Two new species and one new record of the genus Tylopilus (Boletaceae) from Indian Himalaya with morphological details and phylogenetic estimations

Abstract Tylopilus himalayanus and T. pseudoballoui are described as new species from two Himalayan states (Sikkim and Uttarakhand) in India. Tylopilus himalayanus is characterised by a unique combination of features: reddish- or brownish-grey to purplish-grey then brown to reddish-brown or darker pileus, absence of olive or violet tinges on stipe surface, angular pores, stipe without reticulum or rarely with a faint reticulum restricted to the very apex, bitter taste of the context and positive macrochemical colour reaction of the stipe context with KOH (dark orange) and FeSO4 (dark green), medium sized (10.9–14.4 × 3.9–4.9 µm) basidiospores and occurrence under coniferous trees; T. pseudoballoui is distinguished by orange-yellow to brown-yellow sticky pileus, pale yellow pore surface with pinkish hues that turns pale to greyish-orange on bruising; angular pores, stipe concolorous to pileus with pruinose but never reticulate surface, ixocutis pattern of pileipellis and occurrence under broadleaf trees. Another species, T. neofelleus, which was reported earlier from China and Japan, was also collected from Sikkim and reported for the first time from India. All three species are described with morphological details and two-locus based (nrLSU and nrITS) phylogenetic data.


Introduction
The genus Tylopilus P. Karst., one of the less attractive to eye-catching ectomycorrhizal taxa (associated mainly with Fagales and Pinaceae) in the family Boletaceae, is featured by its dry, glabrous to subvelvety pileus, white to greyish pore surface usually becoming flesh pink to purple-brown at maturity, immutable to slightly brownish or becoming blue-green context on bruising, solid stipe with pruina or reticulation over the surface, absence of annulus or veil, flesh-pink to dull flesh-ochre spore print, smooth pink-coloured basidiospores, presence of pleurocystidia and absence of clamp-connections (Smith and Thiers 1971, Wolfe 1979, Singer 1986, Wu et al. 2014). Further, this genus was divided into two subgenera namely, T. subg. Tylopilus and T. subg. Porphyrellus Thiers 1971, Singer 1975). The former subgenus is characterised by "spores with pale cinnamon-yellow to pale yellow walls in KOH and IKI; context usually unchanging or rust coloured on injury; context not turning red-brown in KOH", whereas the latter is characterised by "spores with dark brown walls in KOH and IKI; context usually turning blue-green on injury then becoming red-brown and, in some taxa, the context becoming red-brown in KOH" (Wolfe 1979). From all over the world (mostly from North America, Australia, Asia, Africa and Europe), about 75 species are reported (Kirk et al. 2008, Magnago et al. 2017. Like some other morphology-based genera in Boletaceae, the traditional concept of Tylopilus (Tylopilus s.l.) was split and has given birth to a few other genera with the recent advancement of multi-locus phylogeny. Tylopilus s.l. appeared as polyphyletic and evolved in 11 different lineages during the course of evolution (Nuhn et al. 2013, Wu et al. 2014). Thus, taxonomic placement of the members of this genus are still floating and many previously considered Tylopilus species are shifted into new genera such as Zangia Yan C. Li & Zhu L. Yang, Australopilus Harrya Halling, Nuhn &Osmundson (Li et al. 2011, Halling et al. 2012). According to Wu et al. (2014), all the 11 clades consisting of the members of Tylopilus come under five subfamilies (Austroboletoideae, Leccinoideae, Boletoideae, Pulveroboletus group and Zangioideae) in Boletaceae and Tylopilus s.s., typified by Tylopilus felleus (Bull.) P. Karst., is placed within the subfamily Boletoideae.
The entire Indian Himalayan region comes under one ("Himalaya") of the globally acclaimed biodiversity hotspots and thus has immensely diverse mycobiota (including macrofungi) apart from its myriad flora and fauna. A wide range of phytogeographic variations with the presence of large numbers of ectomycorrhizal host plants, cold to warm monsoon, favourable humidity and plenty of rainfall, supports the growth of ectomycorrhizal mushrooms of Boletaceae. However, due to the lack of mushroomexplorers or mushroom-taxonomists, most of the areas of Indian Himalaya remain unexplored in terms of Boletaceae (only 80 species belonging to 23 genera, while more than 1050 species from 66 genera are reported from the world) ). During macrofungal surveys to different forested areas of Eastern Himalaya (Sikkim) and Western Himalaya, three interesting members of Tylopilus were collected separately. Detailed macro-and micromorphological studies followed by phylogenetic analyses based on nrLSU and nrITS sequences, confirm the novelty of two of them and are proposed here as T. himalayanus and T. pseudoballoui, whereas the third one appeared as conspecific to T. neofelleus (a species so far reported from Japan and China, Gelardi et al. 2015) and is reported as a new record for Indian mycobiota.

