ORIGINAL ARTICLE

Three new species of Boletaceae from the Heishiding NatureReserve in Guangdong Province, China

Fang Li1 & Kuan Zhao2,3 & Qing-Li Deng1 & Ming Zhang4 & Christian Staehelin1&

Xiao-Xia Chen1& Hong-Ying Chen1

& Geng-Shen Wang1 & Bing-Sheng Li1

Received: 2 April 2016 /Revised: 13 September 2016 /Accepted: 21 September 2016 /Published online: 14 October 2016# German Mycological Society and Springer-Verlag Berlin Heidelberg 2016

Abstract Based on morphological evidence and DNA se-quencing, we propose three new species of the basidiomycetefamily Boletaceae, namely Aureoboletus venustus,Leccinellum onyx, and Pulveroboletus sinensis. The specieswere collected from the Heishiding Nature Reserve inGuangdong Province, China. The descriptions are illustratedby photographs and line drawings, and comparisons weremade with related species. Morphologically, A. venustus ischaracterized by its mucilaginous pileus with red to reddishorange color, a vivid yellow hymenophore surface, and smallellipsoid basidiospores (7) 7.5–10.5 (12) × 5–6 (7) μm in size.Leccinellum onyx is characterized by its slightly areolatingpileus with grayish orange to brownish orange color, a grayishbrown to light grayish orange hymenophore surface, shortyellowish white tubes, a yellowish white to brownish graystipe covered with dark brown warts, and small ellipsoid ba-sidiospores with a size of (8) 9–11 (12) × (4) 5–6 μm. Itsnucleotide sequence of the internal transcribed spacer (ITS)region is particularly long (1080 bp) in comparison to relatedspecies. Pulveroboletus sinensis is characterized by its yellow

to vivid yellow pulverulent pileus covered with deep orange tobrownish orange conico-pyramidal scales, an appendiculatemargin, greenish yellow tubes easily becoming cyanescentupon bruising, a yellow to vivid yellow stipe covered withyellow to brownish orange conico-floccose scales, and smallsmooth ellipsoid basidiospores with a size of (6) 6.5–8.5(11) × (4.5) 5–6 (6.5) μm. Phylogenetic analysis of nucleotidesequences of the ITS region and the nuclear large subunitribosomal RNA gene (nrLSU) provided further evidence thatthe described mushrooms represent new species.

Keywords Boletaceae . Aureoboletus . Leccinellum .

Pulveroboletus . Heishiding Nature Reserve . New taxa .

Systematics

Introduction

Mushrooms in the basidiomycete family Boletaceae are main-ly characterized by a fleshy context and a tubulose, rarelylamellate or loculate hymenophore. There are tremendous va-rieties in this family; about 50 genera with 800 species havebeen identified so far (Kirk et al. 2008; Wu et al. 2014).However, there are most likely more species and generaexisting in many tropics and subtropic areas which have hadlittle study and research. The Heishiding Nature Reserve (ab-breviated thereafter as Heishiding) is a part of those areas.This national nature reserve is located in the south central partof Fengkai County, Guangdong Province, about 250 km westof Guangzhou in the south of China. It is 4200 hectares in areaand the Tropic of Cancer runs through its center (111°49′09″–111°55′01″E, 23°25′15″–23°30′02″N). Heishiding has a cov-erage of 95.5%with subtropical evergreen broad-leavedmon-soon forest. The average annual precipitation is 1744 mm,with a humid season in the summer months (Yu et al. 2000).

Section Editor: Zhu-Liang Yang

* Fang Lilsslif@mail.sysu.edu.cn

1 School of Life Sciences, Sun Yat-sen University,Guangzhou 510275, China

2 Key Laboratory for Plant Diversity and Biogeography of East Asia,Kunming Institute of Botany, Chinese Academy of Sciences,Kunming 650201, China

3 University of Chinese Academy of Sciences, No. 19AYuquan Road,Beijing 100049, China

4 State Key Laboratory of Applied Microbiology (Southern China),Guangdong Institute of Microbiology, Guangzhou 510070, China

Mycol Progress (2016) 15:1269–1283DOI 10.1007/s11557-016-1233-5

Since November 2008, the first author of this article has beensurveying the mushrooms of Heishiding: over 500 specimensfrom 50 species and 18 genera of Boletaceae have been col-lected. Three of them, Aureoboletus venustus, Leccinellumonyx, and Pulveroboletus sinensis, are described and illustrat-ed herein as new species.

Aureoboletus, Leccinellum, and Pulveroboletus are threesmall genera of the Boletaceae. The genus Aureoboletus wascircumscribed by Z. Pouzar (1957) with A. gentilis (Quél.)Pouzar as the type species. The basidiomata of this genusare characterized by a mucilaginous pileus and a bright yellowand unchanging hymenophore surface when bruised. The ge-nus comprises about 20 known species, six of which wereoriginally described from China (Patouillard 1895; Klofac2010; Yang et al. 2003; Shi and Liu 2013; Zhang et al.2014; Zeng et al. 2015; Zhang et al. 2015a, b). The genusLeccinellum was circumscribed by A. Bresinsky and M.Binder in 2003 to contain Leccinum species with a yellowhymenophore surface and a trichoderm-like cap cuticle, withLeccinellum nigrescens (Singer) Bresinsky & M. Binder asthe type species. The genus contains about ten known species(Bresinsky and Besl 2003; ht tps: / /en.wikipedia .org/wiki/Leccinellum; http://www.Indexfungorum.org).Finally, the genus Pulveroboletus was first described by theAmerican mycologist W.A. Murrill in 1909, withPulveroboletus ravenelii as the type species. Species of thisgenus are characterized by a basidioma with a pulverulent dryveil sheathing the stipe, covering the mature pileus, andleaving a ring-like zone near the stipe apex (Murrill 1909;Corner 1972). The genus contains about 26 species (Kirket al. 2008; Raspé et al. 2016), and about ten of them havebeen reported to occur in China (Chiu 1957; Teng 1996; Liand Song 2003; Zang 2006; Wu et al. 2014).

