Suillus indicus sp. nov. (Boletales, Basidiomycota), a new boletoid fungus from northwestern Himalayas, India

The new species Suillus indicus is described based on the morpho-anatomical description and molecular analysis of basidiomes found in conifer forests of the northwestern Himalayas, India. Morphologically, the key diagnostic characteristics of the new taxon are brownish-orange to reddish-brown pileus with low obtuse umbo, brownish-red to reddish-brown fibrillose squamules over the pileal surface, and absence of fibrillose squamules and glandular dots on the stipe surface. Sequences derived from the internal transcribed spacer region of basidiomes and culture demonstrated that the species is clearly distinct from other known taxa of Suillus and new to science.


Introduction
The genus Suillus Gray is one of the most prominent genera of ectomycorrhizal fungi in the order Boletales with about 50 species described worldwide (Kirk et al. 2008). Suillus species are confined to the temperate, boreal, and Mediterranean regions, although there are few reports (Natarajan & Raman 1983;Halling & Mueller 2002) of their occurrence from tropical areas. Most of Suillus species known so far are documented from the northern hemisphere, but some have also been reported from southern hemisphere (McNabb 1968;Watling & Gregory 1989;Dunstan et al. 1998) associated with the introduced pine species. Suillus species exhibit narrow host range forming ectomycorrhizae mainly with the members of the family Pinaceae and also with some deciduous species (Singer 1986;Kretzer et al. 1996;Wu et al. 2000). Based on the internal transcribed spacer (ITS) sequence analysis, Kretzer et al. (1996) have demonstrated that in the genus Suillus, Larix association seems to be primitive and associations with pines, Douglas-fir, and hardwoods seem to be derived in the genus Suillus.
The present knowledge of Suillus diversity from India, particularly from northwestern Himalayan region, is scanty. Suillus brevipes (Peck) Kuntze, S. pallidiceps A. H. Sm. and Thiers, S. punctatipes (Snell and E.A. Dick) Singer and S. subluteus (Peck) Snell have been reported from Pinus patula Schltdl. and Cham. forests of Tamilnadu, South India (Natarajan & Raman 1983). Suillus sibiricus (Singer) Singer and S. granulatus (L.) Roussel have been shown to be ectomycorrhizal with Pinus wallichiana A.B. Jacks. (Sagar & Lakhanpal 2005;Dar et al. 2010), whereas S. triacicularis Verma and Reddy has been described fruiting exclusively in association with Pinus roxburghii Sarg. (Verma & Reddy 2014) from the northwestern Himalayan region of India. In the last few years, we have conducted extensive surveys to the conifer forests of this region with the aim to document and preserve the diversity of Suillus and detect some new species of this genus. During our collection trips to northwestern Himalayas, Suillus species were found mostly in early monsoon season and Suillus sibiricus was observed to be the most frequently encountered and widely distributed Suillus species. During our expeditions to Himachal Pradesh in 2010 and 2011, two different specimens of a putative new species showing close morphological resemblance to S. decipiens (Peck) Kuntze were collected from mixed conifer forests. In the present study, morphological and molecular data obtained for these specimens are used to clarify the systematic position of the new species Suillus indicus within the genus.
Malt Extract (ME) agar (2% w/v) media supplemented with streptomycin (50 µg/ml) was used to isolate pure culture. Surface of fresh basidiomes collected was sterilized with rectified spirit and cut along the pileal surface with sterile surgical blades to expose the inner pileal context. Two to three pieces of clean fresh pileal context were transferred to each agar plate and incubated at 25°C for one month. Plates were checked weekly for any contamination and sub-culturing was done, if required. The pure culture isolated for S. indicus has been designated as "Isolate SNW02" and submitted to Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India, under the accession number MTCC 11955.

Morphological description of basidiomes
Standard methodology and terminology used for describing the basidiomes followed Corner (1972). Macro-morphological description, macro-chemical reactions, habitat, and plant association are based on detailed field notes of fresh basidiomes. Color codes in the macroscopic descriptions are from Kornerup and Wanscher (1978). Anatomical features were observed from dried material by reviving the sections either in water or in 3% potassium hydroxide (KOH). Measurements were made at 1000× magnification with a calibrated ocular micrometer on an Olympus light microscope (Olympus, Tokyo, Japan) by mounting the preparations in lactophenol cotton blue. Basidiospores and basidia were measured from the hymenophore of mature basidiomes. The spore measurements exclude the length of apiculus and the basidium length excludes the length of sterigmata. Quotient value (Q = L/ W) was calculated considering the mean value of length and width of 20 basidiospores. Microscopic line drawings of microstructures were made from rehydrated material with the aid of a camera lucida.

