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Research Article
Two new species of Boletopsis (Bankeraceae, Thelephorales) from Southwest China
expand article infoHong-Min Zhou‡, Qi Zhao§, Qi Wang|, Fang Wu‡, Yu-Cheng Dai‡
‡ Beijing Forestry University, Beijing, China
§ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
| Unaffiliated, Fushun, China

Corresponding author: Fang Wu ( fangwubjfu2014@bjfu.edu.cn )

Corresponding author: Yu-Cheng Dai ( yuchengdai@bjfu.edu.cn )

Academic editor: Rui-Lin Zhao
© 2022 Hong-Min Zhou, Qi Zhao, Qi Wang, Fang Wu, Yu-Cheng Dai.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhou H-M, Zhao Q, Wang Q, Wu F, Dai Y-C (2022) Two new species of Boletopsis (Bankeraceae, Thelephorales) from Southwest China. MycoKeys 89: 155-169. https://doi.org/10.3897/mycokeys.89.83197
Open Access

Abstract

Two new species of Boletopsis, B. macrocarpa and B. tibetana, are described and illustrated from Southwest (SW) China based on morphology, ecology and phylogenetic analyses by the internal transcribed spacer regions (ITS) and the large subunit of nuclear ribosomal RNA gene (nLSU). Boletopsis macrocarpa is characterized by big basidiocarps (up to 18 cm in diam), guttulate basidiospores, and the presence of gloeoplerous hyphae in context and growing in pure forest of Pinus yunnanensis. Boletopsis tibetana is characterized by smaller pores (3–4 per mm), the presence of gloeoplerous hyphae in pileipellis, and the growth in forests of Picea. Phylogenetically, the two new species are grouped in two independent lineages nested in Boletopsis. In addition, one sample from Northeast China is temporarily treated as Boletopsis sp. 1 because of the single sample; another Chinese sample from SW China is sister to B. grisea in phylogeny, and it is treated as B. cf. grisea because the morphological difference between B. cf. grisea and B. grisea is indistinct. Furthermore, the main characteristics of Boletopsis species are listed, and a key to accepted species of Boletopsis is provided.

Keywords

Ectomycorrhizal fungi, phylogeny, taxonomy

Introduction

Boletopsis Fayod was established by Fayod based on B. leucomelaena (Pers.) Fayod originally described from Europe (Niemela and Saarenoksa 1989) and is characterized by annual basidiocarps with poroid hymenophore and central to lateral stipes, generative hyphae with clamp connections, and angular to tubercular, hyaline to pale brownish basidiospores which are negative in Melzer’s reagent (Ryvarden and Melo 2017). Previously, seven species, B. grisea (Peck) Bondartsev & Singer, B. leucomelaena, B. mediterraneensis G. Moreno et al., B. watlingii Blanco-Dios (=B. perplexa Watling & Jer. Milne, Nom. inval., Blanco-Dios 2018), B. smithii K.A. Harrison, B. nothofagi J.A. Cooper & P. Leonard and B. atrata Ryvarden, were accepted in the genus, and the first four species have a distribution in Europe (Ryvarden and Melo 2017), B. grisea, B. watlingii and B. smithii occur in North America (Watling and Milne 2008), and B. nothofagi and B. atrata were described from New Zealand and Thailand, respectively (Hjortstam and Ryvarden 1982; Cooper and Leonard 2012). Five of these seven species were analyzed by molecular techniques (Watling and Milne 2008; Cooper and Leonard 2012; Crous et al. 2019). In addition, Boletopsis subsquamosa (L.) Kotl. & Pouzar and B. subcitrina Corner were recorded in Boletopsis (Kotlába and Pouzar 1957; Corner 1989), but the former was considered as a synonym of Albatrellus ovinus (Schaeff.) Kotl. & Pouzar (Donk 1974; Ryvarden and Gilbertson 1993), and the latter was treated as Corneroporus subcitrinus (Corner) T. Hatt. (Hattori 2001).