Materials and methods

Morphology

The description of the new species in this article is based onmorphological studies of fresh material and exsiccata.Specimens were annotated and photographed in the field.Collected material was dried by an electric stove. For micro-scopic examination, tissues were mounted in 5 % KOH,ddH2O, and 1 % Congo Red. Microscopy was performedusing a Motic BA310-T at 100×, 400×, or 1000× magnifica-tion. The color code reported by Kornerup and Wanscher(1978) was used. For the description of basidiospores, theabbreviation (n/m/p) stands for n basidiospores measuredfrom m basidiomata of p collections. The dimensions for ba-sidiospores are given using a range notation of the form (a) b–c (d). The range b–c contains a minimum of 90 % of themeasured values. Extreme values (a and d) are given in

parentheses. Q = length/width ratio of a basidiospore in sideview; Qm = average Q of all basidiospores measured ± samplestandard deviation. Holotype collections of the new specieswere deposited at the Herbarium of Cryptogams, KunmingInstitute of Botany, Chinese Academy of Sciences,Kunming, China (HKAS). Furthermore, additional specimenswere deposited at the HKAS and the Fungal Herbarium ofGuangdong Institute of Microbiology, Guangzhou, China(GDGM).

DNA extraction, PCR amplification, and DNA sequencing

Genomic DNAwas extracted from silica-gel-dried basidiomataaccording to a modified hexadecyltrimethylammonium bro-mide (CTAB) protocol (Doyle 1987). Universal primers,ITS1F or ITS5 in combination with ITS4, were used for theamplification of the internal transcribed spacer (ITS) region.DNA of the nuclear large subunit ribosomal RNA gene(nrLSU) was amplified with the primers LROR and LR5(Vilgalys and Hester 1990). The polymerase chain reaction(PCR) amplifications were performed according to Zeng et al.(2013) and references therein by using an ABI 2720 ThermalCycler (Applied Biosystems, Foster City, CA, USA). The PCRproducts were purified using a Gel Extraction and PCRPurification Combo Kit (Spin-column) (Bioteke, Beijing,China). Sequencing was performed with an ABI 3730 DNAanalyzer (Applied Biosystems, Foster City, CA, USA), usingthe same primer pairs used for the PCRs.

Phylogenetic analyses

According to the sequences already existing in the GenBankand their ability to recognize and distinguish species, nrLSUwas selected as the gene marker to conduct phylogenetic anal-yses on Aureoboletus and Leccinum, and ITS was used forPulveroboletus. A total of 104 sequences were used for phy-logenetic analysis in this study: 92 were retrieved from thenucleotide database of GenBank (http://www.ncbi.nlm.nih.gov), while 12 sequences pertain to this study. The scientificnames, collection information, and GenBank accessionnumbers for the sequences used in the datasets are presentedin Table 1.

DNA sequences were aligned with MAFFT v6.8 (Katohet al. 2005) and then manually optimized in BioEdit v7.0.9(Hall 1999). Bayesian inference (BI) andmaximum likelihood(ML) methods were used to analyze the datasets. Substitutionmodels of partition in the dataset were determined using theAkaike Information Criterion (AIC) implemented inMrModeltest v2.3 (Nylander 2004). BI and ML were per-formed using MrBayes v3.1.2 (Ronquist and Huelsenbeck2003) and RAxML v7.2.6 (Stamatakis 2006), respectively.The partitioned mixed model was used to estimate the modelparameters for each gene separately. K80 + I and GTR + I + G

1270 Mycol Progress (2016) 15:1269–1283

Table 1 Voucher information and GenBank accession numbers of specimens included in the molecular phylogenetic analyses of this study

Taxon Voucher Location GenBank accession numbers References

nrLSU ITS

Aureoboletus auriporus BDCR0431 Costa Rica HQ161871 – Dentinger et al. (2010)

Aureoboletus auriporus MAN020 Costa Rica JQ003659 – Neves et al. (2012)

Aureoboletus citriniporus REH8719 USA KF030298 – Nuhn et al. (2013)

Aureoboletus clavatus GDGM42984 Hainan, China KR052047 – Zeng et al. (2015)

Aureoboletus clavatus HKAS59802 Hainan, China KR052044 – Zeng et al. (2015)

Aureoboletus clavatus GDGM42963 Hainan, China KR052046 – Zeng et al. (2015)

Aureoboletus formosus GDGM 44441 China KT291749 – Zhang et al. (2015a, b)

Aureoboletus formosus GDGM 44444 China KT291750 – Zhang et al. (2015a, b)

Aureoboletus gentilis MG372a Italy KF112344 – Wu et al. (2014)

Aureoboletus gentilis Pug1 Bavaria, Germany DQ534635 – Binder and Hibbett (2006)

Aureoboletus innixus MB 03-104 USA KF030239 – Nuhn et al. (2013)

Aureoboletus innixus 136 USA KF030240 – Nuhn et al. (2013)

Aureoboletus longicollis HKAS 59808 Hainan, China KR052048 – Zeng et al. (2015)

Aureoboletus longicollis HKAS 59810 Hainan, China KR052050 – Zeng et al. (2015)

Aureoboletus longicollis GDGM42849 Guangdong, China KR052051 – Zeng et al. (2015)

Aureoboletus marroninus GDGM43288 China KJ488958 – Zhang et al. (2015a, b)

Aureoboletus mirabilis REH 8717 USA KF030299 – Nuhn et al. (2013)

Aureoboletus mirabilis REH 9765 USA KP327661 – Halling et al. (2015)

Aureoboletus mirabilis HKAS 57776 Yunnan, China KF112360 – Wu et al. (2014)

Aureoboletus moravicus MG374a China KF112421 – Wu et al. (2014)

