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Article

Four Novel Species and Two New Records of Boletes from India

by
Kanad Das
1,
Aniket Ghosh
1,
Dyutiparna Chakraborty
2,
Sudeshna Datta
1,
Ishika Bera
3,
Ranjith Layola MR
1,
Farheen Banu
1,
Alfredo Vizzini
4,* and
Komsit Wisitrassameewong
5,*
1
Central National Herbarium, Botanical Survey of India, Howrah 711103, India
2
Eastern Regional Centre, Botanical Survey of India, Shillong 793003, India
3
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
4
Department of Life Sciences and Systems Biology, University of Torino, 10124 Torino, Italy
5
National Biobank of Thailand, National Science and Technology Development Agency, Pathum Thani 12120, Thailand
*
Authors to whom correspondence should be addressed.
J. Fungi 2023, 9(7), 754; https://doi.org/10.3390/jof9070754
Submission received: 22 June 2023 / Revised: 10 July 2023 / Accepted: 13 July 2023 / Published: 17 July 2023
(This article belongs to the Special Issue Fungal Diversity and Systematics in the Digital Era)
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Abstract

:
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.

1. 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.

2. Materials and Methods

2.1. 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.

2.2. 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 NanoDrop 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 (tef1-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 (Figure 1, Figure 2 and Figure 3) are listed in Table 1, Table 2 and Table 3.

2.3. Phylogenetic Analysis

The newly generated nrITS, nrLSU, rpb2, and tef1-α sequences of Leccinellum binderi, Cyanoboletus paurianus, C. macroporus, Xerocomus fraternus, X. uttarakhandae, and Xerocomellus himalayanus, plus similar ones, were retrieved from a nBLAST search against GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 9 May 2023), UNITE database (https://unite.ut.ee, accessed on 9 May 2023) and relevant published phylogenies [2,5,32,33,34,35,36,37]. Four datasets (nrITS, nrLSU, rpb2, and tef1-α) were created separately. All the datasets were aligned separately using the online version of the multiple sequence alignment program MAFFT v. 7 (https://mafft.cbrc.jp/alignment/software/, accessed on 21 June 2023) with the L-INS-I strategy [38]. The alignment was checked and trimmed manually with MEGA v. 7 [39]. To eliminate ambiguously aligned positions in the alignment as objectively as possible, the online program Gblocks 0.91b [40] was used. The program was run with settings allowing for smaller blocks, gaps within these blocks, and less strict flanking positions. Species delimitation was first examined using single-locus phylogenies. When significant conflict was not observed among the single-locus phylogenies, we concatenated them into one multi-locus dataset using BioEdit v. 7.0.9 [41]. The combined dataset was phylogenetically analyzed using maximum likelihood (ML). ML was performed using raxmlGUI 2.0 [42] with the GTRGAMMA substitution model. ML analysis was executed using the rapid bootstrap algorithm with 1000 replicates to obtain nodal support values. Maximum likelihood bootstrap (MLbs) values ≥70% are shown in the phylogenetic tree (Figure 1, Figure 2 and Figure 3).

3. Results

3.1. Phylogenetic Inferences

In the present study, multi-locus (nrLSU, rpb2, and tef1-α) phylogenetic analysis showed that the sequences obtained from Leccinellum binderi (voucher nos. KD 22-007 and KD 22-015) clustered with the Leccinellum lineage; however, our specimens were recovered as distinct species within the phylogenetic tree (Figure 1). On the other hand, combined three-locus (nrITS, nrLSU and rpb2) phylogenetic analysis revealed that the two collections of our second species, Cyanoboletus paurianus (voucher nos. KD 22-008 and KD 22-0009) clustered with an unidentified Cyanoboletus sp. (voucher nos. HKAS 90208-1 and HKAS 90208-2) from China, however, our specimens are recovered as distinct species within the phylogenetic tree (Figure 2), whereas our third species, Cyanoboletus macroporus (voucher nos. DC 21-02 and DC 21-24) are nested within the C. macroporus clade consisting of sample vouchers (AN-2020a and sarwar1) collected from Pakistan and suggesting its strong similarity or conspecificity with the Asian species C. macroporus with a strong (MLbs = 100%) support (Figure 2). Phylogenetic analysis based on two-locus (nrITS and nrLSU) sequences exhibits that our fourth species, Xerocomus uttarakhandae (voucher nos. KD 22-002 and KD 22-005), is nested (indicated in blue arrow) with an unidentified Xerocomus sp. (voucher nos. HKAS 90208-1 and HKAS 90208-2) with strong support (MLbs = 98%), being sister to X. doodhcha and X. reticulostipitatus (voucher nos. KD 13-082 and MEH 16_B-7) collected from India and X. fulvipes (voucher no. HKAS52556) from China; however, our specimens are recovered as distinct species within the phylogenetic tree (Figure 3). On the other hand, our fifth species, Xerocomus fraternus (voucher nos. KD 22-025 and KD 22-027), is nested within the X. fraternus clade consisting of Chinese collections (voucher nos. HKAS52526 and HKAS69291), suggesting its conspecificity with the Asian species, X. fraternus, with strong (MLbs = 100%) support (Figure 3). Phylogenetic analysis based on nrITS and nrLSU sequences revealed that the two collections of our sixth species, Xerocomellus himalayanus (voucher nos. DC 21-12 and DC 21-56) clustered with Xerocomellus sarnarii from Europe (voucher nos. MCVE 28571, MCVE 28577, and ML900101XE) with strong support (MLbs = 100%) and is sister to Xerocomellus chrysenteron collected from Europe and China; however, our specimens are recovered as distinct species within the phylogenetic tree (Figure 3).

