Four Novel Species and Two New Records of Boletes from India

Repeated macrofungal explorations, followed by thorough examination of species through morphology and molecular phylogeny, have made it clear that European and American names of wild mushrooms were inadvertently misapplied quite often to Asian lookalikes by mycologists/taxonomists in the past. Therefore, in order to reveal this mushroom treasure, in recent years, taxonomical research on wild mushrooms has been intensified in Asian countries, including India, by undertaking a combined approach of morpho-taxonomy and multigene molecular phylogeny. Boletoid mushrooms (Boletaceae) are no exception. While working on boletoid mushrooms of the Indian Himalayas, authors recently came across six interesting species of boletoid mushrooms. In the present communication, four novel species, namely Leccinellum binderi, Cyanoboletus paurianus, Xerocomus uttarakhandae, and Xerocomellus himalayanus, are established based on morphology and molecular phylogenetic estimations. Moreover, Cyanoboletus macroporus and Xerocomus fraternus are also reported here for the first time in India.


Introduction
Boletes represent fleshy, readily decaying (putrescent) poroid macrofungi (mushrooms) in the order Boletales of Agaricomycetes (Agaricomycotina, Basidiomycota). Mainly, these mushrooms belong to four families, namely: Boletaceae, Boletinellaceae, Suillaceae, and Gyroporaceae. They are the most popular wild-edible fleshy mushrooms and are appreciated widely across the globe. As ectomycorrhizal fungi, they play a crucial role in forest ecosystems by establishing mutual associations with forest trees. Presently, boletoid mushrooms comprise over 1270 species from around the world, belonging to 108 genera [1][2][3][4][5][6][7][8][9][10]. Earlier, the systematics of these mushrooms was mainly established based on their macro-and micromorphology. But this scenario has drastically changed during the past decade, when single-to multigene molecular phylogeny was applied in combination with morphology to revise the systematics of these mushrooms. This resulted in the discovery of several novel genera and numerous new species, especially in Asian countries.
The fungi (Mycobiota) of the state of Uttarakhand and the state of Himachal Pradesh (western Himalaya, India) are exceptionally diverse in terms of ectomycorrhizal macrofungi, as evidenced in numerous relevant literatures [11][12][13][14][15]. A focused and dedicated approach to a few other groups of ectomycorrhizal mushrooms (Russulaceae, Amanitaceae) has already been undertaken [16][17][18][19][20][21][22]. But unfortunately, serious investigation, i.e., the combined approach of molecular phylogeny and morpho-taxonomy of boletoid mushrooms, has not yet been undertaken in these states. Earlier, morphology-based part work [23] that has been undertaken in the Garhwal Himalaya (only a part of Uttarakhand) was completely mishandled. Species were wrongly identified, and the names of North American/European mushrooms were misapplied to almost all the collected specimens of the Garhwal Himalaya. Moreover, molecular phylogeny, which is the backbone for classifying boletoid mushrooms, has unfortunately not been applied to these taxa of the Garhwal Himalaya. Similarly, an account of Boletes from Himachal Pradesh was published by Lakhanpal [11], where 56 species were described under 7 genera. Inadvertently, the names of North American or European species were applied to these Indian taxa. Therefore, a holistic approach of thorough exploration followed by dedicated investigation (molecular phylogeny and morpho-taxonomy) on boletoid mushrooms distributed in the west to east of the Indian Himalayas will undoubtedly reveal several novel taxa and will also resolve many taxonomic issues in years to come.
Recently, while undertaking routine macrofungal explorations in different parts of Uttarakhand and Himachal Pradesh, the authors came across some interesting boletoid mushrooms. A thorough examination of these collections through macro-and micromorphology, followed by multigene molecular phylogenetic estimations, uncovered four new species and two new records for Indian mycobiota. Leccinellum binderi sp. nov., Cyanoboletus paurianus sp. nov., Xerocomus uttarakhandae sp. nov., and Xerocomellus himalayanus sp. nov. are described in detail. Moreover, Cyanoboletus macroporus (originally reported from Pakistan) and Xerocomus fraternus (originally reported from China) are also reported here for the first time from India. Xerocomus is abbreviated as 'X.' whereas Xerocomellus is abbreviated as 'Xe.' in this paper.