Aureoboletus moravicus PARMA 1544/11 – KJ676958 – Unpublished

Aureoboletus moravicus Xle1 Germany DQ534641 – Binder and Hibbett (2006)

Aureoboletus projectellus NYBG13392 USA KP327622 – Binder and Hibbett (2006)

Aureoboletus projectellus MB 03-118 USA NG_027638 – Halling et al. (2015)

Aureoboletus roxanae DS626-07 NY, USA KF030311 – Nuhn et al. (2013)

Aureoboletus tenuis N.K.Zeng1296 Hainan, China KR052052 – Zeng et al. (2015)

Aureoboletus tenuis GDGM 42601 China KF534789 – Zhang et al. (2015a, b)

Aureoboletus thibetanus GDGM43283 China KJ907380 – Zhang et al. (2015a, b)

Aureoboletus thibetanus GDGM43284 China KJ907381 – Zhang et al. (2015a, b)

Aureoboletus thibetanus HKAS41151 Yunnan, China AY700189 – Dentinger et al. (2010)

Aureoboletus venustus HKAS 77700 Guangdong, China KU321703 KU321702 This study

Aureoboletus venustus HKAS 82180 Guangdong, China – KU321704 This study

Aureoboletus venustus HKAS 82183 Guangdong, China KU321705 – This study

Aureoboletus venustus HKAS 87635 Guangdong, China – KU360242 This study

Aureoboletus zangii HKAS 63219 Guizhou, China JQ734422 – Shi and Liu (2013)

Aureoboletus zangii HKAS 63217 Shanxi, China JQ734420 – Shi and Liu (2013)

Aureoboletus zangii HKAS 63220 Guizhou, China JQ734421 – Shi and Liu (2013)

Boletellus ananas TH8819 Guyana HQ161853 – Dentinger et al. (2010)

Boletellus ananiceps REH9690 Queensland, Australia KP327660 – Halling et al. (2015)

Borofutus dhakanus HKAS 73785 Bangladesh NG_042663 – Hosen et al. (2013)

Leccinellum aff. griseum KPM-NC-0017381 Hyogo, Japan JN378508 – Orihara et al. (2012)

Leccinellum aff. griseum KPM-NC-0017832 Tottori, Japan JN378509 – Orihara et al. (2012)

Leccinellum albellum TH6968 – AY612811 – Drehmel et al. (2008)

Leccinellum corsicum 931101/1GL France AF139693 – Binder and Besl (2000)

Mycol Progress (2016) 15:1269–1283 1271

Table 1 (continued)

Taxon Voucher Location GenBank accession numbers References

nrLSU ITS

Leccinellum corsicum Buf 4507 – KF030347 – Nuhn et al. (2013)

Leccinellum crocipodium RC/F-94.103 France HQ161870 – Dentinger et al. (2010)

Leccinellum crocipodium 930809/1 France AF139694 – Binder and Besl (2000)

Leccinellum quercophilum M Kuo 07120801 Charleston, USA KC691208 – Kuo et al. (2013)

Leccinellum onyx HKAS 92188 Guangdong, China KU321699 KU360243 This study

Leccinellum onyx HKAS 82191 Guangdong, China KU321701 KU321700 This study

Leccinum aurantiacum La1 Germany AF139689 – Binder and Besl (2000)

Leccinum cerinum MK11800 Finland AF139692 – Binder and Besl (2000)

Leccinum duriusculum GL 4676 Germany AF139699 – Binder and Besl (2000)

Leccinum duriusculum 880904/4 GL France AF139695 – Binder and Besl (2000)

Leccinum duriusculum GR92103 France AF139690 – Binder and Besl (2000)

Leccinum flavostipitatum 24/98 USA AF139696 – Binder and Besl (2000)

Leccinum holopus 9109303 France AF139700 – Binder and Besl (2000)

Leccinum holopus Lh2 Germany AF139697 – Binder and Besl (2000)

Leccinum manzanitae TDB-969 California, USA DQ534613 – Binder and Hibbett (2006)

Leccinum melaneum Lm 1 Regensburg, Germany DQ534616 – Binder and Hibbett (2006)

Leccinum scabrum KPM-NC-0017840 Scotland, UK JN378515 – Orihara et al. (2012)

Leccinum variicolor Lvar1 Germany AF139706 – Binder and Besl (2000)

Leccinum versipelle Lv2 Germany AF139707 – Binder and Besl (2000)

Octaviania asahimontana KPM-NC-0017823 Hokkaido, Japan JQ619185 – Orihara et al. (2012)

Octaviania asahimontana KPM-NC-0017824 Hokkaido, Japan JN378489 – Orihara et al. (2012)

Octaviania asterosperma Oct. 1 Bavaria, Germany DQ534619 – Binder and Hibbett (2006)

Octaviania asterosperma AQUI3899 Italy KC552052 – Unpublished

Octaviania cyanescens OSC58498 British Columbia, JN378503 – Orihara et al. (2012)

Octaviania nigrescens mes270 Maine, USA JN378498 – Orihara et al. (2012)

Phylloporus bellus HKAS 56763 Yunnan, China JQ967196 – Zeng et al. (2013)

Pulveroboletus ravenelii 165-56 Japan – AB509511 Unpublished

Pulveroboletus ravenelii 165-113 Japan – AB509552 Unpublished

Pulveroboletus ravenelii 165-116 Japan – AB509555 Unpublished

Pulveroboletus ravenelii 158-132 Japan – AB509565 Unpublished

Pulveroboletus ravenelii 3699 Canada – KM248937 Unpublished

Pulveroboletus sinensis HKAS 75767 Guangdong, China KT824250 KT824248 This study

Pulveroboletus sinensis HKAS 89483 Guangdong, China KT824251 KT824249 This study

Pulveroboletus brunneoscabrosus HKAS 90212 Guangdong, China KT923682 KT923678 This study

