New Species of Boletellus Section Boletellus (Boletaceae, Boletales) from Japan, B. aurocontextus sp. nov. and B. areolatus sp. nov.

We describe and illustrate two new species of Boletellus section Boletellus, B. aurocontextus sp. nov. and B. areolatus sp. nov., which are generally assumed to be B. emodensis. In this study, we reconstructed separate molecular phylogenetic trees of section Boletellus using the nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, the largest subunit (RPB1) and the second-largest subunit (RPB2) of nuclear RNA polymerase II gene and mitochondrial cytochrome oxidase subunit 3 (cox3) gene. We also examined the morphologies of B. emodensis sensu lato (s.l.) and other related species for comparison. The molecular phylogenetic tree inferred from the sequences of nuclear DNA (ITS, and combined dataset of RPB1 and RPB2) indicated that three genetically and phylogenetically well-separated lineages were present within B. emodensis s.l. These three lineages were also distinguished on the basis of the molecular phylogenetic tree constructed using the sequences of mitochondrial DNA (cox3), suggesting distinct cytonuclear disequilibria (i.e., evidence of reproductive isolation) among these lineages. Therefore, these three lineages can be treated as independent species: B. aurocontextus, B. areolatus, and B. emodensis. Boletellus aurocontextus and B. areolatus are also distinct from B. emodensis by the macro- and microscopic morphologies. Boletellus aurocontextus is characterized by a pileus with bright yellow to lemon yellow context, which can be observed through a gap in the scales, and basidiospores with relatively large length (mean spore length, 21.4 μm; quotient of spore length and width, 2.51). In contrast, B. areolatus is characterized by a pileus with floccose to appressed thin scaly patches, a stipe with pallid or pale cream color at the upper half, and basidiospores with relatively small length (mean spore length, 16.5 μm; quotient of spore length and width, 1.80).


Introduction
The genus Boletellus was originally described by Murrill in the family Boletaceae [1], and ca. 50 species of this genus have since been described worldwide [2]. Boletellus is an ectomycorrhizal fungus that forms a mutualistic relationship with host trees [3], although some species of this genus are often habitat on tree stumps or rotten wood. It is usually characterized by a yellow hymenophore and olive brown elongate to fusoid basidiospores with longitudinally winged basidiospores [4,5]. Seven sections were introduced in this genus by Singer [5], including those characterized by longitudinally winged spores (sections Boletellus, Chrysenteroidei, Ixocephali, and Dictyopodes), smooth spores (section Mirabilis), spores with imbedded short spines (section Allospori), and reticulate spores (section Retispori). Recent molecular phylogenetic studies indicate that Boletellus is polyphyletic [6][7][8] and thus the definition of this genus remains controversial.
Boletellus section Boletellus, in which the type species of the genus, i.e., B. ananas (M.A. Curtis) Murrill, is included, is one of the most well-defined sections among those defined by Singer [5]. This section unites taxa those have dry, reddish-pink to vinaceous-purple pileus finely covered with floccose or squamose scales and elongate to fusoid basidiospores with longitudinally winged ridges. Several species in this section have been reported from tropical areas of Southeast Asia [9], Central America [10,11], the warm temperate and subtropical areas of East Asia [12][13][14][15], North America [10], as well as Australia [16]. However, only a single species B. emodensis (Berk.) Singer, in this section was reported from Japan [12,13]; this species was first described as B. floriformis Imazeki [17], but later synonymized with B. emodensis [9].
Notably, several distinct morphological variations of B. emodensis have been reported [12,14]. Among those identified as B. emodensis in Japan [12], some specimens were clearly characterized by a pileus covered with relatively small scales, showing yellowish context through a gap in the scales. Moreover, distinct variations in basidiospores length and width have been reported within this species [14]. Therefore, we hypothesized that the species currently named as B. emodensis actually represents a complex composed of several different species.
In the present study, we reconstructed molecular phylogenetic trees using nucleotide sequences of both nuclear and mitochondrial DNA from B. emodensis s. l. and the related species to detect genetically and phylogenetically separated lineages within B. emodensis s. l. We also compared the morphological features of B. emodensis s. l. and the holotypes of related species, including B. ananas, B. dissiliens (Corner) Pegler & Young, B emodensis, and B. paradoxus (Massee) E.-J. Gilbert.