Boletopsis is the ectomycorrhizal fungal genus in the family Bankeraceae, phylogenetically, Boletopsis is sister to Hydnellum P. Karst. and Sarcodon Quél. ex P. Karst (Cooper and Leonard 2012; Mu et al. 2021; Crous et al. 2019). Morphologically, Boletopsis is distinguished from other genera in the family by poroid hymenophore.

Species of Boletopsis are edible mushrooms in SW China, and they are sold in the local markets as “black bear’s-paw fungi”, but their scientific names are unknown. During an investigation on forest macrofungi in China, sampling efforts of Boletopsis were especially focused on, and the ecology of these samples was recorded. The aim of this study is to clarify the species of Boletopsis in China and to expound phylogenetic relationships among members in the genus.

Materials and methods

Molecular phylogenetic studies

Eleven samples of Boletopsis were collected from Liaoning Province, Xizang Autonomous Region (Tibet) and Yunnan Province in China and deposited in the Herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Potential host trees of Boletopsis spp. were observed from field trips. The macro-morphology was based on fresh and dried specimens. The color terms in descriptions followed Anonymous (1969) and Petersen (1996). Micro-morphology was studied at magnifications 1000×, using a Nikon Eclipse 80i microscope with phase contrast illumination. The Melzer’s reagent, Cotton Blue and 5% KOH were used in the study. Drawings were made with the aid of a drawing tube. In the text the following abbreviations were used: IKI = Melzer’s reagent, IKI– = non-dextrinoid and non-amyloid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB– = acyanophilous, L = mean basidiospores length (arithmetic average of all basidiospores), W = mean basidiospores width (arithmetic average of all basidiospores), Q = variation in the L/W ratios between the specimens studied, n = number of basidiospores measured from number of specimens.

DNA extraction and amplification

A cetyltrimethyl ammonium bromide (CTAB) rapid plant genome extraction kit (Aidlab Biotechnologies Co. Ltd., Beijing, China) was used to extract DNA from dried specimens following the manufacturer’s instructions with some modifications (Chen et al. 2015, 2016). PCR reactions were performed in the 0.2 mL tubes, along with 1 µL DNA, 29 µL specified primers. The ITS primers pairs were ITS5 and ITS4 (White et al. 1990); the nLSU primers pairs was LR0R and LR7 (Vilgalys and Hester 1990). The optimal annealing temperature and cycles were generated as: an initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, annealing at 54 °C (sometimes at 56 °C) and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR procedure for nLSU was: initial denaturation at 94 °C for 1 min, followed by 34 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min and extension at 72 °C for 1.5 min, and a final extension at 72 °C for 10 min.

Phylogenetic analyses

Fifty-three sequences used in phylogenetic analyses are listed in Table 1, including 24 sequences generated by this study and another 29 downloaded from the National Center for Biotechnology Information (NCBI) which mainly adapted from Cooper and Leonard (2012) and Crous et al. (2019). Sarcodon imbricatus (L.) P. Karst. was used as outgroup (Crous et al. 2019).

Table 1.
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Information on the sequences used in this study.