Pulveroboletus brunneoscabrosus HKAS 90213 Guangdong, China KT923683 KT923677 This study

Pulveroboletus brunneoscabrosus HKAS 90214 Guangdong, China KT923684 KT923676 This study

Pulveroboletus sp. HKAS 74926 Yunnan, China KT923685 KT923680 This study

Pulveroboletus sp. HKAS 74933 Yunnan, China KF112407 KT923679 This study

Pulveroboletus sp. Chu21 Yunnan, China – DQ407259 Unpublished

Pulveroboletus sp. 222 Zhejiang, China – HE814131 Gao et al. (2013)

Pulveroboletus sp. 268 Zhejiang, China – HE814134 Gao et al. (2013)

Pulveroboletus sp. 2042 Zhejiang, China – HE814141 Gao et al. (2013)

Pulveroboletus sp. – Zhejiang, China – HE817957 Gao et al. (2013)

Pulveroboletus sp. TH8232 Guyana – JN168736 Smith et al. (2011)

Pulveroboletus sp. TH9154B Guyana – JN168737 Smith et al. (2011)

Pulveroboletus sp. TH9221 Guyana – KT339273 Unpublished

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were selected as substitution models for ITS and nrLSU, re-spectively. All parameters in the ML analysis were kept as thedefault set t ings, except for the model chosen asGTRGAMMAI, and statistical support values were obtainedusing nonparametric bootstrapping with 1000 replications. BIanalyses using selected models and four chains were conduct-ed by setting generations to 3 million and the stoprul com-mandwith the value of stopval set to 0.01. Trees were sampledevery 100 generations. The first 25 % of generations werediscarded as burn-in, and Bayesian posterior probabilities(PP) were then calculated from the posterior distribution ofthe retained Bayesian trees.

For phylogenetic analysis, outgroups were chosen based onrecent studies on the phylogeny of Boletaceae and the relatedgenera, i.e., Aureoboletus, Leccinum, and Pulveroboletus(Binder and Besl 2000; Nuhn et al. 2013; Wu et al. 2014;Zeng et al. 2015; Zhang et al. 2015a, b). Thus, for the phylo-genetic analysis of Aureoboletus, the outgroups wereBoletellus ananas (M.A. Curtis) Murrill, Boletellus ananiceps(Berk.) Singer, Phylloporus bellus (Massee) Corner,Hourangia cheoi (W.F. Chiu) Xue T. Zhu & Zhu L. Yang,and Xerocomus subtomentosus (L.) Quél. For the phylogenet-ic analysis of Leccinellum, the outgroups were Borofutusdhakanus Hosen & Zhu L. Yang, Retiboletus griseus (Frost)Manfr. Binder & Bresinsky, Tylopilus sp. (HKAS50281), andXerocomus subtomentosus (L.) Quél. Neither Leccinum nor

Leccinellum were monophyletic without integratingRossbeevera and Octaviania (Wu et al. 2014). Therefore,these two gasteroid genera were also included in the phyloge-netic study of Leccinellum. For the phylogenetic analysis ofPulveroboletus, the outgroups were Rubroboletus sinicus(W.F. Chiu) Kuan Zhao & Zhu L. Yang and Suillellus aff.amygdalinus (HKAS57262).

Results

The nrLSU datasets of Aureoboletus and Leccinellumcontained 874 and 924 nucleotide sites (including gaps), re-spectively. For the dataset of Aureoboletus, 205 characterswere variable, of which 146 characters were parsimony-informative; for the dataset of Leccinellum, 270 characterswere variable, of which 209 characters were parsimony-informative.

The ITS dataset of Pulveroboletus had an aligned length of898 nucleotide sites (including gaps), of which 427 characterswere variable and 332 characters were parsimony-informative.

The phylogenetic trees inferred from the ML analysis wereconsistent with those obtained from the BI analysis, and, thus,only the ML trees are shown in Figs. 3, 6, and 9. These phy-logenetic data together with thorough morphological analyses

Table 1 (continued)

Taxon Voucher Location GenBank accession numbers References

nrLSU ITS

Pulveroboletus sp. ECM148 Zhejiang, China – JQ991773 Unpublished

Retiboletus griseus 202/97 MA, USA AF456834 – Binder and Bresinsky (2002)

Rossbeevera eucyanea KPM-NC-0023895 Japan KP222896 – Orihara et al. (2012)

Rossbeevera eucyanea KPM-NC-0023911 Japan KP222897 – Orihara et al. (2012)

Rossbeevera griseovelutina KPM-NC-0018094 Japan KJ001101 – Orihara et al. (2012)

Rossbeevera griseovelutina KPM-NC-0018044 Japan KC552033 – Orihara et al. (2012)

Rossbeevera sp. KPM-NC-0023387 Japan KP222899 – Orihara et al. (2012)

Rossbeevera vittatispora MEL2321058 Australia KP222895 – Orihara et al. (2012)

Rossbeevera vittatispora MEL2329434 Australia KJ001097 – Orihara et al. (2012)

Rossbeevera westraliensis MEL2231712 Australia HQ647162 – Orihara et al. (2012)

Rossbeevera yunnanensis HKAS 70601 China KF112448 – Wu et al. (2014)

Tylopilus sp. HKAS 50281 Yunnan, China KF112451

Xerocomus subtomentosus Xs1 Germany AF139716

Rubroboletus sinicus HKAS 56304 Yunnan, China – KJ605666 Zhao et al. (2014)

Suillellus aff. amygdalinus HKAS 57262 Tibet, China – KT923681 This study

Tylopilus sp. HKAS 50281 China KF112451 – Wu et al. (2014)

Hourangia cheoi HKAS 74744 Yunnan, China KF112384 – Wu et al. (2014)

Xerocomus subtomentosus Xs1 Germany AF139716 – Binder and Besl (2000)

Sequences produced in this study are shown in bold

Mycol Progress (2016) 15:1269–1283 1273

(see below) show that the three newly described taxa of thisstudy are significantly distinguished from other knownspecies.

