﻿Two new species of Boletopsis (Bankeraceae, Thelephorales) from Southwest China

﻿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). Boletopsismacrocarpa 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 Pinusyunnanensis. Boletopsistibetana 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.

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.

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 macromorphology 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 nonamyloid, 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(Chen et al. , 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).
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 1000 th 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).

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   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 claybuff (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.
Additional specimens examined ( 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.

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).
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.  Milne 2006, 2008;Ryvarden and Melo 2017 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 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