Species Sample Location Hosts GenBank Accession No.
ITS nLSU
Boletopsis grisea UPS F-120382 Sweden Pinus sylvestris MN536751 MN535646
Boletopsis grisea UPS F-153996 Sweden Pinus sylvestris MN536742 MN535641
Boletopsis grisea AB 16-09-113 France Abies alba MN536743 –
Boletopsis grisea AB 17-09-52 France Abies alba MN536744 –
Boletopsis grisea AH 42971 Spain Pinus pinea MN536747 MN535642
Boletopsis grisea AH 44091 Spain Pinus pinaster MN536748 MN535643
Boletopsis grisea Rec 227658 USA Tsuga canadensis EF457899 –
Boletopsis grisea Rec 227659 USA Pinus sylvestris EF457902 –
Boletopsis cf. grisea Dai 23070 China Pinus, Quercus OL673003 OL672990
Boletopsis leucomelaena UPS F-173290 Sweden Picea abies MN536739 MN535638
Boletopsis leucomelaena UPS F-575617 Sweden Picea, Populus MN536740 MN535639
Boletopsis macrocarpa Dai 21780 China Pinus yunnanensis OL673004 OL672991
Boletopsis macrocarpa Dai 22727 China Pinus yunnanensis OL673007 OL672994
Boletopsis macrocarpa Dai 22728 China Pinus yunnanensis OL673005 OL672992
Boletopsis macrocarpa Dai 22729 China Pinus yunnanensis OL673006 OL672993
Boletopsis macrocarpa Dai 22748 China Pinus yunnanensis OL673008 OL672995
Boletopsis macrocarpa Dai 23064 China Pinus yunnanensis OL673009 OL672996
Boletopsis macrocarpa Dai 23065 China Pinus yunnanensis OL673010 OL672997
Boletopsis mediterraneensis AB 06-10-343 France Cedrus atlantica MN536717 –
Boletopsis mediterraneensis AB 15-11-97 France Cedrus atlantica MN536736 –
Boletopsis mediterraneensis AH 44070 Spain Pinus nigra MN536724 MN535630
Boletopsis mediterraneensis AH 44080 Spain Pinus MN536723 MN535629
Boletopsis mediterraneensis FR 2016250 France Pinus halepensis MN536726 –
Boletopsis mediterraneensis ML 410112B Cyprus Pinus nigra MN536719 –
Boletopsis nothofagi PDD 96007 New Zealand Nothofagus fusca JQ417193 –
Boletopsis sp. 1 Dai 22172 China Pinus OL673011 OL672998
Boletopsis tibetana Dai 20896 China Picea balfouriana OL673012 OL672999
Boletopsis tibetana Dai 20897 China Picea balfouriana OL673013 OL673000
Boletopsis watlingii Holden E150627 (E) UK Pinus sylvestris DQ408766 –
Boletopsis watlingii Wat. 28788 (E) UK Pinus sylvestris DQ408767 –
Boletopsis watlingii SMI 350 Canada Unknown FJ845401 –
Sarcodon imbricatus Dai 20314 China Unknown OL676807 OL678542
Sarcodon imbricatus NIFoS 1676 – Unknown MF421106 –

New sequences are shown in bold.

Raw chromatograms were aligned and edited using BioEdit Sequence Alignment Editor (Hall 1999), especially those chromatograms with double peaks at the start and the end of sequences. The ITS and nLSU sequences were aligned using MAFFT 7 online (https://mafft.cbrc.jp/alignment/server/), and applying the interative refinement method of G-INS-I (Katoh and Standley 2013). For aligned sequences, the ambiguous regions at the start and the end were deleted. Sequence alignment was deposited at TreeBASE (http://purl.org/phylo/treebase/; submission ID 29052).

The Maximum likelihood (ML) and Bayesian inference (BI) methods were used to conduct phylogenetic trees with ITS + nLSU matrix. The best-fit model was selected by ModelFinder (Kalyaanamoorthy et al. 2017), adopting Akaike information criterion (AIC). The model GTR + F + I + G4 was selected as the best-fit model for the ITS + nLSU matrix, lset nst = 6, rates = invgamma in Bayesian analysis. ML analysis was constructed by RaxmlGUI 1.2 (Stamatakis 2006). We performed default parameters in the ML analysis. A Bayesian tree was produced by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using the same model as ML analysis. Four Markov chains were run for 2 million generations for the analysis. Trees were sampled every 1000th generation. The first 25% of sampled trees were discarded as burn-in, whereas others were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).

Results

Molecular phylogeny

A total of 33 ITS and 20 nLSU sequences were used in the phylogenetic analyses. The Bayes analysis and Maximum likelihood analysis resulted in a similar topology with an average standard deviation of split frequencies = 0.006494. All samples of Boletopsis form a monophyletic clade. Among the Chinese materials, the specimen Dai 23070 is sister to B. grisea samples from Europe and North America with a stable support (100/1). The specimen Dai 22172 has singleton position as a lineage, specimens Dai 20896 & 20897 and Dai 21780, 22727, 22728, 22729, 22748, 23064 & 23065 are grouped respectively in two lineages with high support (97/1, 94/1). So, two species are described from nine specimens grouped in two independent linages nested in Boletopsis clade, and specimens Dai 23070 and Dai 22172 are treated as Boletopsis cf. grisea and Boletopsis sp. 1, respectively (Fig. 1).