Taxonomy

Aureoboletus venustus Fang Li, Kuan Zhao&Qing-Li Deng,sp. nov. (Figs. 1, 2, and 3)

MycoBank: MB816327Etymology: Bvenustus^ means charming, lovely, or grace-

ful in Latin, referring to the pretty basidiomata.

Holotype: China, Guangdong Province, Heishiding(111°49′09″–111°55′01″E, 23°25′15″–23°30′02″N), alt.180 m, 14 August 2012, Fang Li 798 (HKAS77700; nrLSUand ITS sequences generated from the holotype are depositedin GenBank under the accession numbers KU321703 andKU321702, respectively).

Basidiomata (Fig. 1) small to medium-sized. Pileus 3–8 cm, convex, plano-convex to plane, with slightlyincurved entire margin projecting ca. 1 mm beyond thetubes; thickly mucilaginous; red to brownish red (9B8–8C8) when young, reddish brown (9D8–9E8) to reddishorange (7A8–7B8) with age; tubes adnate to adnexed, upto 10 mm long, pale yellow (3A3) to yellow (3A7); pores

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Fig. 1 Basidiomata of Aureoboletus venustus. a, b, d, e Mature basidiomata (holotype). c, f Young basidiomata (HKAS87635). Scale bars: 1 cm

ca. 0.5–1 mm in diam., vivid yellow (3A8) when young,light yellow (3A5) to pastel yellow (3A4) at maturity,slightly depressed around stipe, no discoloring. Stipe 5–13 × 0.8–1.7 cm, thickly mucilaginous, cylindrical or cla-vate, solid, lightly or obviously enlarged downwards, ta-pering towards the base, pastel red, pale red to reddishwhite (7A4–7A2) when young, grayish red (7B4–7B6) toreddish orange (7A7–7B7) when mature, with apical partlight colored, basal mycelia white. Context firm, up to10 mm in the center of the pileus, white to white with lightreddish (7A2) tinge, no discoloring. Taste mild.

Spores (Fig. 2a) [200/10/3] (7) 7.5–10.5 (12) × 5–6 (7) μm,Q = (1.21) 1.42–1.85 (2.24), Qm = 1.59 ± 0.19, mostly ellip-soid, occasionally broadly ellipsoid or elongate, grayish yel-low, with slight to no suprahilar depression, 0.5–0.8 μm thick-walled, apiculus small. Basidia 20–28 × 8–10 μm, clavate, 4-spored. Pleurocystidia 40–62 × 9–13 μm, with walls 0.5–1 μm thick, lageniform to subventricose. Cheilocystidia 13–24 × 7–10 μm, as sterile basidia, spheropedunculate orsubventricose. Tube-trama subparallel to bilateral, with hy-phae 4–10 μm wide, hyaline, yellowish white, scarcely diver-gent. Pileal trama composed of interwoven hyphae 4–16 μmwide, yellowish white (4A2), thin-walled, divergent. Stipetrama composed of parallel hyphae 4–14 μm wide, yellowish

white (4A2), thick-walled (0.5–1 μm thick), scarcely diver-gent. Pileipellis (Fig. 2e) ca. 70–110 μm thick, anixotrichodermium, composed of cylindrical thick-walled(0.5–1 μm thick) hyphae 3–10 μm wide, orange (5A8–6A8), thickly glutinous, branched, with end cells tapering,bullet-form to clavate, 15–40 × 5–10 μm. Stipitipellis(Fig. 2f) composed of parallel thick-walled (0.5–1 μm thick)hyphae 4–14 μm wide, giving rise to clusters of caulocystidiaand caulobasidia mixed with excrescent hyphae 4–8 μmwide.Caulocystidia 16–40 × 5–24 μm, clavate, fusiform tospheropedunculate, with wall 0.5–3 μm thick. Clamp connec-tions absent in all parts of basidioma.

Habitat and distribution: Scattered to gregarious on soil inforests dominated by Fagaceae, at 180 m alt.; presently onlyknown from Heishiding, Guangdong Province, China.

Additional specimens examined: China, GuangdongProvince, Heishiding, alt. 180 m, 11 August 2013, Fang Li1425 (HKAS82180); the same place, alt. 180 m, 12 August2013, Fang Li 1432 (HKAS82183); the same place, alt.180 m, 12 July 2014, Fang Li 1810 (HKAS87635).

Leccinellum onyx Fang Li, Kuan Zhao & Qing-Li Deng,sp. nov. (Figs. 4, 5, and 6)

MycoBank: MB816339Etymology: Bonyx^ means yellowish marble in Latin, and

precious stone in English, referring to the stone-like color andform of the pileus.

Holotype: China, Guangdong Province, Heishiding(111°49′09″–111°55′01″E, 23°25′15″–23°30′02″N), alt.500 m, 20 July 2012, Fang Li 700 (HKAS92188; nrLSUand ITS sequences generated from the holotype are depositedin GenBank under the accession numbers KU321699 andKU360243, respectively).