3.2. Taxonomy

Leccinellum binderi K. Das, A. Ghosh & Vizzini, sp. nov., Figure 1, Figure 4 and Figure 5.
MycoBank: MB 848631
GenBank: OQ858380 (nrLSU, holotype), OQ858379 (nrLSU); OQ914386 (rpb2, holotype), OQ914387 (rpb2); OR102315 (tef1-α, holotype), OR102316 (tef1-α).
Etymology: Commemorating Dr. Manfred Binder for his significant contribution to the systematics of Boletaceae.
Type: INDIA, UTTARAKHAND: Pauri District, Chaubatta, 15 August 2022, alt. 1904 m, N 30°09.676′ E 78°51.240′, KD 22-007 (CAL 1923, holotype!)
Diagnosis: Distinguished from other known Asian species by long slender stipe, stipe context that is changing brown to black in the lower half, and the relatively large basidiospores measuring 13.8–18.22–22 × 5.4–5.96–7 μm, presence of pseudocystidia with brown to white content, occurrence under Quercus sp. and nrLSU, rpb2, and tef1-α sequence data.
Description: Pileus 40–50 mm in diam., hemispherical to convex or planoconvex, then applanate; subtomentose, cracked (towards margin) at maturity; yellowish brown or Snuff (Brown) (5F6) or paler in combinations, greyish yellow (4B3) to champagne (4D4) towards margin; surface viscid when wet; margin entire, initially decurved then somewhat uplifted with a very narrow (up to 0.5 mm) sterile flap of tissue; turning Persian orange (6A7) with KOH but unchanging with NH4OH. Pore surface is pale yellow (2A3) when young, becoming orange-white (5A2) with maturity, and pale brown when bruised; pores are rounded, rarely compound, 2/mm. Tubes adnexed, 7–9 mm long, yellowish white to pale yellow (3A2–2A3), unchanging. Stipe 70–92 × 9–10 mm, more or less cylindrical with tapering apex, pithy, white to buff colored; surface subtomentose to appressed-fibrillose and scabrous; scabrous squamules white when young, becoming darker (brownish black) with maturity or handling; basal mycelia white (1A2). Context in pileus up to 8 mm thick, white to yellowish white; the context in the stipe, cream-colored, changing brown-black when exposed. The odor is mild. Spore prints were not obtained.
Basidiospores 13.8–18.22–22 × 5.4–5.96–7 μm, [Q = 2.55–3.07–3.50], subfusoid to elongate and inequilateral in side view with distinct suprahilar depression, light yellow, smooth, inamyloid. Basidia 32–40 × 9–11.5 μm, subclavate to broadly clavate, 4-spored; sterigmata up to 6 μm long. Pleurocystidia 45–62 × 10–12.5 μm, abundant, fusoid-ventricose with subcapitate to appendiculate apex showing somewhat wrinkled in outline, thin-walled, emergent 25–30 μm. Pseudocystidia are 3–8 μm wide, abundant, filamentous, with an irregular outline, and content colorless to yellow brown. Tube edge fertile. Cheilocystidia 35–49 × 9–12 μm, abundant, fusoid-ventricose with subacute to appendiculate apex, thin-walled, emergent 9–23 μm. Hymenophoral trama boletoid, hyphae cylindrical, 4–8 μm wide. Pileipellis 100–130 μm thick, a trichoderm submerged under 15 μm thick gluten, composed of erect, frequently septate branched elements with chains of subglobose, clavate to pyriform cells; terminal cells 16–33 × 8–18 μm, cylindrical to clavate; oleiferous hyphae present. Stipitipellis is composed of a layer of slender, parallel to loosely arranged adpressed hyphae (4–10 μm wide) and frequently protruding hymenial tufts composed of basidiole, basidia, caulocystidia, and oleiferous hyphae. Caulobasidia 17–26 × 6–10 μm, clavate, 2- or 4-spored. Caulocystidia 22–30 × 9–12 μm, clavate, pyriform, ventricose to fusiform with rounded to subappendiculate or rarely mucronate apex. Clamp connections are absent.
Habitat: solitary or scattered, under Quercus sp. (Fagaceae) in temperate to subalpine Himalaya.
Additional Specimens Examined: INDIA, UTTARAKHAND: Pauri District, Khirsu, 17 August 2022, alt. 1774 m, N 30°10.150′ E 78°52.128′, KD 22-015 (CAL 1924); Rudraprayag District, Baniakund, 21 August 2022, alt. 2518 m, N 30°29.131′ E 79°11.653′, KD 22-032 (CAL 1925).
Commentary: The genus Leccinellum Bresinsky & Manfr. Binder was established to accommodate Leccinum Gray species with a yellow hymenophore and a trichodermium pileipellis [43,44,45]. Presently, this genus is represented worldwide by about 18 taxa [1,32,36,43,44,46,47,48,49,50]. Leccinellum binderi, the proposed new species, is characterized by the yellowish brown to snuff (brown) to greyish yellow pileus, the cream-colored stipe context that changes to brown-black on exposure, the yellowish white to pale yellow hymenophore unchanging when injured, rounded pores, long slender stipe, white unchanging stipe context, the relatively large basidiospores measuring 13.8–18.22–22 × 5.4–5.96–7 μm, and the presence of pseudocystidia with yellow-brown content.
Now, it is realized that only with morphological features is it very difficult to separate the genus Leccinellum (abbreviated as L.) from Leccinum (abbreviated as Le.). But phylogenetically, our Indian collections fall into the genus Leccinellum based on a three-locus dataset (nrLSU, tef1-α, and RPB2) (Figure 1). Combining the molecular data with the morphological features of stipitate-pileate basidiomata, we place our collections in the genus Leccinellum.
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].
Cyanoboletus paurianus K. Das and A. Ghosh, sp. nov., Figure 2, Figure 6 and Figure 7.
MycoBank: MB 848632
GenBank: OQ859919 (nrLSU, holotype); OQ859920 (nrLSU); OQ914388 (rpb2, holotype); OQ914389 (rpb2).
Etymology: Referring to the type locality (Pauri district) in the state of Uttarakhand (India).
Type: INDIA, UTTARAKHAND: Pauri District, Phedkhal, 15 August 2022, alt. 1871 m, N 30°09.579′ E 78°51.313′, KD 22-009 (CAL 1926, holotype!)
Diagnosis: Distinguished from other known Asian species by smaller basidiospores (9.1–11.51–13.2 × 4.3–4.85–5.5 μm), absence of gluten on the hymenial surface and basidia, and occurrence under Quercus sp. and nrITS, nrLSU, and rpb2 sequence data.
Description: Pileus 40–50 mm in diam., convex when young, broadly convex with maturity (but never plane), glabrous to leathery, dark brown (8E–F4); surface viscid to sticky when wet; margin entire, incurved with a narrow flap of tissue (up to 1 mm wide); turning reddish brown (9F8) with KOH, reddish brown (9E7) with NH4OH, dull green (25E3) with FeSO4. Pore surface maize (yellow) (4A6), becoming grayish green (25E5) when bruised, then turning brownish after 2 h; pores rounded, 2/mm. Tubes adnexed, 2–2.5 mm long, maize (yellow) (4A6), becoming greyish green (25E5) when bruised. Stipe 45–55 × 7–10 mm, more or less cylindrical with slightly tapered at apex, pithy; apex (5–8 mm) concolorous to pore surface, i.e., maize (yellow) (4A6); below onwards along the length of the stipe, concolorous to pileus, i.e., reddish brown (8E–F7); glabrous to leathery, with white basal mycelia. Context in pileus up to 4–9 mm thick, cream to buff colored, changing instantly to bluish grey to greyish blue (22–23B3–5); the context in stipe buff to yellow at apex, gradually greyish black towards the base, changing bluish on exposure. Spore print is olivaceous brown.
