Introduction

The family Boletaceae (Basidiomycota, Boletales) represents mushrooms (macrofungi) that are mainly characterized by soft, fleshy, pileate, centrally stipitate and tubulose to rarely lamellate or loculate hymenophore1,2. Being ectomycorrhizal associates of angiospermous and gymnospermous trees (Quercus, Lithocarpus, Castanopsis, Betula, Shorea, Abies, Pinus, Picea, Larix, Tsuga, etc.) 1,3 they are key components of terrestrial ecosystems and one of the dominant wild mushrooms in Indian Himalaya. However, due to complex and overlapping morphological features among its genera and the limited phylogenetic information kept this important mushroom family unresolved for many years in terms of its systematics and evolution. About 50 genera and 800 species were recognised in this family by the Dictionary of Fungi4. Morphotaxonomy, when used alone or combined with molecular phylogenetic analyses using less informative ribosomal genetic markers such as LSU, SSU etc., failed to resolve several issues. Several genera remained polyphyletic, delimitation among many genera was obscured, and evolutionary relationships remained unclear. However, over the last decade, a combined approach, utilizing multilocus molecular phylogeny alongside morphology, revealed crucial insights. Three protein-coding genes, namely rpb1(RNA polymerase II largest subunit), rpb2 (RNA polymerase II second largest subunit), and tef 1-α (translation elongation factor 1α), played the key role to give the proper phylogenetic framework for Boletaceae2. These revolutionary changes lead to the discovery of more than 100 genera and ca 1200 species5 from the world. Moreover, this mode of investigation redefined seven major clades within this family, namely, subfamilies Austroboletoideae, Boletoideae, Chalciporoideae, Leccinoideae, Xerocomoideae, Zangioideae, and Pulveroboletus group2.

In subtropical to subalpine forests of India, the three major mushroom-producing orders are Agaricales Underw., Russulales Kreisel ex P.M. Kirk, P.F. Cannon & J.C. David, and Boletales E.-J. Gilbert (Basidiomycota). Himalaya and adjacent hilly ranges, the home (type locality) of numerous wild mushrooms, are still unexplored to poorly explored. Hidden diversity is much awaited. Boletaceae is no exception of it. Presently, 85 species belonging to 24 genera are known from Indian Himalaya5,6,7,8. Recently, in the month of August (2023), the authors have taken macrofungal exploration to three districts (Rudraprayag, Chamoli and Bageshwar) of the state Uttarakhand in western Himalaya and East Khasi Hills of Meghalaya in Northeast India. Intensive surveys were undertaken to four forested areas namely, Baniyakund: temperate mixed (broadleaf and coniferous) forest in Rudraprayag district (Uttarakhand), Didna: temperate broadleaf forest in Chamoli district (Uttarakhand), Dhakuri: temperate to subalpine mixed forest in Bageshwar district (Uttarakhand) and Sohra, sub-temperate broad leaf forests in East Khasi Hills District (Meghalaya). A large number of boletoid mushrooms were collected. Thorough observation of morphological features followed by a multigene molecular phylogeny using ITS, LSU, rpb2 and/or tef 1-α markers uncovered five novel species and two first records in Boletaceae from this country. Leccinellum bothii sp. nov., Phylloporus himalayanus sp. nov., Phylloporus smithii sp. nov., Porphyrellus uttarakhandae sp. nov., Retiboletus pseudoater sp. nov. are proposed herein. Moreover, Leccinellum sinoaurantiacum (M. Zang & R.H. Petersen) Yan C. Li & Zhu L. Yang and Xerocomus rugosellus (W.F. Chiu) F.L. Tai which were known earlier from China are also recorded for the first time from India.

Results

Phylogenetic inferences

In our present study, the three-locus dataset (LSU + rpb2 + tef 1-α) of Leccinellum consisted of 62 taxa and 2,311 nucleotide sites, including gaps. Borofutus dhakanus Hosen & Zhu L. Yang and Spongiforma thailandica Desjardin, Manfr. Binder, Roekring & Flegel were selected as outgroup taxa. Phylogenetic analysis revealed that sequences from our first species, Leccinellum bothii (voucher nos. KD 23-005 and KD 23-008) clustered with the species of L. crocicum (voucher no. Buff 4507), L. lepidium (voucher no. K(M)-142974), L. fujianense (voucher nos. FHMU2219 & FHMU2223), L. alborufescens (voucher nos. FHMU1908 & FHMU1758), L. aff. griseum (voucher no. KPM-NC-0017381) and L. pseudoscabrum (voucher no. CFMR:DPL-11432, 930808, F300 & MICH-60301 R.Watling-6725) with moderate support (MLbs = 85%), forming a distinct clade within the Leccinellum lineage. However, our specimens were recovered as distinct species within the phylogenetic tree (Fig. 1). Conversely, our second species, Leccinellum sinoaurantiacum (voucher nos. DC ML-52 and DC ML-77) is nested within the L. sinoaurantiacum clade consisting of sample vouchers (Li2770 and Zang13486) collected from China and suggesting its strong similarity or conspecificity with the Asian species of L. sinoaurantiacum with a strong (MLbs = 100%, BPP = 1) support (Fig. 1).

Figure 1
figure 1

Phylogram generated by Bayesian analysis based on combined sequence data of LSU, rpb2 and tef 1-α for Leccinellum bothii, L. sinoaurantiacum and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown on the left of “/” and Bayesian posterior probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Leccinellum bothii and L. sinoaurantiacum are placed in bold red and blue font respectively to highlight their phylogenetic positions in the tree.

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Again, the three-locus dataset (ITS + LSU + tef 1-α) for Phylloporus comprised of 60 taxa and 2400 nucleotide sites, including gaps. Xerocomus magniporus M. Zang & R.H. Petersen and X. subtomentosus (L.) Quél. were selected as outgroup taxa following. In the phylogram, sequences from our third and fourth species, Phylloporus himalayanus (voucher nos. KD 24-046 and KD 23-047) and P. smithii (voucher nos. KD 22-012 and KD 22-022), clustered with the P. yunnanensis clade with strong support (MLbs = 98%, BPP = 0.96), being sister to the P. imbricatus clade. However, our two species were identified as distinct novel taxa within the phylogenetic tree (Fig. 2).

Figure 2
figure 2

Phylogram generated by Bayesian analysis based on combined sequence data of ITS, LSU and tef 1-α for Phylloporus himalayanus, P. smithii and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown on the left of “/” and Bayesian posterior probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Phylloporus himalayanus and P. smithii are placed in bold red font to highlight their phylogenetic positions in the tree.

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The two-locus (ITS + LSU) dataset of Xerocomus, comprising 44 taxa and 1393 nucleotide sites, including gaps, used Hourangia nigropunctata (W.F. Chiu) Xue T. Zhu & Zhu L. Yang as outgroup taxa following. The combined (ITS + LSU) phylogenetic analysis showed that the two collections of our fifth species, Xerocomus rugosellus (voucher nos. KD 23-019 and KD 23-055) is nested within the X. rugosellus clade, consisting of sample vouchers (HKAS 67749 and HKAS68292) collected from China and suggesting its strong similarity or conspecificity with the Asian species of X. rugosellus with a strong (MLbs = 87%) support. (Fig. 3).

Figure 3
figure 3

Phylogram generated by Bayesian analysis based on combined sequence data of ITS and LSU for Xerocomus rugosellus and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown on the left of “/” and Bayesian posterior probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Xerocomus rugosellus is placed in bold blue font to highlight its phylogenetic position in the tree.