Basidiomata (Fig. 4) medium-sized. Pileus 1.3–8 cm,globose, hemispherical at first, expanding to truncateconico-convex, convex, or plano-convex, with entire mar-gin slightly incurved projecting ca. 1 mm beyond thetubes; orange (5A6) when young as a small pearl, lightorange (5A4), grayish orange (5B4) to brownish orange(7 F3) when mature, dark brown (7 F4–7 F5) in exsiccates;dry, very minutely appressed tomentose, finely rugulose,slightly areolate in small irregular patches from margin tocenter, showing pale context in between; discoloring or-ange (6A8) to dark greenish blue (24 F8) after bruised infield with age. Tubes short, up to 6 mm long, adnexed toadnate, never ventricose, yellowish white (4A2). Pores ca.0.2–0.5 mm in diam., grayish brown (5D3), yellowishbrown (5D5) to light grayish orange (lighter than 5B3),discoloring dark greenish blue (24 F8) in wounds inducedby arthropods in the field with age. Stipe 5–13 × 0.8–2.7 cm, cylindrical to clavate, tapering towards the base,dark orange (5A8) and minutely tomentose at first, yellow-ish white (4A2), brownish gray (11C3) when matured,

Fig. 2 Microscopic characters of Aureoboletus venustus (holotype). aBasidiospores. b Basidia. c Pleurocystidia. d Cheilocystidia. ePileipellis. f Stipitipellis and caulocystidia. Scale bars: 10 μm

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entirely covered with grayish brown (6E3) to grayish ma-genta (14E3) longitudinal reticular ridges on which accu-mulated dark brown (6 F8) warts which are small and rareon the apex and base, but coarse and dense in the middlepart; mycelia at base light yellow (4A4). Context firm, upto 10 mm in the center of the pileus, white to yellowishwhite (2A2), slowly staining pinkish (7A2) near thepileipellis after being cut apart for more than 10 min.Spore deposit brown (5 F4). Taste mild.

Spores (Fig. 5a) [200/5/4] (8) 9–11 (12) × (4) 5–6 μm,Q = (1.64) 1.72–2.19 (2.3), Qm = 1.95 ± 0.11, ellipsoid withslight to no suprahilar depression, thin-walled, apiculus small,pale brown, often gathering in small (13–45 μm in diam.)yellowish brown (5 F8) particles in tubes in maturedbasidioma. Basidia 25–33 × 8–13 μm, clavate, 4-spored.Pleurocystidia 30–40 × 8–10 μm, lageniform. Cheilocystidia13–34 × 7–10 μm, as sterile basidia, subventricose orlageniform. Tube-trama subparallel to bilateral, with hyphae5–10 μm wide, hyaline, yellowish white, thin-walled, diver-gent. Pileal trama composed of interwoven hyphae 3–15 μmwide, yellowish white (4A2), thin-walled, divergent. Stipe

trama composed of parallel hyphae 4–20 μm wide, yellowishwhite (4A2), thin-walled, divergent. Pileipellis (Fig. 5b) infully matured ones ca. 100–200 μm thick, a typicaltrichoderm, composed of thin-walled hyphae 3.5–15 μmwide, yellowish brown (5D8) to colorless, short septated, oc-casionally branched, with end cells tapering, bullet-form, fu-siform, subventricose to clavate, 32–90 × 4–25 μm; pileipellisin younger ones an intricate trichoderm often with a cutis-likesuprapellis, composed of thin-walled hyphae 4–15 μm wide,yellowish brown (5D8). Stipitipellis (Fig. 5f) a disruptedhymeniform, composed of parallel hyphae 5–15 μm wide,giving rise to clusters of caulocystidia and caulobasidia.Caulocystidia 20–60 × 7–25 μm, lageniform, clavate, fusi-form to spheropedunculate, with yellowish brown content inKOH, walls smooth and thin. Clamp connections absent in allparts of basidioma.

Habitat and distribution: Gregarious on soil in forestsdominated by Fagaceae, at 300–500 m alt. Presently knownonly from Heishiding, Guangdong Province, China.

Additional specimens examined: China, GuangdongProvince, Heishiding, alt. 450 m, 4 November 2008, Fang

Fig. 3 Phylogenetic tree inferredfrom maximum likelihood (ML)analysis based on nrLSU se-quences of Aureoboletus species.ML bootstraps (BS) over 50 %and Bayesian posterior probabili-ties (PP) over 0.95 are shown onthe branches

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Fig. 4 Basidiomata of Leccinellum onyx. a–f Holotype. g HKAS89483. Scale bars: 1 cm

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Li 2107 (HKAS93725); the same place, alt. 320 m, 20July 2009, Fang Li 2108 (HKAS89483); the same place, alt.320 m, 20 July 2009, Fang Li 46 (GDGM26729, duplicateHKAS 89483); the same place, alt. 500 m, 19 August 2013,Fang Li 1497 (HKAS82191).

Pulveroboletus sinensis Fang Li, Ming Zhang & KuanZhao, sp. nov. (Figs. 7, 8, and 9)

MycoBank: MB814485Etymology: Bsinensis^ means China, referring to the type

of the species collected from China.Holotype: China, Guangdong Province, Heishiding

(111°49′09″–111°55′01″E, 23°25′15″–23°30′02″N), alt.180 m, 7 September 2012, Fang Li 1039 (HKAS75767;nrLSU and ITS sequences generated from the holotype aredeposited in GenBank under the accession numbersKT824250 and KT824248, respectively).

Basidiomata (Fig. 7) medium-sized. Pileus 5–7 cm indiam., globose at first, hemispherical when expanding, laterconvex to plano-convex; surface dry, yellow to vivid yellow

(3A6–3A8), covered with firm conico-pyramidal pulverulentscales 2–6 mm in height and 2–4 mm in diam. at base, yellowto vivid yellow at base, becoming deep orange (5A8–6A8) tobrownish orange (6C8) towards apex, with some orange(4A8) to reddish orange (7A6–8A8) guttates among or onwarts when wet; margin appendiculate. Hymenophore slightlydepressed around the stipe. Tubes 4–6 mm long, pale yellow-ish green (30A5–30A7) when young, brown (7D8) when overmaturity, quickly cyanescent on exposure (Fig. 7g); poressmall, concolorous with the tubes, quickly cyanescent onbruising (Fig. 7h). Stipe 5–8 cm × 4–7 mm, cylindric, slightlyenlarged at base, sheathing with orange yellow (4A7–4B7)pulverulent conico-floccose scales, with a powdery orangeyellow (4A6–4A8) undersurface, leaving a clear ring zone15–20 mm below the stipe apex, yellow to vivid yellow nextto the tubes; mycelia at base pale yellow, often in finerhizomorphs. Pileus context 4–8 mm thick, pale cream(1A3), cyanescent when cut (Fig. 7g); stipe context 4–7 mmthick, becoming orange (5B8) towards the base, unchangedwhen cut (Fig. 7g).