Basidiospores 9.1–11.51–13.2 × 4.3–4.85–5.5 μm, [Q = 1.65–2.38–2.86], subfusoid to elongate and inequilateral in side view with distinct suprahilar depression, light yellow, smooth, inamyloid. Basidia 25.3–35 × 8–9 μm, subclavate to broadly clavate, 4-spored; sterigmata up to 6 μm long. Pleurocystidia 25–35 × 5.5–7.5 μm, abundant, fusoid-ventricose with rounded to subacute apex, thin-walled, emergent 9–14 μm. The tube edge is sterile. Cheilocystidia 21–32 × 6.5–9 μm, abundant, fusoid-ventricose or rarely clavate, thin-walled, emergent 9–15 μm. Hymenophoral trama boletoid, hyphae divergent, cylindrical, glutinous, 4–5 μm wide. Pileipellis 200–300 μm thick, an ixocutis to ixotrichoderm, submerged under very thin gluten, composed of suberect to erect, frequently septate interwoven hyphae with elongate (never inflated) cells; terminal cells 32–55 × 5–8 μm, cylindrical with a rounded to subfusoid apex. Stipitipellis is composed of erect to suberect, somewhat interwoven hyphae forming trichodermium, often with tufts of abundant cystidia and some basidia. Caulocystidia 28–35 × 8–10 μm, clavate to subclavate or fusoid-ventricose, aseptate to septate with rounded to subfusoid apex. Caulobasidia 22–30 × 7–9 μm, rare, narrowly to broadly clavate, 2- or 4-spored; sterigmata up to 8 μm long. Clamp connections are absent.
Habitat: scattered to gregarious, under Quercus sp. (Fagaceae), in temperate broadleaf forest.
Additional Specimen Examined: INDIA, UTTARAKHAND: Pauri District, Phedkhal, 15 August 2022, alt. 1736 m, N 30°08.723′ E 78°51.212′, KD 22-008 (CAL 1927).
Cyanoboletus macroporus Sarwar, Naseer & Khalid, Figure 2, Figure 8 and Figure 9.
GenBank: OQ860238 (nrITS), OQ860240 (nrITS); OQ860239 (nrLSU), OQ860241 (nrLSU); ON364552 (rpb2), OQ876894 (rpb2).
Description: Pileus 15–60 mm in diam., mostly convex, sometimes planoconvex with maturity, applanate with uplifted margin, reddish brown (8–9F6–8) when young to dark brown (8–9D8); surface viscid when moist, velvety, often with patches of appressed small squamules; margin entire to undulated, initially incurved then uplifted; turning greenish black (20F8) when bruised, brownish yellow (5C8) with NH4OH, and dark red (10C8) with KOH. Pore surface: yellowish brown (2A5), becoming blue-black (20D5) when bruised; pores: angular, pore stuffed when young; pore mouth: red (10C–D8), 0.7–0.9/mm. Tubes are adnate, 3–6 mm long, light yellow (2A5), becoming greyish blue (20D5) when bruised. Stipe 30–55 × 5–8 mm, more or less cylindrical, solid, yellow at apex, brownish black towards the base, white to buff colored at base; surface faintly pruinose. Context in pileus up to 10 mm thick, yellowish white, instantly turning bluish green (25B6) when exposed. Odour mild. Taste none. Spore print is olive brown.
Basidiospores 11–12.5–13.4 × 4.6–5.2–6 μm, [n = 30, Q = 2.1–2.2–2.6], ellipsoid to fusoid and inequilateral in side view, hyaline, smooth under light microscopy. Basidia 26–34 × 7–11 μm, clavate, 4-spored; sterigmata 2–4 × 1–2 μm. Pleurocystidia 45–67 × 10–12 μm, subcylindric to fusiform or subventricose with rounded apex, thin-walled, emergent up to 35 μm. Subhymenial layer 10–20 μm thick. Tube edge fertile. Cheilocystidia were not found. Hymenophoral trama divergent, hyphae cylindrical, septate, unbranched, 2–4 μm wide. Pileipellis is 60–150 μm thick, a trichoderm composed of erect chains of cells; terminal cells are 6–15 × 4–8 μm, broadly cylindrical to subventricose. Stipitipellis fertile, composed of slender, subparallel hyphae (3–5 μm wide); sometimes protruding hymenial tufts composed of basidia and cystidia. Caulobasidia 26–35 × 6–8 μm, subcylindrical to clavate, 2- or 4-spored. Caulocystidia 29–35 × 7–9 μm, subcylindric to subfusoid. Clamp connections are absent.
Habitat: Solitary to gregarious, in temperate coniferous forests of Cedrus deodara.
Specimens Examined: INDIA, HIMACHAL PRADESH: Chamba District, Kalatop, 18 July 2021, alt. 2398 m, N 32°32.076′ E 76°00.931′, DC 21-02 (CAL 1934); Kalatop, 19 July 2021, alt. 2374 m, N 32°33.051′ E 76°01.138′, DC 21-24 (CAL 1935).
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 hymenial 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.
Xerocomus uttarakhandae K. Das, Sudeshna Datta, and A. Ghosh, sp. nov., Figure 3, Figure 10 and Figure 11.
MycoBank: MB 848633
GenBank: OQ748036 (nrITS, holotype); OQ748035 (nrITS); OQ748037 (nrLSU, holotype); OQ748038 (nrLSU).
Etymology: Referring to the type locality (the state of Uttarakhand), India.
Type: INDIA, UTTARAKHAND: Pauri District, Teka, 14 August 2022, alt. 1843 m, N 30°06.878′ E 78°45.485′, KD 22-005 (CAL 1928, holotype!).
Diagnosis: Distinguished from other closely allied Asian species by cracked to areolate pileus surface showing reddish context, shorter stipe, absence of reticulation on stipe surface, occurrence under Quercus sp., and nrITS and nrLSU sequence data.
Description: Pileus 37–70 mm in diam., hemispherical to convex or planoconvex; subtomentose to velvety, becoming cracked to areolate at maturity; greyish orange to greyish brown (5B–D3) or paler in combinations, showing reddish areas through cracks and cut or injured areas beneath the cuticle; surface never viscid when wet; margin entire, initially decurved then somewhat uplifted with a very narrow (up to 0.5 mm) sterile flap of tissue; turning brown (6E7) with KOH and olive gray (3E2) with FeSO4. Pore surface: pastel yellow (2A4) or lemon yellow, becoming greyish turquoise (24D5–6) when bruised, then brownish after some time; pores: angular, often compound, 1/mm. Tubes adnexed, 6–8 mm long, pastel yellow (2A4) or lemon yellow, color reaction same as pore surface. Stipe 24–60 × 5–13 mm, more or less cylindrical, gradually tapering towards the base, longitudinally striate to fibrillose at the upper half, yellowish white at the apex, orange white (6A2) towards the middle and lower half, with white (1A2) basal mycelia. Context in pileus up to 12 mm thick, white to yellowish white; the context in the stipe, yellowish white, turning pale yellow (4A3) with KOH and greyish green (25C3) with FeSO4. Odour mild. Spore prints were not obtained.
Basidiospores 9–10.6–12.6 × 3.8–4.5–5.1 μm, [n = 30, Q = 2–2.36–2.74], ellipsoid to fusoid and inequilateral in side view, hyaline, smooth under light microscope but under SEM spore surface bacillate. Basidia 24–32 × 8–8.5 μm, clavate, 4-spored; sterigmata 2–4 × 1–2 μm. Pleurocystidia 38–62 × 7–10 μm, subcylindric to ventricose, or subfusoid, thin-walled, few with incustrations on wall, emergent up to 39 μm. Subhymenial layer 12–15 μm thick. Tube edge fertile. Cheilocystidia 34.5–47 × 7.5–11 μm, abundant, clavate to subfusoid or fusoid with tapering apex, thin-walled, emergent 12–20 μm. Hymenophoral trama phylloporoid, hyphae cylindrical, septate, branched, thin-walled, non-gelatinous, 5–10 μm wide. Pileipellis up to 180 μm thick, as a trichodermium, composed of erect cylindrical septate hyphae; terminal cells 25–51 × 7–10 μm, cylindrical, sometimes tapered at the apex; pigmented. Stipitipellis fertile composed of a layer of slender, parallelly arranged adpressed hyphae (5–8.75 μm wide) and frequently protruding hymenial tufts composed of basidia, basidioles, and caulocystidia. Caulobasidia 24–33 × 7–11 μm, rare, clavate, 4-spored. Caulocystidia 21–33 × 7–13 μm, ventricose-fusoid, clavate to bulbous, or pyriform to subcapitate. Clamp connections are absent.
Habitat: solitary or scattered, in temperate forests under Quercus sp. (Fagaceae).