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Three-locus dataset (LSU + rpb2 + tef 1-α) of Porphyrellus, comprised of 37 taxa and 1980 nucleotide sites, including gaps. Butyriboletus pseudospeciosus Kuan Zhao & Zhu L. Yang and B. regius (Krombh.) D. Arora & J.L. Frank were selected as outgroup taxa following. Combined three-locus phylogenetic analyses revealed that two collections of our sixth species, Porphyrellus uttarakhandae (voucher nos. KD 23-028 and KD 23-056), clustered with Por. orientifumosipes (voucher nos. HKAS84710 and HKAS53372) from China without a strong support, being sister to the Por. pseudocyaneotinctus and Por. griseus clade. However, our specimens were recovered as distinct species within the phylogenetic tree (Fig. 4).

Figure 4
figure 4

Phylogram generated by Bayesian analysis based on combined sequence data of LSU, rpb2 and tef 1-α for Porphyrellus uttarakhandae and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown on the left of “/” and Bayesian posterior probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Porphyrellus uttarakhandae is placed in bold red font to highlight its phylogenetic position in the tree.

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On the other hand, the three-locus dataset (ITS + LSU + tef 1-α) of Retiboletus consisted of 40 taxa and 1999 nucleotide sites, including gaps. Pseudoaustroboletus valens (Corner) Yan C. Li & Zhu L. Yang was used as outgroup taxa following. Combined three-locus phylogenetic analyses revealed that two collections of our seventh species, Retiboletus pseudoater (voucher nos. KD 23-040 and KD 23-048), nested with R. ater (voucher nos. Li1215, Li1224 and HKAS 56069) from China with strong support (MLbs = 100%, BPP = 1). However, our specimens were identified as distinct species within the phylogenetic tree (Fig. 5).

Figure 5
figure 5

Phylogram generated by Bayesian analysis based on combined sequence data of ITS, LSU and tef 1-α for Retiboletus pseudoater and allied species. Maximum likelihood bootstrap support values (MLbs) ≥ 70% are shown on the left of “/” and Bayesian posterior probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Retiboletus pseudoater is placed in bold red font to highlight its phylogenetic position in the tree.

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Taxonomy

Leccinoideae

Leccinellum bothii K. Das, A. Ghosh, Sudeshna Datta, U. Singh & Vizzini sp. nov. Mycobank: MB 851128. Holotype INDIA, Uttarakhand, Rudraprayag district, Baniyakund, 30° 29.000′ N 79° 10.743′ E, alt. 2622 m, temperate mixed forests, under Quercus sp., 3 August 2023, K. Das, KD 23-005 (CAL 1953, holotype!) (Figs. 1, 6, 7).

Figure 6
figure 6

Leccinellum bothii. (KD 23–005, holotype). (a,b) Fresh basidiomata. (c,d) Pileipellis. (e) Caulocystidia. (f) Basidiospores. Scale bars: (c–f) = 10 µm.

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Figure 7
figure 7

Leccinellum bothii. (KD 23-005, holotype). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidia. (e) Elements of pileipellis. (f) Cheilocystidia. Scale bars: (a–f) = 10 µm.

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Etymology Commemorating E.E. Both for his important contribution to the systematics of Boletaceae.

Diagnosis Distinguished from other allied species of Leccinellum by a rugulose or pitted, brown to dark brown pileus, a brown to greyish orange colour changes of hymenophore, unchanging pileus context, greyish black (with greyish orange near base) colour changes of stipe context, pileipellis composed of chains of elongate, subglobose to pyriform elements, the occurrence in temperate Himalaya and LSU, rpb2, and tef 1-α sequence data.

Basidiomata small to medium-sized. Pileus 29–54 mm in diam., hemispherical to conic or convex; surface somewhat rugulose to pitted, non-viscid, with a narrow flap of tissue at margin, caramel brown to brownish orange (6C4–5), yellowish white (3A2) or paler near margins, mostly unchanging with maturity; turning reddish brown (8D7) with KOH and greenish with FeSO4. Hymenophore slightly depressed near stipe apex, adnexed; pore surface yellowish white (3A2) to yellow, becoming brown, greyish orange (5B3) then linoleum brown (5E7) with maturity or on bruising; pores are rounded, 2–3/mm. Tubes adnexed, 2–6 mm long, yellowish white to pale yellow (2–3A2–3), slowly becoming brownish on exposure. Stipe 60–98 × 18–30 mm, clavate when young, more or less cylindrical with tapering apex and a swollen base; surface with striations near apex, distinctively scabrous up to middle or slightly below, yellowish white to pale yellow (3A2–3) with brown (6D6) to dark brown (7F8) squamules that becomes grey black to black when bruised, becoming pale orange towards base on handling. Context in pileus, up to 8 mm thick, white to yellowish white, unchanging; context in stipe, chalky, slowly becoming yellowish then greyish black on exposure, greyish orange (5B4) near base; turning dull green (25E4), then slowly even darker with FeSO4 and pale yellow (3A3) with KOH. Basal mycelium white. Taste mild and odour indistinct. Spore print not obtained.

Basidiospores 8.0–12.2–16.7 × 4.5–5.1–6.0 µm, (n = 30, Q = 1.78–2.41–3.2), subfusoid to elongate and inequilateral in side view with distinct suprahilar depression, light yellow, smooth, inamyloid. Basidia 29–34 × 11–12 µm, clavate, 4-spored; sterigmata 3–5 × 0.5–1 µm. Pleurocystidia 31–49 × 7–11 µm, abundant, fusoid-ventricose with subcapitate to appendiculate apex, thin-walled, hyaline, emergent up to 20 µm. Tube edge fertile. Cheilocystidia 26–39 × 9–12 µm, abundant, fusoid-ventricose with rounded to subcapitate apex, thin-walled, hyaline. Hymenophoral trama divergent, hyphae cylindrical, septate, 3–6 µm wide. Pileipellis 100–120 µm thick, a trichodermium, composed of branched chains of subcylindric, subglobose, clavate to pyriform elements; terminal elements 7–26 × 5–11 µm, cylindrical to clavate, with brown intracellular pigmentation, thromboplerous hyphae present. Stipitipellis up to 100 µm thick, a trichodermium, composed of loosely arranged, erect, branched, septate hyphae, terminal elements 23–40 × 5–9 µm, clavate to cylindric; with frequent clusters of basidia and cystidia (caulohymenium); caulocystidia 41–53 × 9–13 µm, clavate, pyriform, ventricose; caulobasidia 31–34 × 9–11 µm, narrowly clavate, 4-spored. Clamp connections absent in all tissues.

Additional specimen examined: INDIA, Uttarakhand, Rudraprayag District, Baniyakund, 30° 28.892′ N 79° 10.761′ E, alt. 2585 m, temperate mixed forests under Quercus sp., 3 August 2023, K. Das, KD 23-008 (CAL 1954).

Leccinellum sinoaurantiacum (M. Zang & R.H. Petersen) Yan C. Li & Zhu L. Yang, The Boletes of China: Tylopilus s.l. (Singapore): 164 (2021) (Figs. 1, 8, 9).

Figure 8
figure 8

Leccinellum sinoaurantiacum (DC ML-52). (a,b) Fresh basidiomata. (c) Pileipellis. (d) Basidia. (e,f) Pleurocystidia. (g) Basidiospores. Scale bars: (c) = 25 μm, (d–g) = 10 μm.