Macrofungal Survey and Morphological Study
Routine macrofungal surveys were undertaken in temperate and subalpine Himalayan forests in Pauri and Rudraprayag districts (temperate mixed forests) of Uttarakhand and Chamba district (temperate coniferous forests) of Himachal Pradesh in India from 2021 to 2022. Several boletoid mushrooms were collected from both states. Macromorphological characters and habitat details were recorded in fresh, young, and mature basidiomata in the field and/or in the base camp. After recording the macromorphological characters, basidiomata were placed in a field dryer for drying. Photographs of these fresh and dry basidiomata and microphotographs were taken with the aid of Canon SX 220 HS and Nikon-DS-Ri1 (dedicated to the Nikon Eclipse Ni compound microscope) cameras. Color codes and terms used are mostly from the Methuen Handbook of Color [24]. Micromorphological characters were observed with compound microscopes (Nikon Eclipse Ni-U and Olympus CX 41). Free-hand sections from dry specimens were mounted in a mixture of 5% KOH, 1% Phloxine, and 1% Congo red or in distilled water. Micromorphological drawings were prepared with a drawing tube (attached to the Olympus CX 41 microscope) at 1000×. The basidium length excludes that of the sterigmata. Basidiospore measurements were recorded in profile view from 30 basidiospores. Basidiospore measurements and length/width ratios (Q) are recorded here as minimum-mean-maximum. Herbarium codes follow Thiers (continuously updated). Field emission scanning electron microscope (FESEM) illustrations of basidiospores were obtained from dry spores (spore prints) that were directly mounted on a double-sided adhesive tape pasted on a metallic specimen stub and then scanned with a gold coating at different magnifications in high vacuum mode to observe patterns of spore ornamentation. This work was carried out with an FEI Quanta FEG 250 model installed at the S.N. Bose National Centre for Basic Sciences in Kolkata, India.

Genomic DNA Extraction, PCR Amplification and Sequencing
The genomic DNA was extracted from 100 mg of dried basidioma from five samples using a modified cetyltrimethylammonium bromide (CTAB) DNA isolation protocol [25]. The DNA quality and quantity were checked by taking absorbance readings in a Nan-oDrop Lite UV spectrophotometer (Thermo Scientific, Waltham, MA, USA). Genomic DNA dilutions were done for highly concentrated DNA accessions up to 50 ng/µL for PCR amplification. The PCR amplification of the Internal Transcribed Spacer region (nrITS), part of the 28S ribosomal RNA (nrLSU), region between conserved domains 6 and 7 of the second largest subunit of RNA polymerase II (rpb2), and part of the translation elongation factor 1-α (tef 1-α) were done using the primer pairs ITS1-F and ITS4; LR0R and LR5; brpb2-6F and frpb2-7cR; and ef1-983F and ef1-1567R, respectively [26][27][28][29][30]. PCR amplification was carried out in a ProFlex PCR system (Applied Biosystems, Waltham, MA, USA) programmed for an initial denaturation at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 1 min, annealing at 50 • C for 30 s, and extension at 72 • C for 1 min. The final extension was kept at 72 • C for 7 min. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Both strands of the PCR products were sequenced at Eurofins Genomics India Pvt. Ltd., Bengaluru, India. The sequence quality was checked using Sequence Scanner Software ver. 1 (Applied Biosystems). Sequence alignment and the required edition of the obtained sequences were carried out using Geneious Pro ver. 5.1 [31]. All newly generated sequences in this study were submitted to GenBank. Accession numbers of species used in phylogenetic analysis (Figures 1-3) are listed in Tables 1-3.
Leccinum parascabrum X. Meng, Yan C. Li, and Zhu L. Yang (originally described from China) mostly exhibit the similar colors of pileus and hymenophore and the similar slender stipes with scabrous squamules; however, in Le. Parascabrum, the stipe context becomes greenish blue when exposed, and pileipellis shows a trichodermium nature with filamentous hyphae (never with chains of subglobose to pyriform cells) [37].
Two recently described species of Leccinellum from China, L. alborufescens and L. fujianense, are also partly related to the present species. But both L. alborufescens and L. fujianense can easily be separated from L. binderi by a rugulose or pitted brown to dark brown pileus, reddening of the hymenophore, and context (on bruising or exposure). Moreover, L. alborufescens occurs in tropical regions, whereas L. fujianense is found in subtropical regions [36].