Additional Specimen Examined: INDIA, UTTARAKHAND: Pauri District, Teka, 14 August 2022, alt. 1893 m, N 30°06.656′ E 78°45.288′, KD 22-002 (CAL 1929).
Xerocomus fraternus Xue T. Zhu and Zhu L. Yang, Figure 3, Figure 12 and Figure 13.
GenBank: OQ776920 (nrITS), OQ776919 (nrITS), OQ771932 (nrLSU), and OQ771933 (nrLSU).
Description: Pileus 35–75 mm in diam., mostly convex, sometimes planoconvex with maturity, subtomentose, greyish orange (5B3–5) to reddish brown; surface often warty; margin entire to undulated, initially incurved then decurved to upturned with a narrow (up to 1.5 mm) sterile flap of tissue; turning light brown or sunburn (6D5) with KOH, violet brown (11E5) with NH4OH, and dull green (25D3) with FeSO4. Pore surface: light yellow or sun yellow (2A5), becoming greyish green (25E5) when bruised; pores: angular, mostly pentagonal to irregular, rarely compound, 1–2/mm. Tubes adnexed, 7–9 mm long, light yellow or sun yellow (2A5), becoming greyish green (25E5) when bruised. Stipe 40–80 × 6–8 mm, more or less cylindrical with tapering base, solid, light yellow or sun yellow (2A5) at apex, greyish yellow (4B4) towards the base, white to buff colored; surface longitudinally fibrillose; basal mycelia white (1A2). Context in pileus up to 10 mm thick, white to yellowish white, slowly becoming greenish grey (25B2) when exposed; the context in the stipe, yellowish white to cream on the upper half but reddish brown on the lower half. Odour mild. Spore print is olive brown.
Basidiospores 8.2–10.5–12.4 × 3.2–4.61–5.6 μm, [n = 30, Q = 1.73–2.29–2.7], ellipsoid to fusoid and inequilateral in side view, hyaline, smooth under light microscope, but under SEM spore surface bacillate. Basidia 24–39 × 6–12 μm, clavate, 4-spored; sterigmata 3–5 × 1–2 μm. Pleurocystidia 52–99 × 7–20 μm, subcylindric to fusiform or subventricose with rounded or rarely mucronate apex, thin-walled, emergent up to 36 μm. Subhymenial layer 12.5–20 μm thick. Tube edge fertile. Cheilocystidia 38–45 × 6–8 μm, rare, subcylindrical to subfusiform, thin-walled, emergent up to 34 μm. Hymenophoral trama divergent, hyphae cylindrical, septate, unbranched, thin-walled, non-gelatinous, 3–5 μm wide. Pileipellis is 150–200 μm thick, a trichoderm composed of erect chains of cells; terminal cells are 23–48 × 6–13 μm, cylindrical, conic, subventricose, or subclavate. Stipitipellis fertile is composed of a layer of slender, parallel hyphae (5–10 μm wide) and frequently protruding hymenial tufts composed of basidia and cystidia. Caulobasidia 26–38 × 6–7 μm, subcylindrical to clavate, 4-spored. Caulocystidia 24–44 × 6–10 μm, subcylindric to clavate. Clamp connections are absent.
Habitat: solitary to gregarious, under Quercus sp. (Fagaceae) in temperate to subalpine Himalaya.
Specimens Examined: INDIA, UTTARAKHAND: Rudraprayag District, Chopta, 19 August 2022, alt. 2846 m, N 30°28.995′ E 79°10.760′, KD 22-025 (CAL 1930); Baniakund, 20 August 2022, alt. 2518 m, N 30°29.131′ E 79°11.653′, KD 22-027 (CAL 1931).
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.
Xerocomus uttarakhandae is characterized by medium-sized basidiomata with a velvety and cracked to areolate pileus surface showing reddish context, a yellow pore surface that becomes bluish when bruised, stipe yellowish white at the apex, orange white at the mid and lower half with white basal mycelia, the presence of variously shaped (subcylindric to clavate to bulbous to pyriform) and septate caulocystidia, and their occurrence under Quercus sp. in temperate Himalaya. Combining morphology and molecular phylogeny, three species in this genus were erected in India in the last decade. They are Xerocomus doodhcha K. Das, D. Chakr., Baghela, S.K. Singh, and Dentinger; X. longistipitatus K. Das, A. Parihar, D. Chakr., and Baghela; and X. reticulostipitatus Hembrom, D. Chakr., A. Parihar, and K. Das. All three species grow under trees belonging to Fagaceae and are partly related to the presently described Xerocomus uttarakhandae (considering morphology and sequence data) (Figure 3). But Xerocomus doodhcha can be separated in the field from X. uttarakhandae by possessing pileus without a cracked surface and microscopically with larger caulocystidia (20–44 × 5–11 µm) [34]. Xerocomus longistipitatus has a distinctively long (70–185 × 10–24 mm) stipe, an ixotrichoderm nature of pileipellis, and larger basidiospores (10.8–14.6 × 3.6–4.5 µm) [33], whereas X. reticulostipitatus has a distinct reticulation on the stipe surface, larger basidiospores (10.3–15.6 × 3.7–5.3 µm), and larger pleurocystidia (45–66 × 9.5–13 µm) [35]. Another species, X. subtomentosus (Fries) Quélet (described from Europe), is somewhat similar to X. longistipitatus (DC 15-056); however, X. subtomentosus differs from X. uttarakhandae in possessing distinctively longer (10.5–15.2 µm) basidiospores, an olive brown to olive yellow pileus, and a longer (40–100 mm) stipe [53,54]. Phylogenetically, X. fulvipes Xue T. Zhu & Zhu L. Yang (originally described from China) is also close to X. uttarakhandae (Figure 3). but X. fulvipes shows a distinctively larger pileus (30–110 mm in diam.), which never shows a cracked or areolate surface (cracked showing reddening of context in X. uttarakhandae), and a distinctively larger stipe (30–90 × 5–13 mm) [32].
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.
Xerocomellus himalayanus D. Chakr and A. Ghosh, sp. nov., Figure 3, Figure 14, Figure 15 and Figure 16.
MycoBank: MB848680
GenBank: OQ847832 (nrITS, holotype); OQ847959 (nrITS); OQ847962 (nrLSU, holotype); OQ847979 (nrLSU).
Etymology: Referring to the type locality (the western Himalayas), India.
Type: INDIA, HIMACHAL PRADESH: Chamba District, Kalatop, 19 July 2021, alt. 2374 m, N 32°33.051′ E 76°01.138′, DC 21-12 (CAL 1932, holotype!)
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.
Basidiospores 13–15.8–17 × 5.8–6.6–7.4 μm, [n = 30, Q = 1.87–2.21–2.68], ellipsoid to fusoid and inequilateral in side view, often with a truncate apex, hyaline, smooth under light microscopy. Basidia 37–48 × 10–14 μm, clavate, 2 to 4-spored; sterigmata 3–4 × 1–2 μm. Pleurocystidia 45–80 × 8–13 μm, subcylindric to fusiform, ventricose with rounded, thin-walled, few are brown pigmented, emergent up to 20 μm. Subhymenial layer 12–20 μm thick. Tube edge fertile. Cheilocystidia 40–45 × 8–10 μm, rare, subventricose to subfusiform, thin-walled. Pileipellis 110–150 μm thick, a palisadoderm composed of erect brown pigmented and highly incrusted hyphae, incrustation in a ladder-like pattern; terminal cells 18–51 × 6–12 μm, cylindrical to fusoid. Stipitipellis fertile near the apex of the stipe, composed of parallel hyphae (4–7 μm wide); few protruding hymenial tufts composed of basidia and cystidia. Caulobasidia 26–38 × 6–7 μm, subcylindrical to clavate, 4-spored. Caulocystidia 33–45 × 11–15 μm, subclavate to clavate, fusoid with rounded to rarely appendiculate apex. Clamp connections are absent.
Habitat: solitary, in temperate forests under Cedrus deodara (Pinaceae).
Additional Specimen Examined: INDIA, HIMACHAL PRADESH: Chamba District, Kalatop, 22 July 2021, alt. 2391 m, N 32°32.550′ E 76°01.317′, DC 21-56 (CAL 1933).
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].