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Figure 9
figure 9

Leccinellum sinoaurantiacum (DC ML-52). (a) Basidiospores. (b) Pleuro- and cheilocystidia. (c) Basidia. (d) Pileipellis. Scale bars: (a–d) = 10 μm.

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Basidiomata small to medium-sized. Pileus 10–40 mm in diam., hemispherical to convex rarely applanate; surface moist, gelatinous when wet, scarlet or crimson red, vivid red (10A8) when young, with maturity orange-red (8A7); turning brownish red (8C7) with KOH. Hymenophore depressed near stipe apex, adnate; pore surface light yellow to yellow (1–2A5–6) no change on bruising; pores angular, 1–1.4/mm. Tubes adnate, 8 mm long, yellow (2A4), unchanging on exposure. Stipe 40–70 × 5–10 mm, more or less cylindrical with tapering apex and a broader base; surface with squamules, denser towards base, pink to scarlet red (10A5–6). Context in pileus up to 5 mm thick, cream yellow to pale pink, unchanging on exposure; context in stipe, solid, cream white to pale pink. Basal mycelium yellow. Taste mild and odour fungoid. Spore print salmon pink.

Basidiospores 14.8–17.5–20 × 3.6–4.5–5.5 [Q = 2.9–3.2–3.8], elongated, light yellow, smooth, inamyloid. Basidia 32–40 × 11–12 µm, clavate, 4-spored. Pleurocystidia 43–65 × 11–19 µm, less in number, fusiform to subfusiform and ventricose, appendiculate apex, thin-walled, emergent up to 22 µm. Tube edge fertile. Cheilocystidia same as pleurocystidia. Hymenophoral trama divergent, hyphae cylindrical, septate, 6–9 µm wide. Pileipellis 120–130 µm thick, an ixohyphoepithelium, composed of two layers; upper layer composed of erect to suberect septate filamentous hyphae submerged under a gluten layer; lower layer composed of branched chains of subcylindric to subglobose or globose elements; terminal elements of upper layer 11–25 × 8–12 µm. Stipitipellis up to 100 µm thick, a trichodermium, with clusters of basidia and cystidia (caulohymenium); caulocystidia 30–50 × 9–19 µm, clavate, pyriform, ventricose; caulobasidia 29–36 × 6–12 µm, narrowly to broadly clavate, 4-spored. Clamp connections absent in all tissues.

Specimens examined INDIA, Meghalaya, East Khasi Hills district, Sohra, 25° 18.736′ N 91° 45.926′ E, alt. 1535 m, sub-temperate broad leaf forests under Castanopsis sp., 8 August 2023, D. Chakraborty and D. Tudu, DC ML-52 (ASSAM F001); ibid., Mawlyndiar, 25° 18.641′ N 91° 45.321′ E, alt. 1535 m, sub-temperate broad leaf forests under Castanopsis sp., 8 August 2023, D. Chakraborty, DC ML-77 (ASSAM F002).

Notes Presence of yellow pore surface, a distinctively scaly stipe surface and a trichodermium (or rarely ixohyphoepithelium) pattern of the pileipellis undoubtedly place these two species under the genus Leccinellum Bresinsky & Manfr. Binder9. In the field, our proposed new species, Leccinellum bothii is quite similar to L. alborufescens N.K. Zeng, R. Xue & S. Jiang and L. fujianense N.K. Zeng, R. Xue & Zhi Q. Liang (both are originally described from China). However, both L. alborufescens and L. fujianense can be differentiated from the present species by showing the change in the overall colour of stipe surface to red (in L. bothii, never changes to red except at base that becomes pale orange), pileus and stipe context to red (in L. bothii, pileus context remains unchanged, stipe context changes to greyish black except near base that changes to greyish orange). Additionally, L. alborufescens and L. fujianense have distinctively smaller basidiospores and are known to occur in tropical and subtropical forests, respectively, whereas L. bothii is found in temperate mixed forests10. Further, L. binderi K. Das, A. Ghosh & Vizzini, another recently discovered species from the same locality easily falls apart from L. bothii by differently looking pileus (hemispherical to convex to applanate pileus with subtomentose to cracked pileus surface, yellowish brown to greyish yellow in colour), differently featured stipe context (never turning greyish orange near base) and distinctively larger basidiospores (13.8–18.22–22 × 5.4–5.96–7 µm)5. The European L. pseudoscabrum (Kallenb.) Mikšíkis [= L. carpini (R. Schulz) Bresinsky & Manfr. Binder] is morphologically quite similar to L. bothii but differs by larger basidiomata [pileus 30–70 (–100) mm; stipe 60–130 × 6–14 mm], stipe that is entirely covered with brownish black dot-like squamules arranged in longitudinal rows, cutis pattern of stipitipellis and the occurrence under Carpinus betulus or Corylus avellana9,11,12.

The second species in this genus, L. sinoaurantiacum which was collected from East Khasi hills of Northeast India, is a very attractive mushroom for its beautiful scarlet to orange red basidiomata. Combination of macro- and micromorphological characters of Indian collections like scarlet to crimson red sticky pileus, yellow hymenophore with angular pores, scabrous stipe surface, comparatively long basidiospores, an ixohyphoepithelium nature of pileipellis confirm their identity as L. sinoaurantiacum13,14. Moreover, phylogenetic analysis of these collections (with LSU and tef 1-α) warrants this conspecificity of the Indian collections with its Chinese counterpart (voucher nos. Zang13486 and Li2770).

Xerocomoideae

Phylloporus himalayanus K. Das, Sudeshna Datta & A. Ghosh sp. nov. Mycobank: MB 851129. Holotype: INDIA, Uttarakhand, Bageshwar district, 30° 04.270′ N 79° 55.229′ E, alt. 2870 m, on Dhakuri to Loharkhet trek close to Dhakuri-top, subalpine mixed forest under Quercus sp., 15 August 2023, K. Das, KD 23-046 (CAL 1955, holotype!) (Figs. 2, 10, 11).

Figure 10
figure 10

Phylloporus himalayanus. (KD 23-046, holotype). (a) Fresh basidiomata in the field. (b) Pileipellis. (c,d) Stipitipellis. (e) Hymenium layer with basidia and pleurocystidia. (f) Basidiospore under SEM. Scale bars (b) = 40 µm, (c–e) = 10 µm, (f) = 1 µm.

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Figure 11
figure 11

Phylloporus himalayanus. (KD 23-046, holotype). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidia. (e) Elements of pileipellis. (f) Elements of stipitipellis. Scale bars: (a–f) = 10 µm.

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Etymology Refers to the Himalayan Mountain range, where the type locality is situated.

Diagnosis Distinguished from the other known Phylloporus species by subdistant lamellae (8–10/10 mm), sub-bulbous and strigose stipe base, extremely varied hyphal terminal elements of stipitipellis and ITS, LSU and tef 1-α sequence data.