Figure 1. 

Phylogeny of species in Boletopsis generated by maximum likelihood based on ITS + nLSU sequence data. Branches are labeled with maximum likelihood bootstrap ≥ 75% and Bayesian posterior probabilities ≥ 0.80, respectively. New species are in bold.

Taxonomy

Boletopsis macrocarpa Y.C. Dai, F. Wu & H.M. Zhou, sp. nov.

MycoBank No: 843792
Figs 2A, 3

Diagnosis

Differs from other Boletopsis species by largest basidiocarps (up to 18 cm in diam) with grayish brown to dark gray upper surface, gloeoplerous hyphae present in context, guttulate basidiospores, and the fact that it grows in forests of Pinus yunnanensis at high altitude with open and slightly dry environments in SW China.

Holotype

China, Yunnan Province, Nujiang, Lanping County, Xinshengqiao National Forest Park, on ground in forest of Pinus yunnanensis, alt. 3000 m, 2 September 2021, Dai 22728 (BJFC037301).

Etymology

Macrocarpa (Lat.): referring to the species having largest basidiocarps.

Fruiting bodies

Basidiocarps annual, terrestrial, centrally stipitate, solitary. Pilei circular or irregular, slightly depressed at center, with undulate and sharp margin, up to 18 cm in diam and 3 cm thick at center when fresh. Pileal surface grayish brown (5/6E4) with cream margin (4A2/3) when fresh, becoming blackish blue (20F8) to black upon drying, smooth, azonate. Pore surface white when fresh, becoming clay-buff (6D4) to fawn (7D/E4) upon drying; pores round to angular, some irregular, 1–3 per mm, mature pores bigger than juvenile ones; dissepiment thin, even to slightly lacerate. Context white when fresh, become pale mouse-gray (7C2) when dry, brittle, up to 2.5 cm thick when fresh. Tubes concolorous with pore surface, brittle, up to 5 mm long when fresh. Stipe pale ash-gray (19C2) when fresh, become mouse-gray (9F3) when dry, up to 6 cm long and 4 cm in diam when fresh.

Figure 2. 

Basidiocarps of Boletopsis in China A B. macrocarpa (Dai 22728) B B. sp. 1 (Dai 22172) C B. cf. grisea (Dai 23070) D–E B. tibetana (Dai 20896 and Dai 20897).

Hyphal structure

Hyphal system monomitic; generative hyphae with clamp connections; gloeoplerous hyphae present, usually 3–11 μm in diam.

Pileipellis

Pileipellis hyphae hyaline, thin- to thick-walled, 4–9 μm in diam; gloeoplerous hyphae rarely present; tissue darkening in KOH.

Context

Contextual hyphae hyaline, thick-walled, rarely branched, interwoven, distinctly inflated, 5–25 μm in diam; gloeoplerous frequently hyphae present, thin-walled, reflective in Melzer’s reagent.

Stipitipellis

Stipitipellis hyphae hyaline, usually thick-walled with a wide lumen, rarely branched, parallel along stipe, straight, uniform, 4–12 μm in diam; gloeoplerous hyphae rarely present.

Tubes

Tramal hyphae hyaline, thin-walled, occasionally branched, interwoven, uniform, 2–4 μm in diam; gloeoplerous hyphae rarely present; cystidia and cystidioles absent; basidia clavate, tetrasterigmatic with a basal clamp connection, 14–19 × 6–7 μm.

Spores

Basidiospores angular to tubercular with irregular ornaments, hyaline, thin-walled, with a guttule, IKI–, CB–, (4.5–)4.8–6(–6.2) × (3.7–)4–5 μm, L = 5.22 μm, W = 4.31 μm, Q = 1.20–1.22 (n = 90/3).