4. 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

  • Stipe surface with scabs that turns brownish black when bruised; stipe context is cream colored, changing brown-black when exposed ……………………Leccinellum
    1a.
    Basidioma with different combinations of features ……………………………2
2
Pileus surface is sticky; pileus surface, pore surface, stipe surface, and context turn dark blue instantly when bruised or exposed ……………………………Cyanoboletus
2a.
Pileus surface velvety; only pore surface turns blue slowly when bruised …3
3
Pileipellis as a trichodermium; spores with bacillate ornamentation (under SEM) …….………………………………………………………………………Xerocomus
3a.
Pileipellis as a palisadoderm; spores smooth ………………………Xerocomellus.

Author Contributions

Conceptualization: K.D.; Methodology: K.D., A.G., D.C., S.D., I.B., F.B. and R.L.M.; Software: A.G.; Formal analysis: A.G., D.C., A.V. and K.W.; Investigation: K.D.; Resource: K.D., A.G., D.C. and S.D.; Writing original draft: K.D. and D.C.; Writing—review and editing: A.V. and K.W.; Visualization: K.D.; Funding acquisition: K.D. All authors have read and agreed to the published version of the manuscript.

Funding

Two of the authors (A.G. and D.C.) were supported by SERB (DST, Govt. of India) for providing the National Post-Doctoral Fellowship (file nos. PDF/2021/000183 and PDF/2019/000917).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The sequences presented in this study are openly available in https://www.ncbi.nlm.nih.gov/, accessed on 21 June 2023. All new taxa were registered in MycoBank (http://www.mycobank.org/, accessed on 21 June 2023).