Morphological study
Macromorphological characters and habitat details were noted from fresh, young to mature basidiomata in the field and in base-camp. After recording the macromorphological characters, basidiomata were dried with a field drier. Photographs of these fresh and dry basidiomata and microphotographs were taken with the aid of Canon Power Shot SX 50HS, Canon SX 220 HS and Nikon-DS-Ri1 (dedicated to Nikon Eclipse Ni compound microscope) cameras. Colour codes and terms are mostly from Methuen Handbook of Colour (Kornerup and Wanscher, 1978). Micromorphological characters were observed with compound microscopes (Nikon Eclipse Ni-U and Olympus CX 41). 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 Nikon Eclipse Ni microscope) at 1000×. The basidium length excludes that of the sterigmata. Basidiospore measurements were recorded in profile view from 30 basidiospores. Spore measurements and length/width ratios (Q) are recorded here as: minimum-mean-maximum. Herbarium codes follow Thiers (continuously updated).

DNA extraction, polymerase chain reaction (PCR) and sequencing
Genomic DNA (for all the species) was extracted from 100 mg of dry basidiomata using the InstaGeneTM Matrix Genomic DNA isolation kit (Biorad, USA) following the manufacturer's instructions. PCR amplification primers were ITS1 and ITS4 (nrITS region) and LR0R and LR7 (nrLSU region) (White et al. 1990). PCR amplification on "ABI Veriti" thermal cycler protocols for nrITS and nrLSU regions were after Das et al. (2017). The PCR products were then purified using the QIAquick PCR Purification Kit (QIAGEN, Germany) before they were sent for sequencing. Both strands of the PCR fragments were sequenced on the 3730xl DNA Analyzer (Applied Biosystems, USA) using the amplifying primers and assembled using Sequencer

Phylogenetic analyses
The nrLSU and nrITS datasets were assembled according to recent previous studies on this genus (Gelardi et al. 2015, Magnago et al. 2017) and from BLAST (Altschul et al. 1997) searches in GenBank (Clark et al. 2016). As most Tylopilus collections in GenBank are not provided with both molecular markers, we were unable to establish a combined nrITS+nrLSU dataset and so have opted for present separate nrLSU and nrITS phylogenetic inferences. These (nrITS and nrLSU) sequences were aligned separately in MAFFT 7.305 (Katoh and Standley 2013). For the nrLSU dataset, Xanthoconium sinense (KT990666 and KT990664) and X. purpureum (KT990663) from Boletaceae were used as outgroup taxa. Similarly, for the nrITS dataset, two sequences from Gyroporus (KX869874, GQ166901), another genus in Boletales (Gyroporaceae), were used as the outgroup. Phylip file formats were created in AliView (Larsson 2014) using default settings. Phylogenies were reconstructed using Maximum Likelihood (ML) in RAxML 7.2.6 (Stamatakis 2006) in GTRGAMMA substitution model. All parameters in the ML analyses used the default settings in RAxML and Maximum Likelihood bootstrap percentage (MLB) were obtained using nonparametric bootstrapping with 1000 replicates. Additionally (to generate supplementary data), nrLSU and nrITS sequences were also phylogenetically analysed using Bayesian analysis. The best-fit models of nucleotide evolution for nrLSU and nrITS datasets (TIMef and TrNef+G, respectively) were obtained in MrModeltest 3.7 (Posada and Crandall 1998). Bayesian inferences were computed independently twice in MrBayes v.3.2.2 (Ronquist et al. 2012), under TIMef (for nrLSU) and TrNef+G (for nrITS) models, respectively. Bayesian posterior probabilities values (BPP) were calculated in two simultaneous runs with the Markov Chain Monte Carlo (MCMC) algorithm (Larget and Simon 1999). Markov chains were run for 1000000 generations, saving a tree every 100th generation. These analyses were terminated when the average standard deviation of split frequencies fell below 0.01. The first 25% of trees was discarded as burn-in (Hall 2004). The convergence of runs was visually assessed using Trace function in Tracer version 1.6.0 (Rambaut et al. 2013).
Similarly, in our nrITS-based ML and BI analyses (Figs 2 and Suppl. material 2, respectively), the two Indian collections of T. himalayanus , along with a collection of China (JN182869, wrongly labelled as "Tylopilus felleus"), appeared sister (MLB = 96%), in the ML analysis, or close, in the BI analysis, to a clade consisting of two T. rubrobrunneus sequences (KM248939 from Canada, GQ166869 from USA) and two "T. felleus" from USA (GQ166878, GQ166904). However, our collection is recovered as a separate species. The two Indian specimens of T. pseudoballoui (DC 17-30 and DC 17-35) clustered strongly (MLB = 100%, BPP = 1) with a Japanese sequence of "T. balloui" (AB509625) and appeared as sister (MLB = 83%), in the ML analysis, to a clade consisting of one Mexican collection (represented by KY859806 and labelled as "Tylopilus ballouii") and Tylopilus leucomycelinus (JF908789) from Guatemala and as sister (BPP = 0.85) whereas, in the BI analysis, to a clade (MLB = 100%) formed by eight Asian sequences of "T. balloui", four from Japan (AB973733, AB973757, AB973758, AB973735) and four from Thailand (KX017304, KX017306, KX017305, KX017307). However, our species is recovered as a distinct species. Finally, as in the nrLSU analysis, here also the two Indian collections of T. neofelleus are strongly clustered (MLB = 100%, BPP = 1) with three Asian counterparts (KM975487 and KM975489 from Japan, KM975486 from China), showing their conspecificity. Diagnosis. Distinct from all allied taxa by a combination of sequence data (nrITS and nrLSU), reddish-or brownish-grey to purplish-grey, then brown to reddish-brown pileus in basidiomata, absence of olive or violet tinges on stipe surface, presence of angular pores, stipe without reticulum or rarely with a faint reticulum restricted to the very apex, bitter taste of the context, positive reaction of the stipe context with KOH (dark orange) and FeSO 4 (dark green) and medium sized (10.9-14.4 × 3.9-4.9 µm) basidiospores.
Etymology. Referring to Indian Himalaya, the type locality.