Commentary: The genus Cyanoboletus is distinct from all other genera of Boletaceae by its yellowish brown, brown to dark brown pileus that shows instant bluing of context on exposure and hymenophore when bruised, cutis, trichoderm pileipellis, and smooth basidiospores [32]. But based only on morphology, it is difficult to separate most of the species. In the field, C. paurianus is quite close to another Asian species, C. sinopulverulentus (also reported below from India); however, the latter one shows larger basidiospores, while our proposed new species is clearly recovered in multigene phylogeny (Figure 2) [51]. Previously, C. hymenoglutinosus D. Chakr., K. Das, A. Baghela, S.K. Singh, and Dentinger from India could easily be distinguished from C. paurianus by its highly glutinous hy-menial layer and basidia, which are distinctively covered with thick gluten and larger basidiospores (12-12.8-15 × 4.8-5.2-5.8 µm) [47]. Our second species, C. macroporus, is a recently established (2021) species from the temperate to subalpine forests of Pakistan. This species can be distinguished by its brownish-red pileus that instantly changes its color to olivaceous black to dark greenish black when handled, much wider pores among the other similar Cyanoboletus species, stipe without reticulation, yellow to yellowish brown hymenophore with angular pores, and nrITS-based phylogeny. Our Indian collection shows morphological similarities and phylogenetic support to establish its conformity with the Pakistani species [52]. Moreover, in this present study, some characters that are missing in the protologue, like pore size, microchemical spot test on pileus, and context, are also recorded here.   MycoBank: MB 848633 GenBank: OQ748036 (nrITS, holotype); OQ748035 (nrITS); OQ748037 (nrLSU, holotype); OQ748038 (nrLSU).
Commentary: The genus Xerocomus Quél. is separated from all other genera of Boletaceae by its long tubes with relatively large pores (1-3 mm in diam.), a trichodermium pileipellis, and usually bacillately warted basidiospores under SEM. Species in this genus are also quite difficult to separate by morphology alone. Therefore, molecular analysis plays a significant role in separating the species.
Our second species, Xerocomus fraternus, is distinguished by a set of characters: a light yellow pore surface and tubes; basidiospores of length measuring ≤13 µm; the lower half of the stipe context that is mostly reddish brown on exposure; and its occurrence in temperate to tropical forests [32]. Present Indian collections completely agree morphologically with the samples (holotypes) reported from the neighboring country, China. For the first time, it is being reported from India.       Diagnosis: Distinguished from its closely allied species by its unchanging pore surface, tube, and context on exposure, yellow subpellis, occurrence under Cedrus sp., and nrITS and nrLSU sequence data.
Description: Pileus 23-80 mm in diam., solitary, mostly convex, sometimes planoconvex with maturity, greyish yellow (3C4-5) when young to brown (6E5-7) with maturity; surface areolate when mature, showing yellow context, turning deep yellow (4A7-8) with KOH, no reaction with NH4OH and FeSO4; margin entire, sterile flap of tissue not present. Pore surface: maize yellow to deep yellow (4A6-8), no change in bruising; pores: angular, compound, 8-10/mm. Tubes adnate, 5-8 mm long, yellowish white (2A2), no change when bruised. Stipe 40-90 × 10-30 mm, mostly cylindrical to narrowly clavate, sometimes with bulbous base, solid but mostly infested with larvae, light yellow or sun yellow (2A5) at upper one third, brownish red (8C6-7) with combination of dark brown near base, greenish blue when bruised (not instantly), then finally blackish brown; surface longitudinally fibrillose; basal mycelia white (1A2), sometimes forming rooting base. Context in pileus up to 8 mm thick, yellow, no change when exposed; the context in the stipe, yellowish white, slightly turning greenish blue with time when exposed. Odor is acidic. Spore print is olive brown.  Diagnosis: Distinguished from its closely allied species by its unchanging pore surface, tube, and context on exposure, yellow subpellis, occurrence under Cedrus sp., and nrITS and nrLSU sequence data.