Basidiomata small to medium-sized, growing solitary to gregarious. Pileus 21–55 mm in diam., planoconvex with shallowly depressed centre, then applanate with depressed center; margin decurved when young, slightly uplifted; surface smooth to finely tomentose, brown (6E5–7) at centre, light brown (6D5) towards and along margin when young, gradually brownish orange (5C4–6) with pale orange to orange-white (5A2–3) along margin and darker centre at maturity, turning reddish brown (9E6–7) with KOH; context yellowish white (2A2) then brownish, turning greyish red (7B3) with KOH, greyish in FeSO4. Hymenophore lamellate, decurrent, subdistant (8–10/10 mm), intervenose and anastomosing, up to 8 mm in height, yellow to vivid yellow (3A6–8), becoming pastel green to turquoise green (25A4–5) very slowly; lamellulae in 5 series, attenuate, ventricose, concolorous with lamellae. Stipe central, 55–70 × 5–8 mm, subcylindric, dry, finely tomentose, with longitudinal striation on upper part, with sub-bulbous and strigose base, solid, pastel yellow to light yellow (3A4–5) towards apex, pale yellow (3A3) at middle, yellowish white (3A2) towards base with yellowish basal mycelium. Context in pileus yellowish white (2A2), becoming brownish, turning greyish red (7B3) with KOH; greyish with FeSO4; in stipe white to pale yellow (2A2–3), turning greyish red (7B3) with KOH, unchanging with FeSO4. Basal mycelium yellowish. Annulus absent. Taste not recorded. Odour indistinct. Spore print olive brown.

Basidiospores 7–9.4–11.5 × 3–3.9–4.5 μm, (n = 30, Q = 1.88–2.45–3), elliptical to oblong, olivaceous in 5% KOH, smooth under light microscope, but with bacillate ornamentation under SEM. Basidia 28–34 × 7.5–11.5 μm, subclavate to clavate, elongate, hyaline, 4-spored; sterigmata 2–4.5 × 0.5–1 μm. Pleurocystidia 36–49 × 9–14 μm, common, clavate with rounded or subfusoid apex, ventricose, emergent up to 24 μm. Lamellae edge fertile, composed of basidia and cystidia. Cheilocystidia 26–39 × 11–13 μm, common, clavate with rounded or subfusoid apex, emergent up to 18.5 μm. Hymenium layer up to 22 μm thick, hymenophoral trama composed of up to 8 μm wide cylindrical, smooth, hyaline, septate, parallel hyphae. Pileipellis 200–300 μm thick, an interwoven, compact trichoderm composed of erect to suberect, hyaline, septate, branched hyphae; terminal elements 22–69 × 6–11 μm, cylindrical, with rounded to obtuse apex. Stipitipellis up to 150 μm thick, a trichodermium, composed of erect to suberect, content dense to slightly granular in many or hyaline hyphae, forming; terminal elements 20–96 × 9–20 μm, inflated, clavate, ventricose to pyriform or bulbous with mucronate or lageniform apex or cylindrical with fusoid apex; subterminal elements of few hyphae inflated; caulobasidia not found. Clamp connections absent in all tissues.

Additional specimen examined INDIA, Uttarakhanad, Bageshwar district, on way between Dhakuri and Loharkhet, 30° 04.084′ N 79° 55.195′ E, alt. 2849 m, subalpine mixed forest under Quercus sp., 15 August 2023, K. Das, KD 23-047 (CAL 1956).

Phylloporus smithii K. Das, Sudeshna Datta, U. Singh & A. Ghosh sp. nov. Mycobank: MB 851130. Holotype: INDIA, Uttarakhand, Rudraprayag district, Baniyakund, 30° 10.146′ N 078° 52.107′ E, alt. 2563 m, temperate mixed forest under Quercus sp., 4 August 2023, K. Das, KD 23-012 (CAL 1957, holotype!) (Figs. 2, 12, 13).

Figure 12
figure 12

Phylloporus smithii. (KD 23-012, holotype). (a) Fresh basidiomata in the field. (b) Pileipellis. (c) Pleurocystidia. (d) Cheilocystidia. (e) Basidiospores under SEM. Scale bars: (b–d) = 50 µm, (e) = 1 µm.

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Figure 13
figure 13

Phylloporus smithii. (KD 23-012, holotype). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidia. (e) Elements of pileipellis. (f) Elements of stipitipellis. Scale bars: (a–f) = 10 µm.

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Etymology Commemorating Alexander H. Smith for his significant contribution to the systematics of Boletaceae.

Diagnosis Distinguished from the other known Phylloporus species by minutely cracked pileus surface, rather crowded lamellae (18–20/10 mm), stipe that is gradually tapering towards base and ITS, LSU and tef 1-α sequence data.

Basidiomata small to medium-sized, solitary to gregarious. Pileus 15–54 mm in diam., convex to planoconvex with shallowly depressed to flat centre, then applanate, finally somewhat funnel-shaped with depressed centre, margin decurved; surface somewhat velvety and minutely cracked with maturity, brown (6D6) to light yellow (4A4) at or near centre, paler towards margin; violet-brown (10E7) with KOH, greenish with FeSO4; context off white, unchanging in color when injured. Hymenophore lamellate; lamellae decurrent, close to rather crowded (18–20/10 mm), intervenose and anastomosing, up to 5 mm in height, yellow (2A6), slowing becoming blue (25D4) when bruised; lamellulae in 4 series, attenuate, ventricose, concolorous with lamellae. Stipe central, 18–50 × 3–10 mm, subcylindric with distinctively tapering base, solid; pale yellow (3A3) when young, gradually brownish from middle to base; surface dry, tomentose upper part sometimes ribbed to striate by the decurrent lines of the lamellae. Context in pileus off-white with pale yellow centre, unchanging on exposure. Basal mycelium whitish. Annulus absent. Taste not recorded. Odour indistinct. Spore print olive brown.

Basidiospores 8.5–9.8–11.5 × 3–4.1–5 μm, (n = 30, Q = 2–2.4–3), elliptical to oblong, olivaceous (1C2) in 5% KOH, smooth under light microscope, but with bacillate ornamentation under SEM. Basidia 33–44 × 6–10 μm, subclavate to clavate, elongate, hyaline, 4-spored; sterigmata 1–4 × 0.5–1 μm. Pleurocystidia 24–62 × 8–19 μm, common, subclavate to broadly clavate with rounded or subfusoid apex, rarely fusiform, septate, emergent up to 20 μm. Lamellae edge fertile, composed of basidia and cystidia. Cheilocystidia 34–50 × 9–14 μm, common, clavate with rounded or subfusoid apex, rarely subventricose, emergent up to 20 μm. Hymenium layer up to 37 μm thick; hymenophoral trama composed of up to 7 μm wide cylindrical, smooth, hyaline, septate, parallel hyphae. Pileipellis 150–200 μm thick, a trichodermium, composed of erect to suberect hyaline, septate, rarely branched hyphae; terminal elements 20–47 × 5–9 μm, cylindrical, with rounded to subfusoid apex. Stipitipellis up to 150 μm thick, a trichodermium, composed of erect to suberect, hyaline hyphae; terminal elements 29–53 × 11–17 μm, cylindrical or clavate, bulbous to pyriform or cylindrical with fusoid apex, subterminal elements occasionally inflated; caulobasidia similar to tube basidia. Clamp connections absent in all tissues.

Additional specimen examined INDIA, Uttarakhand, Chamoli district, Didna top, 30° 09.922′ N 79° 38.042′ E, alt. 2536 m, temperate mixed forest under Quercus sp., 8 August 2023, K. Das, KD 23-022 (CAL 1958).