Molecular characterization
For phylogenetic analysis of S. indicus, genomic DNA from dried basidiome and culture was extracted based on Zhou et al. (1999). DNA extracts were then quantified with a Nanodrop 1000 spectrophotometer (Thermo Scientific, USA) and stored at −20°C until use. The ITS region of nrRNA was amplified with the universal primers ITS1 and ITS4 (White et al. 1990) using a polymerase chain reaction (PCR) Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions applied for the ITS region included an initial denaturation for 5 min at 94°C followed by 34 cycles of 1 min at 94°C, 1 min at 50°C, and 1.5 min at 72°C and a final extension of 7 min at 72°C. PCR products were purified with Qiaquick columns (Qiagen, Hilden, Germany), following the manufacturer's instructions and sequenced. The ITS sequences obtained from isolate SNW02 and basidiome of S. indicus have been deposited in the GenBank under the accession numbers KJ675500 and KJ675502, respectively.

Phylogenetic analysis
The ITS sequences acquired in this study were compared with those available in the GenBank database by using the BLASTn search algorithm. To find out the possible sister groups for newly sequenced taxon, a preliminary phylogenetic analysis was performed using MrBayes v.3.2.2 (http:// mrbayes.sourceforge.net/; Ronquist et al. 2012) by considering the ITS sequences representing all Suillus species as mentioned by Bruns et al. (2010). On the basis of preliminary analysis, a group comprising 27 Suillus species including our taxon was selected for phylogenetic analysis. Sixty five ITS sequences for other 26 Suillus species were retrieved from GenBank. Suillus triacicularis (KF977189), which phylogenetically belongs to the S. granulatus group (Bruns et al. 2010;Verma & Reddy 2014), was taken as an outgroup taxon. Alignment of the sequences was constructed using MAFFT ver. 7.0 (http://mafft.cbrc.jp/alignment/server/; Katoh & Standley 2013) and edited with BioEdit 5.0.6 (http://www.mbio.ncsu.edu/bioedit/; Hall 1999). The aligned data set has been deposited in TreeBASE (15673). Phylogenetic analysis on the resulting alignment was performed using Bayesian Inference (BI). A Bayesian analysis was implemented in MrBayes v.3.2.2 with two parallel runs each one consisting of four incrementally heated Monte Carlo Markov Chains. The analysis was run using Metropolis-coupled Markov Chains Monte Carlo search algorithm over 2,000,000 generations and the convergence of Bayesian analysis was observed by examination of the standard deviation of split frequencies <0.01. Trees were sampled every 100th generations resulting in total of 20,000 trees. The first 5000 trees, representing the burn-in phase of the analysis, were discarded and the remaining 15,000 trees were used to calculate posterior probabilities (PP) from the 50% majority rule consensus trees.

Phylogenetic inference
The PCR products amplified with ITS1 and ITS4 were 702 bp in length. Sequence analysis by BLAST revealed 93% similarity (query coverage of 99%) with unidentified Suillus sp. K91S8 (GQ267488) from New Zealand (Walbert et al. 2010) and 92% similarity (99% query coverage) with S. flavidus isolate FFP962 (JQ711908) from Canada (Jones et al. 2012). The alignment of ITS sequences of selected Suillus species resulted in a data matrix comprising 28 taxa and 713 characters including gaps. Bayesian analysis of ITS region for the selected Suillus group yielded a consensus tree (Figure 1) and divided the species broadly into five major clades (SuillusI, SuillusII, SuillusIII, SuillusIV, and SuillusV). The clade SuillusV consisted of two ITS sequences of the present study (S. indicus) and forms an independent clade in the group, which is well supported by the Bayesian posterior probability percentage (100%). The ITS sequences of Suillus spraguei (Berk. and Curt.) Kuntze derived from American (M91617, AF166524, and AF166525) and Chinese collections (AF166518, AF166520, and AF166522) are paraphyletic and subdivided into two different subclades. Chinese S. spraguei isolates were found to be sister to American S. decipiens isolates (L54079, AF166508, and AF166510) rather than American S. spraguei isolates. Contrastingly, a few pairs of Suillus species (S. flavidus and S. megaporinus, S. cothurnatus and S. subluteus, S. laricinus and S. grisellus) are not distinguished by the ITS locus.

Habitat
Solitary on humose soil under Cedrus deodara trees in mixed forest of Pinus wallichiana and C. deodara.

