Circumscription and Taxonomic Arrangement of Nigroboletus roseonigrescens Gen. Et Sp. Nov., a New Member of Boletaceae from Tropical South–Eastern China

Nigroboletus is proposed as a novel genus in family Boletaceae, subfamily Boletoideae, to include N. roseonigrescens, a new boletoid species from tropical environment in south–eastern China. Detailed morphological description, color pictures of both fresh basidiomes in habitat and dried material along with photomicrographs and line drawings of the main anatomical features are provided, supported by a comprehensive phylogeny based on multigene molecular analysis (nrITS, nrLSU, rpb1, rpb2 and tef1-α datasets). Taxonomic placement and evolutionary relationships of Nigroboletus are investigated.


Introduction
In recent times Chinese bolete diversity has turned out to be quite impressive and much richer than formerly thought. As a result, several unnamed genera and species belonging to the order Boletales have been documented as new to science over the past decade, based on bio-geographic evidence and mostly supported by multi-locus molecular phylogenetic inferences . However, as outlined by a number of recent publications, boletes heritage in China is currently far from being fully understood and a large amount of fungal taxa are still waiting to be uncovered or formally recognized [24][25][26]. As a matter of fact it is relatively easy to find out boletoid species which have been previously misidentified or even completely overlooked, especially in tropical or subtropical climates where fungi are underdocumented and mycological research has significantly been less extensive than in northern temperate and boreal regions [27][28][29]. Based on recent sampling from tropical south-eastern China (Guangdong province), a new member of family Boletaceae, subfamily Boletoideae ( [26]; corresponding to the "anaxoboletus" group in Nuhn et al. [30]), has been recovered and carefully examined; the new taxon centrifuged again for 2 min and dried. It was finally resuspended in 200 μL ddH 2 O. PCR amplification was performed with the primers ITS1F and ITS4 [33,34] for the nrITS region, while LR0R and LR5 [35] were used to amplify the nrLSU region, reverse of bRPB2-6R2 [36] and bRPB2-7.1R2 (5' -CCCATNGCYTGYTTVCCCATDGC -3') or RPB2-B-F1 and RPB2-B-R [26] for the RNA polymerase II second largest subunit, rpb2, RPB1-Af and RPB1-Cr [37] or RPB1-B-F and RPB1-B-R [26] for the RNA polymerase II largest subunit, rpb1, and finally EF1-983F and EF1-1567R [38] or EF1-B-F1 and EF-B-R [26] for the translation elongation factor 1-α (tef1-α) gene. PCR reactions were performed under a program consisting of a hot start at 95°C for 5 min, followed by 35 cycles at 94°C, 54°C and 72°C (45, 30 and 45 s respectively) and a final 72°C step for 10 min. PCR products were checked in 1% agarose gel, and positive reactions were sequenced with primer ITS4. Chromatograms were checked searching for putative reading errors, and these were corrected. The sequences were deposited in GenBank (Table 1).