Acknowledgments

We are grateful to the Director of the Botanical Survey of India (BSI), for facilitating the present research. Scientist ‘E’ and Head of Office, Central National Herbarium (Howrah) and Eastern Regional Centre (Shillong), BSI, are thanked for their support and encouragement. Help rendered by U. Singh (H.N.B. Garhwal University) during the macrofungal survey in different parts of Uttarakhand are deeply acknowledged.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrLSU, RPB2, and tef1-α for Leccinellum binderi and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Leccinellum binderi is placed in bold red font to highlight its phylogenetic position in the tree.
Figure 1. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrLSU, RPB2, and tef1-α for Leccinellum binderi and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Leccinellum binderi is placed in bold red font to highlight its phylogenetic position in the tree.
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Figure 2. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrITS, nrLSU, and RPB2 for Cyanoboletus paurianus, C. macroporus, and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Cyanoboletus paurianus and C. macroporus are placed in bold red and blue font, respectively, to highlight their phylogenetic positions in the tree.
Figure 2. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrITS, nrLSU, and RPB2 for Cyanoboletus paurianus, C. macroporus, and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Cyanoboletus paurianus and C. macroporus are placed in bold red and blue font, respectively, to highlight their phylogenetic positions in the tree.
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Figure 3. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrITS and nrLSU for Xerocomellus himalayanus, Xerocomus uttarakhandae, X. fraternus, and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Xerocomellus himalayanus and Xerocomus uttarakhandae, X. fraternus, are placed in bold blue and red font, respectively, to highlight their phylogenetic positions in the tree.
Figure 3. Phylogram generated by Maximum Likelihood analysis based on combined sequence data of nrITS and nrLSU for Xerocomellus himalayanus, Xerocomus uttarakhandae, X. fraternus, and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown above or below the branches at nodes. Xerocomellus himalayanus and Xerocomus uttarakhandae, X. fraternus, are placed in bold blue and red font, respectively, to highlight their phylogenetic positions in the tree.
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Figure 4. Photographic illustrations Leccinellum binderi sp. nov. (KD 22-007, holotype). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Basidiospores. Scale bars: (C,D) = 10 μm.
Figure 4. Photographic illustrations Leccinellum binderi sp. nov. (KD 22-007, holotype). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Basidiospores. Scale bars: (C,D) = 10 μm.
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Figure 5. Micromorphological drawings of Leccinellum binderi sp. nov. (KD 22-007, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Pleurocystidia. (D) Cheilocystidia. (E) Elements of pileipellis. Scale bars: (A–E) = 10 μm.
Figure 5. Micromorphological drawings of Leccinellum binderi sp. nov. (KD 22-007, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Pleurocystidia. (D) Cheilocystidia. (E) Elements of pileipellis. Scale bars: (A–E) = 10 μm.
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Figure 6. Photographic illustrations Cyanoboletus paurianus sp. nov. (KD 22-009, holotype). (A,B) Fresh basidiomata in field and basecamp (C) Elements of pileipellis (D) Pleurocystidia (E) Basidiospores. Scale bar: (C,D) = 10 μm.
Figure 6. Photographic illustrations Cyanoboletus paurianus sp. nov. (KD 22-009, holotype). (A,B) Fresh basidiomata in field and basecamp (C) Elements of pileipellis (D) Pleurocystidia (E) Basidiospores. Scale bar: (C,D) = 10 μm.
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Figure 7. Micromorphological drawings of Cyanoboletus paurianus sp. nov. (KD 22-009, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Caulobasidia. (D) Pleurocystidia. (E) Cheilocystidia. (F) Elements of pileipellis. Scale bar: (A–F) =10 μm.
Figure 7. Micromorphological drawings of Cyanoboletus paurianus sp. nov. (KD 22-009, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Caulobasidia. (D) Pleurocystidia. (E) Cheilocystidia. (F) Elements of pileipellis. Scale bar: (A–F) =10 μm.
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Figure 8. Photographic illustrations of Cyanoboletus macroporus (DC 21-02). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bars: C = 20 μm; (D,E) = 10 μm.
Figure 8. Photographic illustrations of Cyanoboletus macroporus (DC 21-02). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bars: C = 20 μm; (D,E) = 10 μm.
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Figure 9. Micromorphological drawings of Cyanoboletus macroporus (DC 21-02). (A) Basidiospores. (B) Basidia. (C) Hymenial cystidia. (D) Elements of pileipellis. Scale bar: (A–D) = 10 μm.
Figure 9. Micromorphological drawings of Cyanoboletus macroporus (DC 21-02). (A) Basidiospores. (B) Basidia. (C) Hymenial cystidia. (D) Elements of pileipellis. Scale bar: (A–D) = 10 μm.
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Figure 10. Photographic illustrations of Xerocomus uttarakhandae sp. nov. (KD 22-005, holotype). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bars: (C–E) = 10 μm.
Figure 10. Photographic illustrations of Xerocomus uttarakhandae sp. nov. (KD 22-005, holotype). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bars: (C–E) = 10 μm.
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Figure 11. Micromorphological drawings of Xerocomus uttarakhandae sp. nov. (KD 22-005, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Cheilocystidia. (D) Pleurocystidia. (E) Elements of pileipellis. Scale bars: (A–E) =10 μm.
Figure 11. Micromorphological drawings of Xerocomus uttarakhandae sp. nov. (KD 22-005, holotype). (A) Basidiospores. (B) Caulocystidia. (C) Cheilocystidia. (D) Pleurocystidia. (E) Elements of pileipellis. Scale bars: (A–E) =10 μm.
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Figure 12. Photographic illustrations of Xerocomus fraternus (KD 22-025). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bar: (C–E) = 10 μm.
Figure 12. Photographic illustrations of Xerocomus fraternus (KD 22-025). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bar: (C–E) = 10 μm.
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Figure 13. Micromorphological drawings of Xerocomus fraternus (KD 22-025). (A) Basidiospores. (B) Caulocystidia. (C) Basidia. (D) Cheilocystidia. (E) Pleurocystidia. (F) Elements of pileipellis. Scale bar: (A–F) = 10 μm.
Figure 13. Micromorphological drawings of Xerocomus fraternus (KD 22-025). (A) Basidiospores. (B) Caulocystidia. (C) Basidia. (D) Cheilocystidia. (E) Pleurocystidia. (F) Elements of pileipellis. Scale bar: (A–F) = 10 μm.
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Figure 14. Photographic illustrations of Xerocomellus himalayanus sp. nov. (DC 21-12). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bar: (C–E) = 10 μm.
Figure 14. Photographic illustrations of Xerocomellus himalayanus sp. nov. (DC 21-12). (A,B) Fresh basidiomata in field and basecamp. (C) Elements of pileipellis. (D) Hymenial cystidia. (E) Basidiospores. Scale bar: (C–E) = 10 μm.
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Figure 15. Micromorphological drawings of Xerocomellus himalayanus sp. nov. (DC 21-12). (A) Basidiospores. (B) Basidia. (C) Hymenial cystidia. (D) Elements of pileipellis. Scale bar: (A–D) = 10 μm.
Figure 15. Micromorphological drawings of Xerocomellus himalayanus sp. nov. (DC 21-12). (A) Basidiospores. (B) Basidia. (C) Hymenial cystidia. (D) Elements of pileipellis. Scale bar: (A–D) = 10 μm.
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Figure 16. SEM images of basidiospores. (A,B) Xerocomus uttarakhandae. (C) Xerocomus fraternus. (D) Xerocomellus himalayanus.
Figure 16. SEM images of basidiospores. (A,B) Xerocomus uttarakhandae. (C) Xerocomus fraternus. (D) Xerocomellus himalayanus.
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Table
Table 1. Leccinellum and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Table 1. Leccinellum and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Species Name (as Reported in GenBank)Voucher No.GenBank Accession No.
nrLSUrpb2tef1-α