Discussion
Our first novel species in Tylopilus s.s. (Wu et al. 2014), i.e. T. himalayanus, is featured by its brown, reddish-brown to purplish-grey, dry pileus, angular pores, stipe usually without reticulum even though sometimes with faintly reticulate apex, but longitudinally striate throughout, white unchanging context on exposure, bitter taste, sterile tube edge and trichodermic structure of pileipellis. Morphologically, T. rubrobrunneus Mazzer & A.H. Sm., T. felleus (Bull.) P. Karst., T. neofelleus Hongo and T. intermedius A.H. Sm. & Thiers resemble T. himalayanus. Tylopilus rubrobrunneus (originally reported from North America) differs from this species by its olive tinge on stipe surface, pileus surface with vinaceous tinges, rounded pores, negative colour reaction with KOH or NH 4 OH on context (Mazzer and Smith 1967, Smith and Thiers 1971, Grund and Harrison 1976, Both 1993, Bessette et al. 2010, 2016. Similarly, T. intermedius differs from the present Indian species by possessing a distinctively whitish pileus that stains pinkish buff to brown with age and context (pileus) that turns pinkish with FeSO 4 , but remains unchanged with KOH (Smith and Thiers 1971, Both 1993, Bessette et al. 2010, 2016. Some other members of this genus, such as T. felleus (originally described from Europe and known from India as well without checking its conspecificity through phylogeny), T. neofellus (originally reported from Japan but reported here for the first time from India), T. plumbeoviolaceus Snell & Dick (originally reported from North America but also known from this country without verifying its conspecificity through phylogeny) and T. violatinctus T.J. Baroni & Both (originally reported from North America), can also be separated from T. himalayanus morphologically: T. felleus has brownish pileus and distinctively reticulate stipe (Lannoy andEstadès 2001, Mu-ñoz 2005); T. neofellus and T. plumbeoviolaceus possess reddish-brown to violaceousbrown pileus and reticulate purplish-violaceous stipe (Snell and Dick 1941, Smith and Thiers 1971, Grund and Harrison 1976, Both 1993, Lakhanpal 1996, Bessette et al. 2010, 2016, Gelardi et al. 2015; T. violatinctus is easily distinguished by the more brightly coloured, bluish-violet to lilac-lavender or purple-greyish pileus, bruising dark rusty-violet when handled, the stipe turning yellowish on bruising, pileus surface and context staining yellowish-brown and negative to pinkish-brown with KOH, respectively, small basidiospores [(5.6-)7-9(-10) × 3-4 µm] and the growth in mixed woodlands possibly with Quercus, Fagus or Picea, in any case not in association pine or cedar trees (Baroni and Both 1998, Ortiz-Santana et al. 2007, Bessette et al. 2010.