Concordance of multigene genealogy along with morphological evidence unveils five novel species and two new records of boletoid mushrooms (fungi) from India

Agaricales, Russulales and Boletales are dominant orders among the wild mushrooms in Basidiomycota. Boletaceae, one of the major functional elements in terrestrial ecosystem and mostly represented by ectomycorrhizal symbionts of trees in Indian Himalaya and adjoining hills, are extraordinarily diverse and represented by numerous genera and species which are unexplored or poorly known. Therefore, their hidden diversity is yet to be revealed. Extensive macrofungal exploration by the authors to different parts of Himalaya and surroundings, followed by through morphological studies and multigene molecular phylogeny lead to the discovery of five new species of wild mushrooms: Leccinellum bothii sp. nov., Phylloporus himalayanus sp. nov., Phylloporus smithii sp. nov., Porphyrellus uttarakhandae sp. nov., and Retiboletus pseudoater sp. nov. Present communication deals with morphological details coupled with illustrations and phylogenetic inferences. Besides, Leccinellum sinoaurantiacum and Xerocomus rugosellus are also reported for the first time from this country.

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).

Taxonomy
Leccinoideae Leccinellum bothii K. Das  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 FeSO 4 .Hymenophore slightly depressed near stipe apex, adnexed; pore surface yellowish white (3A2) to yellow, becoming brown, greyish orange (5B3) then linoleum brown (5E7)   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.Binder 9 .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 forests 10 .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× 5.4-5.96-7µm) 5   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 avellana 9,11,12 .
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, www.nature.com/scientificreports/yellow hymenophore with angular pores, scabrous stipe surface, comparatively long basidiospores, an ixohyphoepithelium nature of pileipellis confirm their identity as L. sinoaurantiacum 13,14 .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).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 FeSO 4 .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    Notes Basidiomata with strong decurrent intervenose to anastomosing lamellae (instead of poroid hymenophore) and bacillate spores place the two proposed species under Phylloporus Quél. 15,16among 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.

Boletoideae
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 www.nature.com/scientificreports/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.Gilbert 1,3 .

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 & Wanscher 28 .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 Thiers 29 .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.www.nature.com/scientificreports/

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/ genba nk) and relevant published phylogenies 5,16,18,[22][23][24][36][37][38] . Four rw 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/ align ment/ softw are/) with L-INS-i strategy and normal alignment mode, respectively.The alignment was checked and trimmed with the conserved motifs manually with MEGA v. 7 39 .To eliminate ambiguously aligned positions in the alignment as objectively as possible, the on-line 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, 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 twolocus dataset (ITS + LSU) of Xerocomus, 553 bp are for ITS and 840 bp for LSU. Inthe 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 2 42,45 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 2.2.2.6 43 , employing the best model for each locus chosen by ModelFinder 42 . Aditionally, ultrafast bootstrap with 1000 replicates was applied to obtain nodal support values.Bayesian inference (BI) was computed in MrBayes v.3.2.6 44 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 models 45 .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 1.5 46

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:// sweet gum.nybg.org/ scien ce/ 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.

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.

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.

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.

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.

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.
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.

Table 1 .
Leccinellum and allied sequences used in phylogenetic analyses of this study.Newly sequenced collections are in bold.
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,

Table 4 .
Porphyrellus and allied sequences used in phylogenetic analyses of this study.Newly sequenced collections are in bold.

Table 5 .
Retiboletus and allied sequences used in phylogenetic analyses of this study.Newly sequenced collections are in bold.