Notes Basidiomata with strong decurrent intervenose to anastomosing lamellae (instead of poroid hymenophore) and bacillate spores place the two proposed species under Phylloporus Quél.15,16 among boletoid fungi. It is realized that due to phenotypic plasticity in this genus, morphology-based species identification is quite impossible. Concordance of multigene genealogy along with morphology is the only solution to separate these species having overlapping morphological features. Present species, P. smithii is distinctively characterised by the pileus surface being minutely cracked, rather crowded lamellae (18–20/10 mm) and stipe that is gradually tapering from apex to base whereas, P. himalayanus is significantly featured by subdistant lamellae (8–10/10 mm), typically sub-bulbous strigose stipe base, diversified terminal elements of stipitipellis hyphae and absence of caulobasidia. These two species can be separated in the field itself.

Phylloporus himalayanus looks like P. yunnanensis N.K. Zeng, Zhu L. Yang & L.P. Tang (originally reported from China) and P. subrubeolus Chuankid, K.D. Hyde & Raspé (originally reported from Thailand). However, both P. yunnanensis and P. subrubeolus are distinguished from P. himalayanus by the absence of strigose sub-bulbous stipe base and microscopically, they lack terminal elements with a mucronate, lageniform to appendiculate apex in the hyphae of stipitipellis15,17. Similarly, P. smithii appears quite close to P. imbricatus N.K. Zeng, Zhu L. Yang & L. P. Tang, another Asian species originally reported from China. However, the later can be separated by the distinctively larger (50–100 × 3–15 mm) stipe, subdistant lamellae and microscopically, by larger basidiospores (10–13 × 4–5 μm), fertile stipitipellis and fusiform pleurocystidia15. Moreover, our multigene molecular phylogenetic estimation clearly separates these two Indian species among themselves and from the other known species of Phylloporus as shown in Fig. 2.

Xerocomus rugosellus (W.F. Chiu) F.L. Tai, Syll. fung. sinicorum: 815 (1979) (Figs. 3, 14, 15).

Figure 14
figure 14

Xerocomus rugosellus (KD 23-019). (a–c) Fresh basidiomata. (d) Pileipellis. (e) Caulocystidia. (f) Cheilocystidia. (g) Basidiospore under SEM. Scale bars: (d–f) = 10 µm, (g) = 1 µm.

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Figure 15
figure 15

Xerocomus rugosellus (KD 23-019). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidia. (e) Elements of pileipellis. (f) Caulocystidia. Scale bars: (a–f) = 10 µm.

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Basidiomata small to medium-sized. Pileus 40–50 mm in diam., convex when young, becoming planoconvex with maturity; surface rugose to subtomentose, non-viscid, greenish yellow (4C5) to pompeian yellow (5C6) or paler; margin entire, with a very narrow sterile flap of tissue; turning brown (7E8) with KOH. Pore surface yellow (3A6–7), initially unchanging when bruised, later becoming blue green; pores ellipsoid to elongate or bacillate, often compound, 1–2/mm. Tubes adnate to adnexed, 4–4.3 mm long, light yellow (2A5), unchanging when bruised or exposed. Stipe 85–95 × 8–10 mm, more or less cylindrical, gradually tapering towards the base; surface reticulate at apex, then longitudinally ridged or striated towards mid, yellowish white at the apex, orange-white (6A2) towards the middle and lower half. Context in pileus up to 12 mm thick, pale to pastel yellow (2A3–4), turning light orange (5A4) in KOH and slightly greenish with FeSO4; context in stipe solid to pithy, light yellow (2A5) on the upper 1/3rd of the stipe length or paler, lower 2/3rd brownish, pith brown (6E5–6). Basal mycelium white. Odour mild. Spore print not obtained.

Basidiospores 8.5–10.5–13 × 3.5–4.7–6 µm, (n = 30, Q = 1.98–2.25–2.89), ellipsoid to fusoid and inequilateral in side view, hyaline, smooth under light microscope but under SEM spore surface bacillate. Basidia 27–40 × 9–11 µm, clavate, 4-spored; sterigmata 2–5 × 0.5–1 µm. Pleurocystidia 36–60 × 8–11 µm, ventricose or fusoid, thin-walled, with finely granular content, emergent up to 37 µm. Tube edge fertile. Cheilocystidia 34–45 × 7–8.5 µm, less frequent, ventricose or fusoid, thin-walled, emergent up to 33 µm. Hymenophoral trama composed of thin-walled, septate, parallel; hyphae up to 10 µm wide, branched, septate. Pileipellis up to 150 µm thick, a trichodermium, composed of erect to suberect, cylindrical, regularly septate hyphae, sometimes branched, thin-walled, few with dense content, olive with 5% KOH; terminal elements 15–40 × 5–11 µm, cylindrical, sometimes clavate, or ventricose to fusiform. Stipitipellis up to 250 µm thick, fertile, composed of thin-walled, branched, septate, parallelly arranged hyphae and few tufts of basidia, basidioles, and caulocystidia (caulohymenium); caulocystidia 27–42 × 10–17 µm, subclavate to clavate, ventricose, pyriform to bulbous; caulobasidia 33–40 × 9–10 µm, 4-spored. Clamp connections absent in all tissues.

Specimens examined INDIA, Uttarakhand, Chamoli district, Didna top, 30° 09.922′ N 79° 38.042′ E, alt. 2536 m, temperate mixed forests under Quercus sp., 8 August 2023, K. Das, KD 23-019 (CAL 1964); ibid., Bageshwar district, on way between Dhakuri and Khati, 30° 04.934′ N 79° 55.080′ E, alt. 2545 m, temperate mixed forests under Quercus sp., 14 August, 2023, K. Das, KD 23-055 (CAL 1965).

Notes Present species is the first report for the Indian mycobiota. In the field, Xerocomus rugosellus is characterized by the rugose pileus surface (when young), the slowly bluing pore surface and context (on bruising), comparatively tall and slender stipe. Our Indian collections are in conformation with the holotype (Chinese material) except the hymenial cystidia and basidiospores which are comparatively small in present collections16,18. Few Asian (Indian) species that share morphological and molecular affinities with the present species are Xerocomus doodhcha K. Das, D. Chakr., A. Baghela, S.K. Singh & Dentinger, X. longistipitatus K. Das, A. Parihar, D. Chakr. & A. Baghela, X. uttarakhandae K. Das, Sudeshna Datta, and A. Ghosh and X. reticulostipitatus Hembrom, D. Chakr., A. Parihar & K. Das. However, X. doodhcha is distinct by a typical “milk–tea” colour of pileus, comparatively shorter stipe (50–68 × 4–10 mm) and angular pores19. Xerocomus longistipitatus has robust basidiomata with brown pileus and exceptionally long stipe (70–185 × 10–24 mm), pore surface that turns greenish grey to dull green slowly on bruising, angular to irregular pores20, whereas X. reticulostipitatus shows very prominent and typical brownish red to reddish brown reticulation on stipe and distinctively larger basidiospores (10.3–12.2–15.6 × 3.7–4.4–5.3 µm)21. Xerocomus uttarakhandae is segregated (from X. rugosellus) by possessing typically cracked to areolate greyish orange to greyish brown pileus surface exposing inner reddish context5.

Boletoideae

Porphyrellus uttarakhandae K. Das, Sudeshna Datta & A. Ghosh sp. nov. Mycobank: MB 851131. Holotype: INDIA, Uttarakhanad, Chamoli district, Lohajung, 30° 27.811′ N 79° 16.178′ E, alt. 2283 m, temperate mixed forests under Quercus sp., 10 August 2023, K. Das, KD 23-028 (CAL 1959, holotype!) (Figs. 4, 16, 17).