Additional specimens examined (paratypes)

China, Yunnan Province, Chuxiong, Wuding County, on ground in forest of Pinus yunnanensis, alt. 2400 m, 23 September 2021, Dai 23064 (BJFC037635), Dai 23065 (BJFC037636); Dali, Jianchuan County, Laojunshan Nature Reserve, on ground in forest of Pinus yunnanensis, alt. 3100 m, 29 August 2020, Dai 21780 (BJFC035681); Nujiang, Lanping County, Luoguqing Nature Reserve, on ground in forest of Pinus yunnanensis, alt. 3000 m, 3 September 2021, Dai 22748 (BJFC037321); Xinshengqiao National Forest Park, on ground in forest of Pinus yunnanensis, alt. 3000 m, 2 September 2021, Dai 22727 (BJFC037300), Dai 22729 (BJFC037302).

Figure 3. 

Microscopic structures of Boletopsis macrocarpa (Holotype) a basidiospores b basidia c stipitipellis hyphae d pileipellis hyphae e hyphae from context f hyphae from trama.

Boletopsis tibetana Y.C. Dai, F. Wu & H.M. Zhou, sp. nov.

MycoBank No: 843793
Figs 2D–E, 4

Diagnosis

Differs from other Boletopsis species by smaller pores (3–4 per mm), the presence of gloeoplerous hyphae in pileipellis and context, and the fact that it grows in the forest of Picea in Tibet, SW China.

Holotype

China, Tibet, Linzhi, on ground in the forest of Picea balfouriana, alt. 2900 m, 23 August 2019, Dai 20896 (BJFC032554).

Etymology

Tibetana (Lat.): referring to the species having a distribution in Tibet.

Fruiting bodies

Basidiocarps annual, terrestrial, centrally stipitate, solitary to confluent. Pilei convex, or irregular, with undulate and incurved margin, up to 7 cm in diam and 1 cm thick at center when fresh. Pileal surface vinaceous buff (4C4) to clay buff (6D4) when fresh, becoming mouse-gray (9F3) to black upon drying, smooth, azonate; margin concolorous with pileal surface. Pore surface white when fresh, become fawn (7D/E4) when bruised, ash-gray (19C2) when dry; pores round to angular, 3–4 per mm; dissepiment thin, entire to slightly lacerate. Context white when fresh, become ash gray (19C2) when dry, rigid, up to 9 mm thick when dry. Tubes concolorous with pore surface, brittle, up to 1 mm long when dry. Stipe concolorous with pileal surface, cylindrical or tapering to the base, up to 6 cm long and 2 cm in diam when fresh.

Figure 4. 

Microscopic structures of Boletopsis tibetana (Holotype) a basidiospores b basidia and basidioles c stipitipellis hyphae d pileipellis hyphae e hyphae from context f hyphae from trama.

Hyphal structure

Hyphal system monomitic; generative hyphae with clamp connections; gloeoplerous hyphae present, usually 3–11 μm in diam.

Pileipellis

Pileipellis hyphae hyaline, thin-walled, with finger-shaped tips, 5–7 μm in diam; gloeoplerous hyphae frequently present, thin-walled, strongly reflective in Melzer’s reagent; tissue darkening in KOH.

Context

Contextual hyphae hyaline, thick-walled, rarely branched, interwoven, distinctly inflated, 6–22 μm in diam; gloeoplerous hyphae present, thin-walled, strongly reflective in Melzer’s reagent.

Stipitipellis

Stipitipellis hyphae hyaline, thin- to thick-walled, frequently branched, subparallel along stipe, straight, uniform, 2–6 μm in diam; gloeoplerous hyphae rarely present.

Tubes

Tramal hyphae hyaline, thin-walled, occasionally branched, loosely interwoven, uniform, 2–4 μm in diam; gloeoplerous hyphae rarely present; cystidia and cystidioles absent; basidia clavate, tetrasterigmatic with a basal clamp connection, 13–25 × 6–8 μm; basidioles clavate, 22–40 × 3–4 μm.