Field survey
From July 9 to August 24, 2009, 132 specimens of B. emodensis s. l. were collected from mixed forests of Castanopsis cuspidata and evergreen Quercus spp. and mixed forests of Pinus densiflora and deciduous Quercus spp. in Honshu and Kyushu, Japan. In addition, one specimen of B. cf. paradoxus was collected from lowland, mixed-dipterocarp forest in the Lambir Hills National Park, Sarawak, Borneo, on January 19, 2011. These specimens are deposited in the National Museum of Nature and Science, Tokyo (TNS).
We state that no specific permits were required for the described field studies in Japan, and the location was not privately-owned or protected in any way. Moreover, field study in the Lambir Hills National Park was conducted in accordance with a Memorandum of Understanding signed between the Sarawak Forestry Corporation and the Japan Research Consortium for Tropical Forests in Sarawak in November 2005. These field studies did not target endangered or protected species.

DNA extraction, PCR amplification, and sequencing
Total DNA was extracted from the tissue of voucher specimens listed in Table 1 using a cetyltrimethylammonium bromide (CTAB) method, as described previously [18]. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was amplified by PCR using the universal primers ITS1 and ITS4 [19]. For the amplifications of the largest subunit (RPB1) and the second-largest subunit (RPB2) of nuclear RNA polymerase II region, primer pairs RPB1-B-F/RPB1-B-R and RPB2-B-F1/RPB2-B-R [8] were used, respectively. Partial sequences of mitochondrial cytochrome oxidase subunit 3 (cox3) gene were also amplified by PCR using the Boletales-specific primers COX3st-F forward and COXst-R reverse [18]. The amplification of these regions was performed in a total 10-μL reaction mixture containing 1× Ampdirect buffer with dNTPs, 5 pmol of both forward and reverse primers, 0.5 U of BIOTAQ Hot Start DNA Polymerase (Shimazu, Kyoto), and 10-50 ng of total DNA. Cycling parameters for PCR: hot start at 95C for 10 min; followed by 40 cycles at 95C for 30 s, at 50-55C for 30 s, and at 72C for 60 s; and a final extension at 72C for 7 min. Before nucleotide sequencing, PCR products were purified using ExoSAP-IT (GE Healthcare) according to the manufacturer's instructions. The purified PCR products were sequenced using the same primers that were used for amplification. For ITS region, the universal primers ITS2 and ITS3 [19] were also used as internal primers for sequencing. Nucleotide sequencing was performed using an ABI 3130 automated sequencer (Applied Biosystems, Foster City, CA) with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), following the manufacturer's instructions.

Molecular phylogenetic analyses
After the sequences from the GenBank database were further added (Table 1), nucleotide sequences of the ITS region, RPB1, RPB2 and cox3 genes were aligned using the multiple sequence alignment program, Muscle [20]. The aligned sequences of the ITS region, RPB1 and RPB2 genes were cleaned using Gblocks v0.91b [21], allowing smaller final blocks and gap positions within the final blocks. The resulting alignments of ITS, RPB1 RPB2 and cox3 were deposited in the TreeBASE (Study ID: 17303; http://www.treebase.org/) and were separately subjected to molecular phylogenetic inference. According the previous report [8], closely related taxa of the ingroup were selected as outgroups ( Table 1).
The most appropriate evolutionary model was determined for each dataset by comparing different evolutionary models via the corrected Akaike information criterion (AICc) [22,23] using the Kakusan 4 [24]. Phylogenetic inference based on the maximum-likelihood (ML) method was performed using TREEFINDER 2011-March version [25] with shotgun searches. Shotgun searches were repeated until no improvement was observed in the likelihood value. The confidence of the internal branches from the resulting tree was tested by bootstrap (BS) analysis [26] with 1,000 replications. Bootstrapping was also performed on the basis of the maximum parsimony method (10,000 replications) using PAUP 4.0b10 (PAUP Ã ) phylogenetic inference package [27]. Pairwise ML distances were calculated using the TREEFINDER based on the most appropriate evolutionary model selected by Kakusan 4.