Figure 16
figure 16

Porphyrellus uttarakhandae. (KD 23-028, holotype). (a,b) Fresh basidiomata. (c,d) Pileipellis. (e) Caulocystidia. (f) Basidiospores. Scale bars: (c–f) = 10 µm.

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Figure 17
figure 17

Porphyrellus uttarakhandae. (KD 23-028, holotype). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidia. (e) Elements of pileipellis. (f) Caulocystidia. Scale bars: (a–f) = 10 µm.

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Etymology referring to the Himalayan state of Uttarakhand, where the type locality is situated.

Diagnosis Distinct from closely allied species i.e., P. orientifumosipes by shorter tubes, absence of a bluish ring like zone on stipe apex, larger basidiospores, shorter hymenial cystidia and LSU, rpb2, and tef 1-α sequence data.

Basidiomata small to medium-sized. Pileus 45–65 mm diam., sub-hemispherical to convex or at the most planoconvex, yellowish brown (5D–E8) to light brown (7D4–5) to reddish brown or umber with slightly darker in the center; surface dry, minutely cracked into small squamules on a whitish background; margin decurved with a flap of tissue of 0.8 mm wide by diam. Hymenophore adnexed to sinuate when young, depressed around apex of stipe when mature; pore surface whitish to pinkish to brownish pink, turning asymmetrically greyish turquoise (24D5–6) or greenish blue when bruised; pores subangular to roundish, 1–2/mm; tubes up to 7 mm long, concolorous to pore surface, turning faint greenish blue when exposed. Stipe cylindrical, 50–75 × 8–13 mm, concolorous to pileus surface; surface minutely cracked. Context in pileus, chalky to greyish white, asymmetrically greenish blue or paler when exposed; in stipe chalky up to mid, greyish white towards base, asymmetrically greenish blue or paler when exposed. Basal mycelium whitish to greyish white, unchanging when bruised. Taste and odour mild. Spore print orange-red to brownish red.

Basidiospores 8.7–11–13.7 × 5–5.4–6.2 µm (n = 30, Q = 1.64–2.01–2.45), broadly subfusiform to ellipsoid, inequilateral in sideview, smooth under light microscope. Basidia 36–45 × 9–14 µm, clavate, elongate, 4-spored; sterigmata 3–5 × 1–2 µm. Pleurocystidia 34–46 × 7–12 µm, fusiform, clavate to subventricose with rounded apex or appendiculate apex, thin walled, hyaline; emergent up to 26 µm. Tube edge fertile, composed of basidia, basidiole and cystidia. Cheilocystidia 24–40 × 9–17 µm, broadly clavate to pyriform, thin-walled, hyaline; emergent up to 22 µm. Hymenophoral trama divergent, composed of compactly arranged, septate, thin-walled hyphae, 5–7 µm wide. Pileipellis up to 150 µm thick, a trichodermium to palisadoderm, composed of compactly arranged, branched, septate, erect, thin-walled hyphae with chains of slightly inflated elements; terminal elements 18–47 × 6–12 µm, cylindrical to subcylindrical, clavate, subfusiform with rounded or tapering apex, rarely bulbous. Stipitipellis up to 50 µm thick, composed of irregularly arranged, branched, septate, erect, thin-walled hyphae, with infrequent tuft of basidia and cystidia; caulocystidia 27–53 × 8–13 µm, ventricose to subfusiform with rounded, subcapitate to appendiculate apex, thin-walled, hyaline; caulobasidia 37–44 × 13–16 µm, broadly clavate, 4-spored. Clamp connections absent in all tissues.

Additional specimen examined INDIA, Uttarakhand, Chamoli district, Kuling, 30° 27.811′ N 79° 16.178′ E, alt. 2296 m, temperate mixed forests under Quercus sp., 10 August 2023, K. Das, KD 23-056 (CAL 1960).

Notes Possession of umber coloured basidiomata, whitish or greyish context without significant discoloration or becoming asymmetrically greenish blue, white to pinkish pore surface that changes asymmetrically greenish blue, palisadoderm pattern of pileipellis, and smooth basidiospores place the present species under Porphyrellus E.-J. Gilbert1,3.

Two Asian species namely, Por. orientifumosipes and Por. pseudocyaneotinctus look quite similar to Por. uttarakhandae in the field. However, Por. orientifumosipes differs mostly from the present species by longer tubes (up to 20 mm), presence of ring-like bluish zone at stipe apex, smaller basidiospores (9.5–10.5 × 4.5–5.5 µm) and much longer hymenial cystidia (58–74 × 15–19 µm) whereas, Por. pseudocyaneotinctus shows distinctively more robust basidiomata (pileus 46–99 mm in diameter, stipe 48–123 × 9–19 mm), larger hymenial cystidia (pleurocystidia 36.8–85 × 8.5–13 µm, cheilocystidia 38.2–60.5 × 10.8–17.9 µm), differently-shaped cheilocystidia (lageniform), differently-shaped terminal elements of pileipellis hyphae and a sterile stipitipellis18,38.

Retiboletus pseudoater K. Das, A. Ghosh, Sudeshna Datta & Vizzini sp. nov. Mycobank: MB 851132. Holotype: INDIA, Uttarakhanad, Bageshwar district, on way between Dhakuri to Khati, 30° 04.934′ N 79° 55.080′ E, alt. 2545 m, temperate mixed forest under Quercus sp., 14 August 2023, K. Das, KD 23-040 (CAL 1961, holotype!) (Figs. 5, 18, 19).

Figure 18
figure 18

Retiboletus pseudoater. (KD 23-040, holotype): (a,b) Fresh basidiomata (c) Pileipellis (d) Cheilocystidia (e) Stipitipellis (f) Basidiospores. Scale bars (c) = 20 µm, (d–f) = 10 µm.

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Figure 19
figure 19

Retiboletus pseudoater. (KD 23-040, holotype). (a) Basidiospores. (b) Basidia. (c) Pleurocystidia. (d) Cheilocystidioid elements. (e) Elements of pileipellis. (f) Hyphal elements of stipitipellis showing terminal and subterminal elements. (g) Caulocystidia. Scale bars: (a–f) = 10 µm.

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Etymology Referring to the morphological similarity of the species with Retiboletus ater another Asian species.

Diagnosis Distinguished from the closely allied R. ater by larger (22–90 mm in diameter) pale orange to greyish orange or brownish orange pileus, presence of cheilocystidioid elements at lamellar edges and different ITS, LSU, and tef 1-α sequence data.

Basidiomata small to medium-sized. Pileus 22–90 mm in diam., hemispherical to convex, becoming planoconvex at maturity; surface dry, velvety, pale orange (5A3) to greyish orange (5B4) with patches of grey to greyish brown (5F1–3) or completely black when young, becoming greyish orange (5B3), brownish orange (5C4), light brown to cinnamon brown (6D4–5) in combination brownish grey to negro (6F2–3) with distinctive blackish dots or darker, unchanging with KOH; margin with a flap of tissue of 1 mm wide. Hymenophore adnate to adnexed; pore surface yellowish white to pale yellow (2–3A2–3), becoming nougat (5D3) initially, then slowly becoming to drab grey (5E3); pores angular, 2/mm. Tubes up to 10 mm long, concolorous with pore surface. Stipe 47–65 × 12–14 mm, elongate, subclavate, solid towards apex stuffed below; surface dry, grey to black prominent and coarse reticulation till more than middle of the stipe (but never extended up to base) on the background of absinthe yellow to olive yellow (3C5–6) or darker; base smooth, pale orange (5A3). Context in pileus, 12 mm thick at the centre, yellowish white (3–4A2), turning to reddish or pinkish white (7A2) with KOH, greenish grey (28B2) with FeSO4, in stipe yellowish white (3A2) at apex, gradually pastel yellow to light yellow (3A4–5) or deep yellow towards mid to base, turning reddish brown to mahogany (8E5–7) with KOH, greenish grey (28B2) with FeSO4. Basal mycelium white (2A1). Taste and odour indistinct. Spore print not obtained.