Description: Pileus 23-80 mm in diam., solitary, mostly convex, sometimes planoconvex with maturity, greyish yellow (3C4-5) when young to brown (6E5-7) with maturity; surface areolate when mature, showing yellow context, turning deep yellow (4A7-8) with KOH, no reaction with NH 4 OH and FeSO 4 ; margin entire, sterile flap of tissue not present. Pore surface: maize yellow to deep yellow (4A6-8), no change in bruising; pores: angular, compound, 8-10/mm. Tubes adnate, 5-8 mm long, yellowish white (2A2), no change when bruised. Stipe 40-90 × 10-30 mm, mostly cylindrical to narrowly clavate, sometimes with bulbous base, solid but mostly infested with larvae, light yellow or sun yellow (2A5) at upper one third, brownish red (8C6-7) with combination of dark brown near base, greenish blue when bruised (not instantly), then finally blackish brown; surface longitudinally fibrillose; basal mycelia white (1A2), sometimes forming rooting base. Context in pileus up to 8 mm thick, yellow, no change when exposed; the context in the stipe, yellowish white, slightly turning greenish blue with time when exposed. Odor is acidic. Spore print is olive brown. Basidiospores  Commentary: The genus Xerocomellus Šutara is separated from its morphologic sister genus Xerocomus by its smooth or longitudinally striate (never bacillate) basidiospore, palisadoderm nature of pileipellis, small or mostly medium-sized, often vividly colored, surface dry, at first velvety and later often rimose-areolate, and a minutely granulose, sometimes longitudinally striate but mostly non-reticulate stipe, which is usually slender and not very firm [55]. Our newly proposed Indian collection features medium-sized basidiomata, brown pileus that turned areolate with maturity, deep yellow pores that remain unchanged when bruised, yellow pileus context, unchanging when exposed, stipe cylindrical to sometimes bulbous at base, yellow stipe with combination of brownish red and dark brown towards base, smooth basidiospore, and occurrence under Cedrus deodara in temperate coniferous forest of Western Himalaya. Xerocomellus himalayanus is morphologically as well as phylogenetically close to the European species Xe. sarnarii Simonini, Vizzini, and U. Eberh, but can be separated in the field as the latter shows a bluish color when context, tubes, and pores are bruised or exposed to air, and its occurrence under Quercus sp. Moreover, Xe. sarnarii shows smaller basidiospores (13.8-15.1 × 5.5-6.1 µm) and pleurocystidia (35-52 × 6-11 µm) [56]. Some morphologically similar and phylogenetically close members of this Indian species are Xe. poederi G. Moreno, Heykoop, Esteve-Rav., P. Alvarado, and Traba, and Xe. chrysenteron (Bull.) Šutara, but Xe. poederi differs from Xe. himalayanus by its reddish epicutis, depressed pores, reddish stipe context, and habitat under Quercus sp. [57]. Similarly, Xe. chrysenteron can be distinguished by its reddish cracks on pileus, context turning faint blue and then finally reddish on exposure, and narrower basidiospores (12.3-16.1 × 4.1-5.6 µm) without any truncation at apices [54,58]. Xerocomellus mendocinensis (Thiers) N. Siegel, C.F. Schwarz, and J.L. Frank; Xe. dryophilus (Thiers) N. Siegel, C.F. Schwarz, and J.L. Frank; and Xe. diffractus N. Siegel, C.F. Schwarz, and J.L. Frank are distinguished from the Indian collection by their geographical location, ecology, and nrITS and nrLSU sequences. Moreover, Xe. Mendocinensis differs by its pink scabrous stipe and instantly bluing tubes when bruised. Similarly, Xe. Dryophilus and Xe. Diffractus both show bluing of the hymenophore and context on exposure, which makes them distinct from Xe. himalayanus in the field [59].

Discussion
India, with its luxuriant forests of coniferous and/or deciduous trees, is immensely diverse in terms of fleshy mushrooms. Boletoid mushrooms (Boletes) are no exception. To date, about 96 species belonging to 27 genera have been reported in Boletes from India [60][61][62][63][64][65][66][67][68]. Major ectomycorrhizal host trees that support the growth and development of these mushrooms belong to genera like Quercus L., Castanopsis (D. Don) Spach, Lithocarpus Blume, Hopea L., Betula L., Shorea Roxb. ex C.F. Gaertn., Abies Mill., Picea A. Dietr., Cedrus Trew, Pinus L., Tsuga (Endl.) Carrière, and Larix Mill. Keeping in view the existing number of genera of Boletes from the globe (108) and the reported number of genera in India (27), it becomes clear that this group is seriously under-explored, and multigene molecular phylogeny (the backbone of systematics in Boletoid mushrooms) has hardly been applied to reveal the diversity of this group. The present contribution with morphotaxonomy and multigene molecular phylogeny is an initiative to uncover this immensely diverse wealth of Boletoid mushrooms in the Indian Himalayas. The reporting of six species of Boletes in the present article brings the total to 102 species from this vast country. The four genera dealt with in this contribution are separated by the key given below. Following the present study, many macrofungal surveys will be conducted in this region to unveil the hidden diversity of Boletoid mushrooms in the near future.
Key to the Studied Genera of Boletes

1.
Stipe surface with scabs that turns brownish black when bruised; stipe context is cream colored, changing brown-black when exposed .