Observation of morphological characteristics
To compare morphological characteristics, specimens of B. emodensis s. l. that were collected from warm temperate forests in Japan were examined. The holotypes of B. ananas, B. dissiliens, B. emodensis, and B. paradoxus were also examined for a comparative study of morphological  features. Macro-and micro-morphological characteristics of basidiomes were described from fresh and dried specimens. The colors on the chart were composed of various percentages of the component colors: cyan, magenta, yellow, and black (CMYK). Microscopic observations were performed under an Eclipse 80i optical microscope (NIKON, Tokyo) with material (sections or fragments of the basidiome tissues) mounted in 5% potassium hydroxide (KOH) solution. Basidiospores measurements were performed at 1000× magnification under an Eclipse 80i optical microscope. The lengths and widths of 10 basidiospores were measured for each collection (for species with few available collections, 20 spores were measured per specimen). Mean and standard deviation (SD) of spore lengths and widths, and mean of the quotient of spore length and width (Q m ) were then calculated for each species. Between-group differences in spore lengths and quotients of spore length and width (Q) were analyzed based on the Steel-Dwass multiple comparison procedure using R ver. 3.0.1 statistical software. Basidiospores were also observed under a scanning electron microscope (SEM) (Philips XL-series; Eindhoven) at a magnification of 5000×.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank, from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix www.mycobank. org/MB. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Molecular phylogenetic inference
The ITS dataset consisting 17 taxa of Boletaceae, included 1041 nucleotide sites for each taxon or sample, of which 252 were parsimony informative, after cleaning the aligned sequences using Gblocks. The most appropriate model for the ITS region determined using Kakusan 4 was the K80 + G model. The combined data of RPB1 and RPB2 (hereafter, RPB1-RPB2) comprised 20 taxa and 1338 total characteristics, of which 200 were parsimony informative, after cleaning the aligned sequences using Gblocks. For the RPB1-RPB2 data, K80, TN93, K80 and TIM+G were selected as the most appropriate models for the first, second, third codons and intron partition of RPB1 gene, whereas J1, F81 and K80+G were selected for the first, second and third codons of RPB2 gene (a codon proportional model was used). The cox3 data set comprised five taxa and 571 total characteristics, of which 28 were parsimony informative. For the cox3 gene, J2 + G, F81, and TVM models were selected as the most appropriate models for the first, second, and third codons (a codon proportional model was used). Topology of the ML trees based on ITS, RPB1-RPB2 and cox3 sequences are shown in Fig 1A, 1B and 1C, respectively. Based on the molecular phylogenetic trees inferred from the ITS sequences (Fig 1A), three genetically and phylogenetically separated lineages were confirmed within B. emodensis s. l. (hereafter, B. emodensis, B. aurocontextus, and B. areolatus). Small variations of the ITS sequences were observed within these three lineages: the mean ML distances within the B. emodensis, B. aurocontextus and B. areolatus were 0.0032, 0.0024, and 0.0075, respectively. Monophyly of each lineage was supported by high BS values (ML/MP BS: 99/100, 100/100, and 83/98, respectively; Fig 1A). The ML tree of the ITS region indicated that B. emodensis and B. areolatus formed a well-supported clade (ML/MP BS: 99/100), although these two lineages were genetically differentiated (mean ML distance: 0.0306). The phylogenetic tree also strongly supported the grouping of B. ananas, B. aurocontextus, and B. cf. paradoxus (ML/MP BS: 99/ 95), although these lineages were genetically separated.
Topology of the ML tree inferred from sequences of the cox3 gene was almost concordant with those of the nuclear DNA (Fig 1C). No variations of the cox3 sequences were observed within B. emodensis, B. aurocontextus, and B. areolatus. Notably, the three genetically separated lineages, i.e., B. emodensis, B. aurocontextus, and B. areolatus, were distinguished by cox3 sequences, concordant with the results of the ITS and RPB1-RPB2 sequences (Fig 1A and 1B), although there were smaller genetic variations in the cox3 sequences. Etymology: aurocontextus, Latin, golden context, referring to the yellowish context. Diagnosis: Boletellus aurocontextus sp. nov. is characterized by the following unique characters: a pileus with bright yellow to lemon yellow context, which can be observed through a gap in the scales, and relatively large and elongated basidiospores, measuring 18.5-24.5 × 7.5-10 μm (Q m = 2.51).
Description: Pileus 6-12 cm in diameter, at first convex then plano-convex, surface dry, finely covered with squamulose to verrucose scales, often rimulose to rimulose-areolate at maturity; scales up to 1.  Habitat: Solitary or gregarious on the ground, tree stumps or rotten wood in mixed forests of Pinus densiflora and Quercus serrata. Putative ectomycorrhizal fungi.
Distribution: Honshu, Japan (presumably distributed in mixed conifer-broad-leaved forests in East Asia).