Basidiospores 10.2–11.29–12.9 × 3.4–4.2–4.9 μm (n = 30, Q = 2.36–2.65–3.1), subfusiform, elliptical to oblong, inequilateral in side view, elliptical to oblong, olivaceous (1C2) in 5% KOH, smooth. Basidia 25–30 × 8–10 μm, clavate, hyaline, 4-spored; sterigmata 2–4.5 × 0.5–1 μm. Pleurocystidia 57–70 × 11–13 µm, ventricose with subcapitate, capitate or appendiculate apex, thin-walled, with brown pigment, emergent up to 31 µm. Lamellae edge fertile, composed of basidia and cystidioid elments. Cheilocystidioid elements 30–38 × 7–10 μm, subfusiform to ventricose, 2–3 septate, terminal elements fusoid. Hymenophoral trama parallel; hyphae thin-walled, septate, cylindrical, 6–9 μm wide, hyaline to yellowish in KOH, yellowish to brownish yellow in Melzer’s reagent. Pileipellis 140–160 µm thick, a trichodermium, composed of erect to suberect, cylindrical, regularly septate branched thin-walled hyphae, mostly with brown intracellular pigmentation; terminal elements 15–40 × 5–11 µm, cylindrical. Stipitipellis up to 80 μm thick, mainly hymeniform with clusters of cystidia and basidia; terminal elements of 10–20 × 8–14 μm, subclavate to broadly clavate, or bulbous, some with brown pigmentation in KOH; subterminal elements often inflated; caulocystidia 34–60 × 9–12 μm, fusiform with lageniform, appendiculate or mucronate apex, ventricose to obclavate; caulobasidia 25–35 × 8–10 μm, clavate, 4-spored. Clamp connections absent in all tissues.

Additional specimens examined INDIA, Uttarakhand, Bageshwar district, Dhakuri to Loharkhet trek, 30° 04.270′ N 79° 55.229′ E, alt. 2870 m, subalpine mixed forest under Quercus sp., 15 August 2023, K. Das, KD 23-048 (CAL 1962); ibid., Dhakuri to Dhur trek, 30° 05.009′ N 79° 53.882′ E, alt. 2538 m, temperate mixed forest under Quercus sp., 16 August 2023, K. Das, KD 23-051 (CAL 1963).

Notes Distinctive features of basidiomata like coarsely reticulate stipe surface and vivid yellow stipe context place this species under the genus Retiboletus22. Moreover, presence of pale orange to greyish brown to black pileus surface places the present species in recently established subgenus: R. subg. Nigroretiboletorum Yan C. Li & Zhu L. Yang23. This species can easily be distinguished from other species of Retiboletus by combination of features like pale orange, brownish orange to greyish orange, black dotted pileus, yellowish white to pale yellow pore surface, greyish black to black coarse reticulation on stipe surface, microscopically, by presence of typically 2–3 septate cheilocystidioid elements, and occurrence under Quercus species in subalpine Himalaya. In the field, this species resembles R. ater Yan C. Li & T. Bau (originally reported from China) however, the latter can be distinguished from the earlier by a smaller pileus (30–50 mm in diam.), comparatively smaller basidiospores “[60/3/2] (7)8–10.5(11) × 3–4.5(5) μm, absence of orange with black-dotted pileus surface, 2–3 septate cheilocystidioid elements, differently shaped (fusiform with lageniform, appendiculate or mucronate apex, ventricose to obclavate) caulocystidia and the presence of differently-shaped terminal elements of pileipellis (“narrowly clavate to subcylindrical or subfusiform, sometimes narrowly mucronate, rostrate”24). Few other species namely, R. fuscus (Hongo) N.K. Zeng & Zhu L. Yang, R. nigrogriseus N.K. Zeng, S. Jiang & Zhi Q. Liang and R. pseudogriseus N.K. Zeng & Zhu L. Yang (all originally reported from China) also share some features with R. pseudoater. However, all three species are characterized by overall reticulate stipe and the absence of orange tinges on pileus surface25,26. Another species reported from China, viz. R. kauffmanii (Lohwag) N.K. Zeng & Zhu L. Yang is easily separated in the field from our present species by grey-brown to brown pileus, yellow pore surface (2A5–6) and yellow reticulation on stipe surface27. Another Chinese species, R. sinogriseus Yan C. Li & T. Bau is also partly similar to the present novel species, however the former has stipe with pale yellow apex, blackish-yellow towards the base and thinner pileipellis (100–120 μm)24.

Materials and methods

Morphological study

Fresh basidiomata were collected during the month of August from different parts of Uttarakhand and Meghalaya. Photographs were taken in the field with a Canon Power Shot SX 50 HS camera. Macromorphological characterizations were done in the field or at basecamp from fresh and dissected basidiomata with the help of daylight. Colour codes and terms mostly follow Kornerup & Wanscher28. After noting down all possible macromorphological and macrochemical spot test details, samples were placed for drying in an aluminium field drier. Micromorphological characters were observed after mounting the freehand sections of dried samples in a solution of 5% KOH, 1% Phloxin, and 1% ammoniacal Congo red with an Olympus CX 41 (installed in Central National Herbarium, Botanical Survey of India, Howrah) or Olympus CX 43 compound microscope (installed in Eastern Regional Centre, Botanical Survey of India, Shillong). Drawings of the micromorphological features were made with the help of drawing tube at 1000 × magnification. Microscopic photographs were taken with an Olympus BX 53 or Magcam DC camera. The basidiospores were measured in lateral view. Basidiospore measurements and length/width ratios (Q) are recorded as: minimum–mean–maximum. Basidium length excludes the length of sterigmata. Herbarium codes follow Thiers29. Field emission scanning electron microscope (FESEM) illustrations of basidiospores were 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 Centre for Research in Nanoscience and Nanotechnology (CRNN) in University of Calcutta, India.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from 100 mg of a dried basidioma (for seven species) with the InstaGeneTM Matrix Genomic DNA isolation kit (Biorad, USA) following the manufacturer’s instructions. The PCR amplification of ITS region, part of the LSU, region between conserved domains 6 and 7 of rpb2 and 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 respectively30,31,32,33,34. PCR amplification was carried out in a ProFlex PCR system (Applied Biosystems) 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, Germany). Both strands of the PCR fragment were sequenced on the ABI 3500 DNA Analyzer (Applied Biosystems, USA) using the amplifying primers. The sequence quality was checked using Sequence Scanner Software ver. 1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro ver. 5.135. All sequences newly generated in this study were submitted to GenBank. Accession numbers of species used in phylogenetic analysis (Figs. 1, 2, 3, 4, 5) are listed in the Tables 1, 2, 3, 4 and 5.