Spores

Basidiospores angular to tubercular with irregular ornaments, hyaline, thin-walled, IKI–, CB–, 5–6.5(–7) × 4–5(–5.2) μm, L = 5.55 μm, W = 4.41 μm, Q = 1.22–1.29 (n = 60/2).

Additional specimen examined (paratype)

China, Tibet, Linzhi, on ground in forest of Picea balfouriana, alt. 2900 m, 23 August 2019, Dai 20897 (BJFC032555).

Discussion

Previously seven species of Boletopsis were accepted mostly based on morphological examination, and five were confirmed by phylogenetic analyses (Cooper and Leonard 2012). In the present study, four distinct taxa of Boletopsis were found in China: B. macrocarpa, B. tibetana, B. cf. grisea and B. sp. 1 based on morphological and molecular evidence, and the phylogenetic relationship of seven Boletopsis taxa is analyzed (Fig. 1). The former two new species are proposed, but the latter two taxa require further collections and analyses.

Morphologically, Boletopsis macrocarpa and B. mediterraneensis share similar pileal surface, almost the same shape and size of basidiospores, and both species take Pinus as a potential host (Table 2), but the former has white fresh context which was unchanged when cut and hyaline basidiospores, while the latter has pale gray fresh context, becoming pale red when cut and hyaline to pale yellow-brown (Crous et al. 2019). In addition, B. macrocarpa has a distribution in SW China, while B. mediterraneensis is known in the Mediterranean area. Boletopsis grisea resembles B. macrocarpa by almost the same size of pores and basidiospores, but the former has uniform grayish tinges for all upper surface, its gloeoplerous hyphae are present at pileipellis (Ryvarden and Melo 2017), while the upper surface is grayish brown to dark gray with cream margin and the gloeoplerous hyphae present in context in B. macrocarpa (Table 2).

Table 2.
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A comparison of morphology, ecology and distribution of Boletopsis species.

Species Type Locality Basidiocarps in diam (cm) Pileal surface when fresh gloeoplerous hyphae Pores/mm Basidiospores (μm) Guttules in basidiospores Hosts Distribution References
B. atrata Thailand 2–5 black – 2–3 4.5–6 in diam – Quercus, Castanea Asia and North America Hjortstam and Ryvarden 1982
B. grisea Norway 5–18 gray–white to silvery gray, gray–brown, or brownish vinaceous frequent in pileipellis 1–3 5–6.2 × 4–5 present Pinaceae Europe and North America Watling and Milne 2008; Ryvarden and Melo 2017
B. leucomelaena Norway Up to 10 deep grayish to black rarely present 1–3 5–6.5 × 4–5 present mostly Picea Europe Ryvarden and Melo 2017
B. macrocarpa China 12–18 grayish brown to dark gray present in context 1–3 4.8–6 × 4–5 present Pinus Asia This study
B. mediterraneensis Spain 4–12 pale gray, brownish gray to ochraceous brown or dark brown – 1–3 4.5–6.7 × 3.3–5.2 – mostly Pinus, Cedrus Europe Crous et al. 2019
B. nothofagi New Zealand 1–8 gray present in context 2–3 5.3 × 4.1 – Nothofagus Oceania Cooper and Leonard 2012
B. smithii USA 4–5 dull orange – 2–3 5.5–7 × 4.5–5.6 absent – North America Harrison 1975
B. tibetana China 5–7 vinaceous buff to clay buff present in pileipellis 3–4 5–6.5 × 4–5 absent Picea Asia This study
B. watlingii UK 4–7 dark fuliginous brown to gray–brown present in pileipellis 1–3 4.5–4.8 × 3.5–4.5 present Pinus Europe and North America Watling and Milne 2006, 2008; Ryvarden and Melo 2017

Boletopsis tibetana resembles B. grisea by almost the same shape and size of basidiospores. However, the latter species has bigger pores (1–3 per mm vs. 3–4 per mm, Table 2), and both species are phylogenetically distantly related. In fact, Boletopsis tibetana has pores as 3–4 per mm, and other Boletopsis species have pores 1–3 per mm, so it is easily distinguished B. tibetana from other Boletopsis species.