Remarks: This species is distinguished from B. emodensis and other related species by the appearance of yellow or lemon yellow pileus context. This feature is particularly evident at the pileus surface on which the bright yellow to lemon yellow context can be observed through a gap in the scales. This species is also characterized by relatively large and elongated basidiospores measuring 18.5-24.5 × 7.5-10 μm (Fig 5A and 5B). Moreover, unlike B. emodensis, rose-red to purplish red coloration of the pileus scales likely persists throughout the stages of basidioma development. Large and elongated basidiospores of the present species are similar to those of B. ananaeceps (Berk.) Singer discovered in Australia, which has basidiospores measuring 16-26 × 6-11 μm [4]. However, the present species is distinguished from B. ananaeceps by the morphological feature of the pileus surface as described above. The lectotype of B. annamiticus (Pat.) E.-J. Gilbert (Basionym: Strobilomyces annamiticus Pat., 1909), which was discovered in Vietnam and is considered a synonym of B. emodensis [14,28], also has relatively large and elongated basidiospores measuring 21-24 × 7-8.5 μm, Q = 2.63-3.29 [14]. However, the lectotype is apparently too young to assess the spore size, and other important morphological characteristics of the present species have not been detected. Therefore, we conclude that the name "B. annamiticus" cannot be applied to the present species.
Distribution: Kyusyu and Honshu in Japan (presumably distributed in evergreen oak forests in East Asia). l. and the related species that were measured at 1000× magnification under an Eclipse 80i optical microscope. The box plot displays median ("bold line"), first and third quartile ("hinges"), and 95% confidence interval ("notches"). The number of spores examined is shown in parentheses. An asterisk after a species name indicates that spore measurement was performed only for the holotype. Different letters above the box plot (e.g., a, b, c, and d) indicate a statistically significant difference between groups. Remarks: This species is morphologically similar to B. emodensis and B. dissiliens, but it shows a pileus with floccose to appressed thin scaly patches and a stipe with pallid or pale cream color at the upper half. This species is also characterized within the Boletellus section by smaller and broader basidiospores measuring 14-20 × 7.5-11 μm (Fig 5A and 5B). context through a gap in the scales; margin widely appendiculate with a membranous veil concolorous with pileus surface (Fig 9). Stipe 6-18 cm long, 10-20 mm thick, almost equal, straight or curved, longitudinally fibrillose, rose-red to fuscous red, sometimes yellowish near the apex (Fig 9). Tubes up to 18    45 × 9-15 μm, clavate, 4-spored; sterigmata 4-6 μm long. Cheilo-and pleurocystidia 9--20 × 30-80 μm, numerous, clavate, utriform to fusoid, hyaline or with brownish to fuscous contents, thin-walled. Scales of pileipellis composed of subradially arranged hyphae, 5-15 μm wide (terminal cells: 6-20 μm), cylindrical, reddish to fuscous brown. Pileus trama composed of interwoven hyphae, 7-20 μm. Hyphae of stipitipellis, cylindrical, 4-12 μm wide. Caulocystidia scattered, 33-80 × 8-15 μm, cylindrical, clavate, to fusiform. All hyphae without clamp connections.  lemon yellow context through gaps in the scales and relatively large and elongated basidiospores. Boletellus areolatus is morphologically similar to B. emodensis particularly in coloration of the pileus surface, but the former is characterized by a pileus with flocosse to appressed thin scaly patches, a stipe with pallid or pale cream color at the upper half, and less elongated basidiospores. These characteristics are also useful to distinguish this species from B. dissiliens, which has relatively small basidiospores, as in this species.
Nucleotide sequences from specimens of B. emodensis collected in the type locality (India, Darjeeling) were not available, and thus there remains a possibility that the Japanese specimens identified as "B. emodensis" might be distinct from B. emodensis. Nevertheless, there are no reasonable grounds to distinguish them in the present state, because no significant morphological differences were detected between the Japanese specimens and the holotypes of this species. Moreover, evergreen oaks (e.g., Quercus and Castanopsis) were predominant in both of tropical upper montane forests around the type locality [33] and evergreen temperate forests in Japan, suggesting that potential host plants were presumably similar between these regions.
The geographical distribution of B. aurocontextus, B. areolatus, and B. emodensis may be restricted to temperate and subtropical regions of East Asia, Southeast Asia, and South Asia, as in case of most ectomycorrhizal fungal species that are observed in Japan [34]. Boletellus aurocontextus can be characterized by the distribution pattern and habitat. Our field survey implies that this species likely inhabits mixed forests of Pinus and deciduous Quercus species in Japan, although most species of the same section have been reported from broad-leaved evergreen forests in warm temperate, subtropical, and tropical areas [8,9]. However, further field surveys are required for revealing the distribution pattern and habitat (or host-fungus association) of this species.
Key to the species of Boletellus section Boletellus