Table 1 Leccinellum and allied sequences used in phylogenetic analyses of this study. Newly sequenced collections are in bold.
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Table 2 Phylloporus and allied sequences used in phylogenetic analyses of this study. Newly sequenced collections are in bold.
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Table 3 Xerocomus and allied sequences used in phylogenetic analyses of this study. Newly sequenced collections are in bold.
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Table 4 Porphyrellus and allied sequences used in phylogenetic analyses of this study. Newly sequenced collections are in bold.
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Table 5 Retiboletus and allied sequences used in phylogenetic analyses of this study. Newly sequenced collections are in bold.
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Alignment and phylogenetic analyses

The ITS, LSU, rpb2 and tef 1-α sequences of the newly generated Leccinellum bothii, L. sinoaurantiacum, Phylloporus himalayanus, P. smithii, Xerocomus rugosellus, Porphyrellus uttarakhandae and Retiboletus pseudoater and their close relatives were retrieved from nBLAST search against GenBank (https://www.ncbi.nlm.nih.gov/genbank) and relevant published phylogenies5,16,18,22,23,24,36,37,38. Four raw datasets (ITS, LSU, rpb2 and tef 1-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/) with L-INS-i strategy and normal alignment mode, respectively. The alignment was checked and trimmed with the conserved motifs manually with MEGA v. 739. To eliminate ambiguously aligned positions in the alignment as objectively as possible, the on-line program Gblocks 0.91b40 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, then we concatenated into multi-locus dataset using BioEdit v. 7.0.941. The introns of protein coding genes (rpb2 and tef 1-α) were excluded entirely in the phylogenetic analyses. In the three-locus dataset (LSU + rpb2 + tef 1-α) of Leccinellum, 953 bp are for LSU, 770 bp for rpb2 and 588 bp for tef 1-α. In the three-locus dataset (ITS + LSU + tef 1-α) of Phylloporus, 421 bp are for ITS, 1377 bp for LSU and 602 bp for tef 1-α. In the two-locus dataset (ITS + LSU) of Xerocomus, 553 bp are for ITS and 840 bp for LSU. In the three-locus dataset (LSU + rpb2 + tef 1-α) of Porphyrellus, 880 bp for LSU, 661 bp for rpb2 and 439 bp for tef 1-α. In the three-locus dataset (ITS + LSU + tef 1-α) of Retiboletus, 550 bp are for ITS, 867 bp for LSU and 582 bp for tef 1-α. To find the best-fit evolutionary models of matrixes for IQ-tree and MrBayes were selected using ModelFinder and PartitionFinder 242,45 respectively. The combined dataset was phylogenetically analysed using both maximum likelihood (ML) and Bayesian inference (BI) methods. Maximum likelihood (ML) analysis was conducted using the IQ-tree tool version 2.2.2.643, employing the best model for each locus chosen by ModelFinder42. Additionally, ultrafast bootstrap with 1000 replicates was applied to obtain nodal support values. Bayesian inference (BI) was computed in MrBayes v.3.2.644 with four Markov chain Monte Carlo (MCMC) algorithm. PartitionFinder2 was used to find the best nucleotide substitution models using the Bayesian information criterion (BIC) with a greedy search over all models45. Two MCMC runs of four chains were executed simultaneously from a random starting tree for 100,000 generations until the standard deviation of split frequencies reached below the 0.01 threshold. Trees were sampled every 100th generation. The first 25% of trees were discarded as burn-in. Chain convergence was determined using Tracer 1.546 to ensure sufficiently large effective sample size (ESS) values (> 200). Gaps in the alignment were treated as missing data in phylogenetic analyses. Both ML and BI analyses resulted in essentially the same tree topologies and our five novel taxa are presented in the phylogenetic trees in bold red font (Figs. 1, 2, 3, 4, 5). Maximum likelihood bootstrap (MLbs) values ≥ 70% and Bayesian posterior probabilities (BPP) values ≥ 0.95 are shown in the phylogenetic trees.

Statements

The present research was undertaken in India, and the authors have obtained all kinds of permission or licences for the respective macrofungal surveys and collections of wild mushrooms for research purpose. Voucher specimens were duly submitted in the public herbaria: CAL and ASSAM (both are indexed in Index Herbariorum, https://sweetgum.nybg.org/science/ih/). The authors herewith confirm that all field studies and corresponding collections of mushrooms are complied with relevant institutional, national, and international guidelines and legislation.

Discussion

Boletaceae, the fastest revealing family among mushroom forming ectomycorrhizal Basidiomycota is now comprising of over 100 genera that are only came into the light with the combined approach of multigene molecular phylogeny and morphology. Considerable studies have been undertaken across the continents especially during last one decade and this family has undergone dramatic taxonomic reassessment. Several novel genera and numerous novel species are continually being uncovered across the continents in general and Asian countries in particular. Only in past five to six years, about 22 genera were discovered in this family from all over the world namely, Acyanoboletus G. Wu & Zhu L. Yang, Afrocastellanoa M.E. Smith & Orihara, Brasilioporus A.C. Magnago, Alves-Silva & T.W Henkel, Cacaoporus Raspé & Vadthanarat, Carolinigaster M.E. Sm. & S. Cruz, Erythrophylloporus Ming Zhang & T.H. Li, Hemiaustroboletus Ayala-Vásquez, García-Jiménez & Garibay-Orijel, Hemilanmaoa Yang Wang, Bo Zhang & Y. Li, Hongoboletus G. Wu & Zhu L. Yang, Indoporus A. Parihar, K. Das, Hembrom & Vizzini, Ionosporus O. Khmelnitsky, Kaziboletus Hosen & Zhu L. Yang, Kgaria Halling, Fechner & Davoodian, Longistriata Sulzbacher, Orihara, Grebenc, M.P. Martín & Baseia, Neotropicomus A.C. Magnago, Alves-Silva & T.W Henkel, Nevesoporus A.C. Magnago & T.W. Henkel, Phylloporopsis Angelini, A. Farid, Gelardi, M.E. Smith, Costanzo, & Vizzini, Rostrupomyces Vadthanarat & Raspé, Rubinosporus Vadthanarat, Raspé & Lumyong, Spongispora G. Wu, S.M.L. Lee, E. Horak & Zhu L. Yang, Tropicoboletus Angelini, Gelardi & Vizzini and Villoboletus L. Fan & N. Mao37,38,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63. It is noteworthy that 11 out of 22 genera are established from Asian countries like, China, Thailand and India. But unlike China and Thailand the megadiverse country like India remains seriously under-focussed in terms of Boletaceae. The stretch of Indian Himalaya and surrounding hilly regions are the hub for the ectomycorrhizal mushrooms including Boletaceae. Except few sporadic works there was no systematic documentary. Therefore, Indian taxa are remained unattended, uncovered or undiscovered. There is a high chance that considerable numbers of these taxa will be extinct due to uncontrolled man-made activities even before they are discovered. Exploration by the trained mushroom-taxonomists and the documentation would be the only hope to create the awareness and save these creatures, Moreover, most of the Indian elements are wrongly known by their European or North American lookalikes. Keeping in view the number of unaddressed species in different forests of Indian Himalaya and its adjacent hills, scarcity of boletologists, only in 2022 the project on boletoid mushrooms were proposed for the first time by Botanical Survey of India, premier research institute of Ministry of Environment, Forest and Climate Change (Govt. of India). Our extensive and intensive survey followed by methodical morphology-based characterization, molecular phylogenetic estimation and documentation will not only open the avenues for research on boletoid mushrooms of India but also resolve the many hidden mystery.