Spores (Fig. 8a) [300/3/3] (6) 6.5–8.5 (10.8) × (4.5) 5–5.8 (6.5), Q = (1.15) 1.29–1.53 (1.80), Qm = 1.42 ± 0.09,orange yellow to reddish yellow (4A6–4B7), ellipsoid tobroad ellipsoid, smooth, slightly thick-walled; apiculussmall. Basidia 22–36 × 7.5–12 μm, clavate; sterigmata 4,5–9 μm long. Pleurocystidia 33–55 × 7–12 μm, grayishyellow (4B4), ventricose with prolonged obtuse or sub-acute apex, with dense contents, thin-walled, often withboth base and apex colorless, abundant. Cheilocystidia19–36 × 7.5–12 μm, grayish yellow, sphaeropedunculateto ventricose, with dense contents, thin-walled, often witha short obtuse to subacute apex. Tube-trama boletoid, withhyphae 4–17 μm wide, hyaline, yellowish white to lightyellowish vacuolar pigmented, thin-walled, scarcely di-vergent. Pileal and stipe trama composed of hyphae 4–17 μm wide, yellowish white (4A2) to yellowish vacuolarpigmented, thin-walled, scarcely divergent. Pileipellis(Fig. 8e) ca. 50–90 μm thick, composed of interwovenhyphae 2–8 μm wide, most short-celled (7–42 μm inlength), hyaline to light yellowish vacuolar pigmented,branching, encrusted with small yellow (3A6) granularmatter soluble in KOH but dissolving very slowly in wa-ter. Scales on pileus (Fig. 8e) composed of tightly inter-woven, subvertically arranged, short-celled hyphae 2.5–8 μm wide and 7–42 μm long, light yellow to yellowishvacuolar pigmented, branching, heavily encrusted withyellow or reddish brown (7A8–8A8) granular matter sol-uble in KOH and dissolving very slowly in water.Stipitipellis similar to pileipellis. Color of pileus and stipecaused by the thick granular incrustation on the hyphaeand by yellow, deep orange (5A8–6A8) to reddish orange(7A8–8A8) cuboid crystals up to 100 μm wide with fas-cicles of acicular crystals, soluble in KOH, and dissolving

Fig. 5 Microscopic characters of Leccinellum onyx (holotype). aBasidiospores. b Pileipellis. c Basidia. d Pleurocystidia. eCheilocystidia. f Stipitipellis and caulocystidia. Scale bars: 10 μm

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very slowly in water, scattered through the tissue of theveil. Clamp connections absent in all parts of basidioma.

Habitat and distribution: Solitary on soil with humus anddebris under conifers mixed with broadleaf trees, at alt. 180–600 m; from July to October; presently only known fromHeishiding, Guangdong Province, China.

Additional specimens examined: China, GuangdongProvince, Heishiding, alt. 180 m, 12 July 2009, Fang Li(GDGM26735); the same place, alt. 600 m, 29 September2013, Fang Li 1530 (HKAS 89483).

Specimens of Pulveroboletus frians examined: Singapore,Bukit Timah, 19 August 1939, E.J.H. Corner, Royal BotanicGarden Edinburgh (E) 93625, type specimen; Malaysia,Brunei, Ulu Belalong, 16 February 1959, E.J.H. Corner,E93626.

Discussion

The phylogenetic tree of Fig. 3 shows that Aureoboletusvenustus belongs, with statistical significance, to the clade of

Aureoboletus and forms a distinct branch. Aureoboletusgentilis is a closely related sister species. Morphologically,A. venustus is also similar to A. gentilis, as well as otherAureoboletus species, such as A. auriporus (Peck) Pouzar,A. clavatus N.K. Zeng & M. Zhang, A. formosus M. Zhanget al., and A. viridiflavus Coker et al.. These species share asimilar size of basidioma, similar colors, an incurved margin,and a mucilaginous stipe. However, A. venustus can be easilydistinguished from the other species by its much smaller el-lipsoid spores (7) 7.5–10.5 (12) × 5–6 (7) μm and other subtlemacroscopic characteristics, as indicated below. Comparedwith A. venustus, A. gentilis has longer fusiform spores(10.5–16 × 4.5–6 μm) and a vivid yellow stipe with a rootingbase (Singer 1945; Pouzar 1957; Klofac 2010), while the stipeof A. venustus has neither vivid yellow tinges nor a rootingbase. Aureoboletus auriporus has longer spores (11–16 × 4–6 μm), and its stipe apex is covered with yellow pruina orfloccosity (Pouzar 1957; Smith and Thiers 1971; Klofac2010), which was never observed for A. venustus.Aureoboletus clavatus has smaller subglobose spores (7–8 ×5.5–6 μm), which is similar to those of A. venustus, but its

Fig. 6 Phylogenetic tree inferredfrom maximum likelihood (ML)analysis based on nrLSU se-quences of Leccinellum species.ML bootstraps (BS) over 50 %and Bayesian posteriorprobabilities (PP) over 0.95 areshown on the branches

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context slowly turning yellowish green when bruised (Zenget al. 2015), which was not observed in A. venustus.Aureoboletus formosus has much longer fusiform spores(15–16.5 × 4.5–5 μm) (Zhang et al. 2015a, b). Aureoboletusviridiflavus has slender fusiform spores (11.5–15 × 4–5 μm)and a dull brick red discoloring hymenophore (Coker andBeers 1943; Both 1993; Klofac 2010).