Borofutus dhakanusHKAS73789JQ928616JQ928597JQ928576
Leccinellum albellumMICH KUO-07241101MK601746MK766308MK721100
Leccinellum alborufescensFHMU1908MK816322MK816333MK816330
Leccinellum alborufescensFHMU1758MK816321MK816332MK816329
Leccinellum binderiKD 22-015OQ858379OQ914387OR102316
Leccinellum binderiKD 22-007OQ858380OQ914386OR102315
Leccinellum corsicumBuf 4507KF030347KF030389KF030435
Leccinellum crocipodiumMICH KUO-07050707MK601749MK766311MK721103
Leccinellum fujianenseFHMU2219MK816319MK816334MK816327
Leccinellum fujianenseFHMU2223MK816320MK816336MK816328
Leccinellum indoaurantiacumDC 14-019KT860059——
Leccinellum lepidumK(M)-142974MK601751MK766312MK721105
Leccinellum pseudoscabrumCFMR:DPL-11432MK601752MK766313MK721106
Leccinellum sp.OR0082—MZ824749MZ803024
Leccinum aff. griseumKPM-NC-0017381JN378508—JN378449
Leccinum aff. scabrumHKAS 57266KF112442KF112722KF112248
Leccinum albumLi1072MW413907—MW439267
Leccinum aurantiacumL:0342207MK601759MK766318MK721113
Leccinum cerinumMK11800AF139692——
Leccinum duriusculumGL4676AF139699——
Leccinum duriusculumYang5971MZ675541MZ707779MZ707785
Leccinum flavostipitatumMENMB10801MH620342——
Leccinum holopusYang5972MW413906MW439258MW439266
Leccinum holopus9109303AF139700——
Leccinum holopusMICH: KUO-09150707MK601763MK766322MK721117
Leccinum manzanitaeNY-14041 REH-6717MK601765MK766324MK721119
Leccinum monticolaHKAS:76669KF112443KF112723KF112249
Leccinum monticolaNY-00815448 REH-8591MK601767MK766326MK721121
Leccinum monticolaNY-760388 REH-8288MK601766MK766325MK721120
Leccinum palustreMK11107AF139701——
Leccinum parascabrumLi1700MW413912MW439265MW439272
Leccinum parascabrumWu1784MW413911MW439264MW439271
Leccinum pseudoborneenseWGS965—MW439263—
Leccinum pseudoborneenseWGS960—MW439262—
Leccinum pseudoborneenseWGS947MW413908MW439261MW439268
Leccinum rugosicepsCFMR BOS-866MK601770MK766329MK721124
Leccinum scabrumHKAS56371KT990587KT990423KT990782
Leccinum scabrumKPM-NC-0017840JN378515—JN378455
Leccinum variicolorLvar1AF139706——
Leccinum versipelleFB27MZ675546MZ707782MZ707790
Leccinum versipelleLJW418MZ675545MZ707781MZ707789
Leccinum versipelleCFMR DLC2002-122MK601778MK766336MK721132
Octaviania japonimontanaKPM-NC-0017812JN378486—JN378428
Octaviania tasmanicaNY-02449788 REH-10066MK601798MK766355MK721152
Rossbeevera bisporaGDGM 45639MK036347MK350309—
Rossbeevera eucyaneaKPM-NC0023895KP222896—KP222915
Rossbeevera griseobrunneaGDGM45913MH537793——
Rossbeevera griseovelutinaTNS-F-36991KC552032—KC552077
Rossbeevera vittatisporaMEL2321058KP222895—KP222911
Rossbeevera westraliensisOSC61480 JN378505—JN378445
Spongiforma thailandicaBBH:DED 7873NG_042464——
Table
Table 2. Cyanoboletus and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Table 2. Cyanoboletus and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Species Name (as Reported in GenBank)Voucher No.GenBank Accession No.
nrITSnrLSUrpb2
Cupreoboletus poikilochromusGS 10070KT157051KT157060KT157068
Cupreoboletus poikilochromusGS 11008KT157050KT157059KT157067
Cupreoboletus poikilochromusAQUI 7195KT157052KT157061—
Cyanoboletus bessetteiARB1393BMW675738—MW737458
Cyanoboletus bessetteiARB1393AMW675737MW662571MW737457
Cyanoboletus brunneoruberHKAS80579-2—KT990569KT990402
Cyanoboletus brunneoruberHKAS80579-1—KT990568KT990401
Cyanoboletus cyaneitinctusJAB_184MW675731MW662584MW737467
Cyanoboletus cyaneitinctusFarid_920MW675744MW662579MW737465
Cyanoboletus hymenoglutinosusDC 14-010KT907355KT860060—
Cyanoboletus instabilisN.K.Zeng2862MG030473MG030466—
Cyanoboletus instabilisHKAS:59554—KF112412KF112698
Cyanoboletus macroporussarwar1MW369503——
Cyanoboletus macroporusAN-2020aMW045557——
Cyanoboletus macroporusDC 21-02OQ860238OQ860239ON364552
Cyanoboletus macroporusDC 21-24OQ860240OQ860241OQ876894
Cyanoboletus mediterraneensisHAI B12-077OM801199OM801212—
Cyanoboletus paurianusKD 22-008—OQ859920OQ914389
Cyanoboletus paurianusKD 22-009—OQ859919OQ914388
Cyanoboletus pulverulentusMG 126aKT157053KT157062—
Cyanoboletus pulverulentusMG 456aKT157054KT157063—
Cyanoboletus pulverulentusMG 628aKT157055KT157064KT157069
Cyanoboletus pulverulentusTUR-A 208930MZ265186—MZ265200
Cyanoboletus sinopulvirulentusHMAS266894KC579402——
Cyanoboletus sp.HKAS90208-2——KT990405
Cyanoboletus sp.HKAS90208-1—KT990571KT990404
Cyanoboletus sp. TUR-A 209199MZ265183MZ265198—
Cyanoboletus sp. TUR-A 208928MZ265179MZ265194—
Cyanoboletus sp. TUR-A 209198MZ265182MZ265197—
Cyanoboletus sp. TUR-A 208929MZ265181MZ265196—
Lanmaoa angustisporaHKAS:74765—KF112322KF112680
Lanmaoa angustisporaHKAS 74752—KM605139KM605177
Lanmaoa asiaticaHKAS63516—KT990584KT990419
Lanmaoa asiaticaHKAS 63603—KM605143KM605176
Lanmaoa flavorubraNY775777—JQ924339KF112681
Rugiboletus brunneiporusHKAS 83209—KM605134KM605168
Rugiboletus brunneiporusHKAS 68586—KF112402KF112719
Table
Table 3. Xerocomus and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Table 3. Xerocomus and allied sequences used in ML analyses of this study. Newly sequenced collections are in bold.
Species Name (as Reported in GenBank)Voucher No.GenBank Accession No.
nrITSnrLSU
Boletus rubellusF:PRL5575MANGQ166888—
Boletus rubellusF:PRL5788MANGQ166883—
Boletus rubellusChL22KX438318—
Boletus rubellusLAH0710KJ802928—
Boletus rubellusLAH0810KJ802929—
Hortiboletus cf. rubellusiNat31879606MN498119—
Hortiboletus indorubellusDC 14-002KT319647—
Hortiboletus indorubellusDC 14-001—KU566807
Hortiboletus indorubellusLS15MK002767MK002872
Hortiboletus kohistanensisAST48MG988192MG988187
Hortiboletus kohistanensisAST22AMG988193—
Hortiboletus napaeusFHMU3325MT646445MT646438
Hortiboletus napaeusFHMU3326MT646440MT646433
Hortiboletus rubellusFLAS-F-61506MH211937—
Hortiboletus rubellusFLAS-F-60513MH211664—
Hortiboletus rubellus52AMN652008—
Hortiboletus rubellusS.D. Russell MycoMap 6338MK560106—
Imleria obscurebrunneaHKAS52557KC215207KC215220
Imleria subalpinaHKAS74712KC215208KC215218
Rheubarbariboletus armeniacusMA:Fungi:47678AJ419221—
Rheubarbariboletus persicolorSOMF28154MH011932—
Rheubarbariboletus persicolorSOMF29860MH011931—
Xerocomellus salicicolaUCSC1028KU144793KU144794
Xerocomellus aff. chrysenteronMushroom Observer #338913ON705310—
Xerocomellus amylosporusJLF3012KM213635KU144742
Xerocomellus behriiOSC_Trappe12988KJ882288—
Xerocomellus chrysenteronHKAS:56494 KF112357
Xerocomellus chrysenteron—UDB000441—
Xerocomellus chrysenteron—UDB000439—
Xerocomellus corneriHKAS90206—KT990669
Xerocomellus corneriHKAS52503—KT990668
Xerocomellus diffractusNS120612KM213650KM213651
Xerocomellus dryophilusJLF4134KX534076KY659593
Xerocomellus himalayanusDC 21-56OQ847959OQ847979
Xerocomellus himalayanusDC 21-12OQ847832OQ847962
Xerocomellus mendocinensisJLF2275KM213653KM213654
Xerocomellus perezmorenoiAV1660OK350679OK350681
Xerocomellus perezmorenoiAVRG1161OK350680OK350682
Xerocomellus poederiAH44050KU355475KU355488
Xerocomellus poederiAH45803KU355480KU355491
Xerocomellus rainisiaeJLF3523KU144789KU144790
Xerocomellus salicicolaB391MW675727MW662569
Xerocomellus salicicolaCS_5Mar2014_1KU144791KU144792
Xerocomellus sarnariiML900101XEMH011930—
Xerocomellus sarnariiMCVE 28571KT271745—
Xerocomellus sarnariiMCVE 28577KT271749—
Xerocomus chrysonemusah2000037DQ066381—
Xerocomus chrysonemusJAM0359—KF040544
Xerocomus doodhchaKD 13-082KR611867KU566806
Xerocomus fennicusVP-10KT692929—
Xerocomus ferrugineusgs0898DQ066403—
Xerocomus ferrugineusat2001071DQ066402—
Xerocomus fraternusKD 22-025OQ776920OQ771932
Xerocomus fraternusKD 22-027OQ776919OQ771933
Xerocomus fraternusHKAS52526—KT990682
Xerocomus fraternusHKAS69291—KT990683
Xerocomus fulvipesHKAS52556—KT990672
Xerocomus illudensMB03-005JQ003658—
Xerocomus longistipitatusDC 16-056KY008398—
Xerocomus microcarpoidesHKAS54753—KT990680
Xerocomus microcarpoidesHKAS53374—KT990679
Xerocomus perplexusMB00-005JQ003657JQ003702
Xerocomus piceicolaHKAS55452—KT990685
Xerocomus piceicolaHKAS76492—KT990684
Xerocomus puniceiporusHKAS 80683—KU974141
Xerocomus reticulostipitatusMEH 16_B-7MF167353—
Xerocomus ripariellus—UDB000485
Xerocomus rubellus—UDB036190—
Xerocomus rubellus—UDB001406—
Xerocomus rubellusIB2004272EF644119—
Xerocomus rugosellusHKAS68292—KT990686
Xerocomus silwoodensisAH2005039 (K(M)137134) DQ438143—
Xerocomus silwoodensisgs1959DQ066375—
Xerocomus sp. AN-2016aKU761593—
Xerocomus sp. AN-2016bKU761592—
Xerocomus subparvusHKAS50295—KT990667
Xerocomus subtomentosusah1997028DQ066370—
Xerocomus subtomentosusK 167686JQ967281JQ967238
Xerocomus uttarakhandaeKD 22-002OQ748035OQ748038
Xerocomus uttarakhandaeKD 22-005OQ748036OQ748037
Xerocomus velutinusHKAS 52575—KF112393
Xerocomus yunnanensisHKAS68420—KT990690
Xerocomus yunnanensisHKAS68282—KT990691
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MDPI and ACS Style