Two species in Boletopsis, B. atrata and B. smithii, have so far no DNA data available, and their relationships with our new species are still unknown. Morphologically, B. atrata can be distinguished from our two new species by its small basidiocarps (2–5 cm in diam), verruculose basidiospores with regular ornaments (Hjortstam and Ryvarden 1982), while our new species have big basidiocarps (5–18 cm in diam), angular to tubercular basidiospores with irregular ornaments. Boletopsis smithii is different from our new species by its dull orange and smaller basidiocarps (4–5 cm in diam), and inflated hyphae (up to 17 μm in diam) in pileipellis and stipitipellis (Harrison 1975).

Although the specimen Dai 22172 forms an independent linage nested in Boletopsis clade in our phylogeny (Fig. 1), it is temporarily treated as Boletopsis sp. 1 because of the single sample. The taxon is characterized by the presence of scales at pileal margin, a bulbous stipe base, dentate pores, the presence of cystidioles, and the fact that it grows in a forest dominated by Pinus sylvestris var. mongolica in NE China.

All European and North American samples of Boletopsis grisea clustered together with a support (88/0.86), and a single Chinese sample Dai 23070 is sister to them (100/1). We treat the sample Dai 23070 as B. cf. grisea because no distinct morphological difference has been found between them to date. More samples and a multi-locus phylogeny are needed to clarify the status of the Chinese Boletopsis cf. grisea.

Species of Boletopsis form ectomycorrhizae with certain host plants, and the potential host trees may help to identify species, for instance, Boletopsis leucomelaena is usually associated with Picea abies (L.) Karst. In Europe (Niemela and Saarenoksa 1989), and B. nothofagi are usually accompanied by Nothofagus in Oceania (Cooper and Leonard 2012). Almost all Boletopsis species are found in the Northern Hemisphere except B. nothofagi; most Boletopsis species grow coniferous trees in temperate areas and two species are known from more than one continent (Watling and Milne 2008; Ryvarden and Melo 2017). According to our field inventory, the two Chinese new species were found in temperate zone, and Boletopsis macrocarpa seems to prefer to pine forest at high altitude with open and slightly dry environments; Boletopsis tibetana was found in coniferous forest dominant by spruce at high altitude with cold and humid environments. Previously numerous new species have been found in SW China (Dai et al. 2021; Wang et al. 2021), and the present paper confirms the fungal diversity is very rich in the montane forests of East Himalayas.

The main morphological characteristics, ecology and distribution of the accepted species of Boletopsis are summarized in Table 2.

A key to accepted species of Boletopsis in the world

1 Basidiospores verruculose with regular ornaments B. atrata
– Basidiospores oblong, angular to tubercular with irregular ornaments 2
2 Basidiospores < 5 μm long B. watlingii
– Basidiospores > 5 μm long 3
3 Pileal surface dull orange when fresh B. smithii
– Pileal surface vinaceous, grayish brown, dark gray or brownish to black when fresh 4
4 Pores 3–4 per mm B. tibetana
– Pores 1–3 per mm 5
5 Basidiospores oblong to tuberculate; associated to Nothofagus forest, distribution in Oceania B. nothofagi
– Basidiospores angular to tuberculate; associated to Picea or Pinus forest, distribution in Northern Hemisphere 6
6 Upper surface grayish brown with cream margin when fresh; distribution in Asia B. macrocarpa
– Upper surface brownish gray to blackish without cream margin when fresh; distribution in Europe and North America 7
7 Context pale gray, becoming pale red when cut B. mediterraneensis
– Context white, becoming darker when cut 8
8 Pileus dark gray to blackish, flesh brittle, usually associated to Picea forest B. leucomelaena
– Pileus grayish to grayish brown, flesh tough, usually associated to Pinus forest B. grisea

Acknowledgements

The research is supported by the Second Tibetan Plateau Scientific Expedition and Research Program (STEP Grant No. 2019QZKK0503) and the National Natural Science Foundation of China (Project Nos. U1802231, 31870007).

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