Leccinellum onyx belongs, with statistical significance, tothe clade of Leccinellum. It forms a distinct branch that is notclosely related to any other species in this clade (Fig. 6). Thesequence alignment revealed that the ITS region (1080 bp) ofL. onyx is much longer than those of related species (600–800 bp). Thus, a simple PCR can be used to distinguishL. onyx from related species. Morphologically, L. onyx is also

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Fig. 7 Basidiomata of Pulveroboletus sinensis at different stages of development. The basidiomata in a–d, f–h are the same single basidioma (holotype)in different growth periods. e Young basidioma (HKAS89483). i An old basidioma (GDGM26735). Scale bars: 1 cm

very unique. Compared to the yellow hymenophore surfacesand the large fusiform spores of most Leccinellum species,L. onyx has a grayish brown to light grayish orangehymenophore surface, much smaller ellipsoid spores (8) 9–11 (12) × (4) 5–6 μm, and its tubes are short (up to 6 mmlong), never ventricose. Leccinellum onyx is somewhat similarto L. crocipodum (Letell.) Bresinsky & Manfr. Binder,L. carpini (R. Schulz) Bresinsky & Manfr. Binder, andL. quercophilum M. Kuo with respect to the brownish pileusand the brownish scabs covered on the yellowish whitishstipes. But compared with L. onyx, L. crocipodium has a ratherbright yellow hymenophore surface and larger fusiform spores(11.5) 12–15 × 5–6.5 μm; L. carpini has subventricose to ven-tricose tubes up to 25 mm long and much larger fusiformspores (12.5) 13.0–18.5 × 4.5–6.0 μm (den Bakker andNoordeloos 2005); L. quercophilum has 10–20mm long tubesand much larger spores 15–18(28) × 5–7.5 μm (Kuo et al.2013).

Based on phylogenetic analysis, P. sinensis is nested, withstatistical significance, within the clade of Pulveroboletus.Pulveroboletus sinensis forms a distinct branch which isclosely related to P. brunneoscabrosusHar. Takah (specimensalso collected from Heishiding) and an as yet undescribedspecies, namely BPulveroboletus sp. HKAS74926 andHKAS74933^ collected from Yunnan Province, China, ofwhich HKAS74933 was mentioned by Wu et al. (2014).

Morphologically, P. sinensis is distinct from all other knownPulveroboletus species by its deep orange to brownish-orangeconico-pyramidal scales. These scales maintain, during themature period, their color and shape on the pileus and stemsurface. Both P. brunneoscabrosus and the undescribed relat-ed species mainly differ from P. sinensis in their appressedpatched scales on the pileal surface. Pulveroboletus frians(Corner) Singer, originally described from Singapore, appearsto be closely related to P. sinensis as far as similar size andcolor of basidiomata, similar cyanescent reactions, and similarshape and size of basidiospores are concerned (Corner 1972).However, we can easily distinguish P. sinensis from P. friansby the scales on the pileus, which are not conical but floccose-squarrose, soft, friable, mealy squamules, weathering pallid,and nearly smooth in P. frians (Corner 1972; Tham andWatling 2010). Unfortunately, we failed to obtain DNAsequences from the specimens E93625 and E93626 ofP. frians. Therefore, we cannot compare these two specieson a mo lecu l a r l eve l . Ano the r As i an spec i e s ,P. viridisquamosus Watling et al., originally described fromMalaysia, also resembles P. sinensis in shape and size ofbasidiomata and basidiospores. However, P. viridisquamosus

Fig. 8 Microscopic characters of Pulveroboletus sinensis (holotype). aBasidiospores. b Basidia. c Pleurocystidia. d Cheilocystidia. e Pileipellisand hyphae in the scales of the pileus. Scale bars: 10 μm

Fig. 9 Phylogenetic tree inferred from maximum likelihood (ML) anal-ysis based on ITS sequences of Pulveroboletus species. ML bootstraps(BS) over 50 % and Bayesian posterior probabilities (PP) over 0.95 areshown on the branches

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is characterized by distinct olive green surfaces of pileus andstipe and a yellowish green stipe context (Watling et al. 2006).

Acknowledgments We are grateful to the herbarium curator Prof. ZhuL. Yang and Dr. Gang Wu at the Kunming Institute of Botany (ChineseAcademy of Sciences) for kindly providing specimens, for preservingspecimens of the three new species, for scientific discussions, and forcritical comments on the manuscript. The authors are indebted to Dr.D.J. Harris (herbarium curator), Suzanne Cubey (deputy herbarium cura-tor), Dr. Stephan Helfer (mycologist), and Lesley Scott (technician) at theRoyal Botanic Garden Edinburgh for temporarily providing specimens ofPulveroboletus frians and for granting permission to extract DNA fromthem. We thank Dr. Takamichi Orihara, curator of Kanagawa PrefecturalMuseum of Natural History (KPM, Japan), for providing Pulveroboletusbrunneoscabrosus on a loan basis and for granting permission to extractDNA from it. Ms. Zheng-Duan Xiao (technician) from the GuangdongInstitute of Microbiology and Dr. Xin-Yu Wang (technician) fromKunming Institute of Botany are acknowledged for providing specimens.We also thank Dr. Christian Wagner (School of Life Sciences, Sun Yat-sen University) for polishing the English of the manuscript. Thanks arealso due to Prof. Yong-Jun Lu, Prof. Wen-Sheng Shu, Prof. ZhouSongyang, Prof. Wen-Qing Zhang, Prof. Yan Zhang, Prof. Yi-ShunZhang, and Dr. Xiang-gang Shi (School of Life Sciences, Sun Yat-senUniversity), who provided peer support. Furthermore, we are grateful toMr. Eiji Nagasawa (curator) of the Tottori Mycological Institute (Japan)and to Prof. Tai-Hui Li (Guangdong Institute of Microbiology) for theirkind support. Finally, the two anonymous reviewers are acknowledgedfor their critical comments and suggestions to improve the manuscript.This study was financed by the School of Life Sciences, Sun Yat-senUniversity and the National Natural Science Foundation of China(J1310025, U1501232).

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