yellow to Suillus luteus (L.) Roussel was used as an outgroup (Li et al. yellowish brown in 5% KOH and yellow brown to dark brown 2011). The tree construction procedure was performed in in Melzer's reagent, thin-walled. Basidia 4-spored, MEGA 5.2 (Tamura et al. 2011). According to the Bayesian in- 20e23 8e10 mm, clavate, yellowish white to hyaline in 5% formation criterion (BIC) score, a Kimura 2-parameter model KOH, yellow to yellowish brown in Melzer's reagent, without of nucleotide substitution with a gamma distributed rate basal clamps; sterigmata 2.5e4 mm long. Pleurocystidia heterogeneity and a proportion of invariant sites (K2þGþI) 35e62 7e11 mm, fusiform, thin-walled, yellowish white to was chosen as the optimal substitution model (Tamura et al. hyaline in 5% KOH. Cheilocystidia infrequent, similar to 2011). Clade robustness was assessed using a bootstrap anal- pleurocystidia. Hymenophoral trama subparallel to nearly ysis with 1000 replicates. Bootstrap values greater than or bilateral, yellow to hyaline in 5% KOH, composed of branching equal to 50% were shown on the ML tree. hyphae 6e8 mm wide. Pipeipellis in an early stage of devel- opment usually an ixotrichodermium, composed of Taxonomy frequently septate, thin-walled hyphae of 4e6 mm in diam. embedded in a gelatinous layer, brownish to yellowish white Aureoboletus marroninus T.H. Li & Ming Zhang, sp. nov. in 5% KOH, yellowish white in Melzer's reagent, terminal cells Figs. 1, 2. 26e37 6e12 mm, cylindrical, clavate or nearly fusoid. Stip- MycoBank no.: MB 809068. ipellis a tangled layer of repent to suberect branching hyphae Pileus small, violet brown, wrinkled, glutinous, with veil 5e7 mm in diam., yellowish white to hyaline in 5% KOH, sub- remnants at margin; flesh white, unchanging to slightly gelatinous; terminal elements 30e50 3e7 mm, long and mycoscience 56 (2015) 481e485 483

Fig. 1 e Basidiomata of Aureoboletus marroninus (holotype, GDGM43288). Bars: 20 mm.

Fig. 2 e Microstructures of Aureoboletus marroninus (holotype, GDGM43288). A: Basidiospores. B: Pleurocystidia. C: Basidia. D: Pileipellis. E: Stipipellis. Bars:10mm. 484 mycoscience 56 (2015) 481e485

slender, cylindrical, rounded-obtuse at apex. Caulocystidia type locality of A. marroninus, have been conducted regularly not observed. Stipe trama made up of densely arranged par- for dozens of times since 1980's(Bi et al. 1993; Deng and Li allel hyphae, 3e12 mm broad. Clamp connections absent in all 2011; Wang et al. 2013; Zhang et al. 2014a), but specimens of tissues. the new species were obtained only once in 2013, and no more Habitat: Under Fagaceae (Cyclobalanopsis spp.) and Lophir- basidiomes appeared again in the type locality during several aceae trees. following investigations in 2013 and 2014, as well as in the Known distribution: Solitary or scattered on soil in a broad- neighboring areas in Guangdong, Jiangxi and Hunan prov- leaved forest dominated by Fagaceae (Cyclobalanopsis spp.) and inces. The small and viscid basidiomes seems to be easily Lophiraceae. Only known from Chebaling National Nature decayed and need strict environmental requirements for Reserve, Guangdong Province, China. growing, which might be the main reasons for its scarcity and infrequency. Molecular results Distinctive features of this taxon includes the small and slender basidiomata, maroon colored, glutinous and wrinkled The phylogenetic tree (Fig. 3) based on the LSU dataset using pileus appendiculate with yellowish white to subhyaline veil ML analyses shows that all the sampled Aureoboletus species remnants at the margin, and relatively short basidiospores of in the tree were resolved as a clade with a strong support (8e)8.5e10 4e4.5(e5) mm. In addition, the slightly cyanes- (Bootstrap value ¼ 93%). The new species A. marroninus was cent nature in the stipe flesh and tubes also seems to be nested in the well-supported Aureoboletus clade, suggesting characteristic, but the taxonomic significance of this char- that A. marroninus should be a member of this genus. This acter should be confirmed based on more material. Such a species formed a sister clade with A. thibetanus, but the combination of characters is quite unique and makes the new phylogenetic distances between the two species were 0.022 taxon easily separable from the other species in the genus. (AY700189 A. thibetanus), 0.018 (KJ907380 A. thibetanus) and Aureoboletus marroninus might be confused with a Chinese 0.018 (KJ907381 A. thibetanus), respectively. These differences species A. thibetanus due to a glutinous and wrinkled pileus are considered to represent interspecific differences, although fringed with yellowish white to subhyaline veil remnants at noting that LSU is generally inferior to the internal transcribed the margin. However, A. thibetanus has more robust basidioma spacer (ITS) region in species discrimination and has a less (pileus up to 5 cm), white ridged reticulation on pileus surface, clearly defined barcode gap (Schoch et al. 2012) Thus, A. mar- longer basidiospores (9.0e15.0 4.0e5.5 mm), and yellowish roninus is genetically and morphologically distinct from all the granular incrustation on cystidia (Patouillard 1895; Yang et al. bolete species. 2003; Klofac 2010). An AsiaePacific taxon A. auriporus (Peck) Pouzar var. novoguineensis (Hongo) Klofac is also similar to A. Remarks marroninus on account of a subrugose, viscid and reddish brown pileus, but different in the more robust basidiomata The new species should be a rare or endangered taxon. Sur- (pileus up to 5 cm diam., stipe up to 7 1 cm), and larger ba- veys of macrofungi in Chebaling national nature reserve, the sidiospores (11.5e15.5 4.5e5.5 mm) (Hongo 1973; Klofac

Fig. 3 e Phylogenetic tree of Aureoboletus marroninus and related species based on LSU sequences generated by ML method in MEGA5.2. Suillus luteus was chosen as an outgroup. Bootstrap values (≥50%) are shown above or beneath individual branches. mycoscience 56 (2015) 481e485 485

2010). Another Chinese species A. zangii although has small Kornerup A, Wanscher JH, 1978. Methuen handbook of colour. Eyre basidiomata, it can be easily distinguished from A. marroninus Methuen, London. by its smooth and greyish orange to yellowish brown pileus, Li YC, Feng B, Yang ZL, 2011. Zangia, a new genus of Boletaceae supported by molecular and morphological evidence. Fungal absence of marginal veil remnants, larger basidiospores Diversity e e e m e e m 49: 125 143; http://dx.doi.org/10.1007/s13225-011- (10.5 11.0 4.5 5.0 m) and basidia (30 38 11 14 m) (Shi 0096-y. and Liu 2013). Patouillard N, 1895. Enumeration des champignons recolt es par les RR. PP. Farges et Soulie, dans le Thibet oriental et Su- tchuen. Bulletin de la Societe Mycologique de France 11: 196e199. Acknowledgments Pouzar Z, 1957. Nova genera macromycetum I. Ceska Mykologie 11: 48e50. 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