Das, K.; Ghosh, A.; Chakraborty, D.; Datta, S.; Bera, I.; Layola MR, R.; Banu, F.; Vizzini, A.; Wisitrassameewong, K. Four Novel Species and Two New Records of Boletes from India. J. Fungi 2023, 9, 754. https://doi.org/10.3390/jof9070754

AMA Style

Das K, Ghosh A, Chakraborty D, Datta S, Bera I, Layola MR R, Banu F, Vizzini A, Wisitrassameewong K. Four Novel Species and Two New Records of Boletes from India. Journal of Fungi. 2023; 9(7):754. https://doi.org/10.3390/jof9070754

Chicago/Turabian Style

Das, Kanad, Aniket Ghosh, Dyutiparna Chakraborty, Sudeshna Datta, Ishika Bera, Ranjith Layola MR, Farheen Banu, Alfredo Vizzini, and Komsit Wisitrassameewong. 2023. "Four Novel Species and Two New Records of Boletes from India" Journal of Fungi 9, no. 7: 754. https://doi.org/10.3390/jof9070754

APA Style

Das, K., Ghosh, A., Chakraborty, D., Datta, S., Bera, I., Layola MR, R., Banu, F., Vizzini, A., & Wisitrassameewong, K. (2023). Four Novel Species and Two New Records of Boletes from India. Journal of Fungi, 9(7), 754. https://doi.org/10.3390/jof9070754

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