Commentary
The species is morphologically close to Suillus decipiens (Peck) Kuntze, but can be distinguished mainly by the absence of fibrillose squamules over the stipe, umbonate pileus and less numerous fibrillose squamules over the pileal surface. Microscopically, the species differs from S. decipiens by the presence of few 2spored basidia on hymenium and absence of caulocystidia on stipe surface.

Etymology
The specific epithet indicus is taken from Latin with reference to the new species being reported from the Indian region.

Discussion
In the Bayesian analysis of the Suillus group, selected for the phylogenetic inference in the present study, a few pairs of Suillus species (S. flavidus and S. megaporinus, S. cothurnatus and S. subluteus, S. laricinus and S. grisellus) are not distinguished by the ITS locus suggesting that there are limitations of using ITS locus for species-level identifications in the genus Suillus. This might be due to over-description (=synonymy) or the lack of ITS divergence between the sibling species (Bruns et al. 2010;Verma & Reddy 2014). The S. spraguei isolates (American and Chinese) analyzed showed remarkable heterogeneity and were divided into two different subclades. Chinese S. spraguei clade is sister to S. decipiens, whereas American S. spraguei is paraphyletic. On the basis of ITS data, Wu et al. (2000) also observed similar heterogeneity among the Chinese and American S. spraguei isolates.
The phylogenetic analysis of selected Suillus taxa clustered them mainly into five different clades (Figure 1). According to Smith and Thiers (1964) and Klofac (2013), these clades can be morphologically distinguished on the basis of few basic morphological characteristics such as presence or absence of veil, annulus, and glandular dots on the basidiomes. All of the clades, except SuillusIV, mainly comprise species exhibiting a well-developed veil or a false veil in young specimens either leaving an annular zone or a true annulus on the stipe or otherwise adhered to the pileal margin at maturity. On the other hand, the clade SuillusIV consists of a single species S. placidus (Bonord.) Singer, which has no veil and annulus at any stage of the development but glandular dots or smears are present on the stipe (Singer 1945;Smith & Thiers 1964). The first four clades comprising Suillus species with a well-developed veil or false veil in young specimens can be further divided on the basis of presence or absence of glandular dots on the stipe. The clade SuillusI mainly comprises species with glandular dots, while the clades SuillusII, SuillusIII, and SuillusV comprise only species that lack glandular dots on the stipe. Furthermore, the species in clades SuillusII, SuillusIII, and SuillusV can be classified on the basis of plant host to which they are found to be associated in nature. All the Suillus species in clade SuillusII are generally associated either with Larix, Pseudotsuga, Abies, or Picea (Smith & Thiers 1964;Klofac 2013) as compared to the species in clade SuillusIII (Kuntze 1898;Smith & Thiers 1964;Wu et al. 2000;Burchhardt et al. 2011;Klofac 2013), which are found to be associated only with Pinus species. Suillus indicus specimens (clade SuillusV) were collected from mixed forests of P. wallichiana and C. deodara under C. deodara trees suggesting its probable association with C. deodara, although its association with P. wallichiana cannot be denied. In fact, host shifts of basidiomycetes are considered to be major driving forces in the evolution process (Refrégier et al. 2008;Li et al. 2009Li et al. , 2011Rochet et al. 2011). Thus, the Suillus species of selected group are phylogenetically grouped on the basis of their basic morphological features and the host specificity. The Bayesian analysis clustered S. indicus as a distinct clade (SuillusV) in the group distinguishing it from all other Suillus species.
Morphologically, S. indicus come close to S. decipiens but differs considerably identifying it as a distinct species. The presence of umbo, less numerous/prominent fibrillose squamules over the pileal surface and absence of squamules over the stipe differentiate it from S. decipiens. Anatomically, the occasional presence of two-spored basidia and absence of caulocystidia distinguish the species from S. decipiens. Suillus spraguei is the next closest species, which is commonly referred to as Suillus pictus A.H. Sm. and Thiers although the name S. spraguei is used for one or even several disjunct populations of S. pictus in Asia (Wu et al. 2000;Burchhardt et al. 2011;Klofac 2013). The pileus color of young and fresh S. spraguei specimens is much redder than S. decipiens and S. indicus but the faded specimens strongly resemble S. decipiens. Furthermore, in contrast to S. indicus, S. spraguei also bears fibrillose squamules on the stipe. The combination of morpho-anatomical features and phylogenetic analysis of ITS sequences derived from fruiting bodies and culture distinguishes S. indicus as a distinct species and we herein report it as new to science.