Sequence alignment, data set assembly and phylogenetic analyses
The sequences obtained in this study were checked and assembled using Geneious v5.3 [39] and compared to those available in GenBank database by using the Blastn algorithm. A general combined Maximum Likelihood tree including all the Boletaceae sequences present in Gen-Bank and UNITE (http://unite.ut.ee/) databases was generated to detect the phylogenetic position of our collections in the major clades of Boletaceae as circumscribed by Wu et al. [26] (tree not shown). Consequently, phylogenetic analyses were restricted to the major clade including Nigroboletus sequences (subfamily Boletoideae, Figs 1 and 2). Alignments were generated for each ITS, LSU, rpb1, rpb2 and tef1-α dataset with MAFFT [40] with default conditions for gap openings and gap extension penalties. Alignments were imported into MEGA 6.06 [41] for manual adjustment. The best-fit substitution model for each alignment was estimated by the Bayesian information criterion (BIC) with jModelTest 2.0 [42] to provide a substitution model for the alignment. GTR+G model was chosen for the ITS alignment, while TrN+G was selected for LSU, 010230+G for rpb1, 010023+G for rpb2 and TIM3ef+G for tef1-α alignments. Two phylogenetic analyses were performed: the first large phylogenetic analysis, based on a combined LSU/rpb1/rpb2/tef1-α dataset, was focused on the intergeneric position of Nigroboletus in the Boletoideae as delimited by Wu et al. [26]. According to the results by Wu et al. [26], species of Austroboletus (Austroboletoideae) were chosen as outgroup taxa for the combined dataset. The second phylogenetic analysis based only on a ITS dataset was restricted to the taxa closely related to Nigroboletus: members of the rubellus clade (Xerocomus rubellus and X. communis) were selected as outgroup taxa.
Phylogenetic hypotheses were constructed with Bayesian inference (BI) and Maximum likelihood (ML) criteria. The BI was performed with MrBayes 3.2.2 [43] with four incrementally heated simultaneous Monte Carlo Markov chains (MCMC) run for 10 000 000 generations, under the selected evolutionary model. Trees were sampled every 1000 generations, resulting in overall sampling of 10 001 trees; the first 2500 trees (25%) were discarded as burn-in. For  [44] with 1000 bootstrap replicates [45] using the GTRGAMMA algorithm to perform a tree inference and search for optimal topology. Support values from bootstrapping runs (MLB) were mapped on the globally best tree using the ''-fa"option of RAxML and ''-b 12345" as a random seed to invoke the novel rapid bootstrapping algorithm. BI and ML analyses were run on the CIPRES Science Gateway web server [46]. Only BPP values exceeding 0.75 and MLB over 50% are reported in the resulting trees (Figs 1 and 2). Branch lengths were estimated as mean values over the sampled trees. Alignments and phylogenetic trees are available at TreeBASE (www.treebase.org, submission number S17886).

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 analysis
Both Bayesian and Maximum likelihood analyses produced the same topology; therefore only the Bayesian trees with both BPP and MLB values are shown (Figs 1 and 2). The combined data matrix (focused on the Boletoideae) comprised 256 sequences (including 247 from Gen-Bank). corresponding to 76 collections. The three newly sequenced collections of Nigroboletus roseonigrescens, together with Xerocomellus sp. HKAS56311 (BPP = 0.75; MLB = 77), occupy a sister position (BPP = 1; MLB = 88) to the clade consisting of Xerocomellus species (BPP = 1; MLB = 80) (Fig 1). The ITS data matrix comprised 57 sequences (including 16 from GenBank and 38 from UNITE). In the ITS analysis Nigroboletus is clearly distinct from Xerocomellus and from two Xerocomellus species, X. armeniacus and X. persicolor, which form an independent clade (here referred as the armeniacus clade) (Fig 2). Original diagnosis: Basidiome stipitate-pileate with tubular hymenophore, epigeal, evelate, medium-small sized; pileus convex to applanate, subtomentose to glabrous; hymenophore very thin, poroid, adnate to subdecurrent, yellow to olive-yellow; stipe solid, dry, smooth to minutely pruinose-punctate, reticulation absent; context firm, yellowish; tissues turning dull grayish to blackish throughout when injured or exposed; taste mild; spore print olive-brown; spores smooth, broadly ellipsoid to subovoid; pleuro-, cheilo-and caulocystidia present; pileipellis consisting of subparallel to loosely interwoven erect hyphae; hymenophoral trama bilateraldivergent of the . Etymology: the specific epithets "roseo" (pink) and "nigrescens" (blackening) are derived from Latin and refer to the pinkish color of pileus and stipe which turn blackish when damaged.

Taxonomy
Original diagnosis: Basidiomes characterized by xerocomoid habit, pinkish pileus, yellowish to pinkish-orange stipe surface, white basal mycelium, yellowish context, tissues turning dull grayish to blackish throughout when injured or exposed, broadly ellipsoid to subovoid, smooth spores, pileipellis consisting of subparallel to loosely interwoven erect hyphae with subtle granular or zebra-pattern epiparietal encrustations and presence of congophilous plaques on cystidial wall.

Nigroboletus phylogeny and intergeneric relationships
The combined and ITS molecular analyses (Figs 1 and 2) clearly indicate that Nigroboletus represents a new and independent phyletic line within the subfamily Boletoideae in sister position to the genus Xerocomellus Šutara, typified by X. chrysenteron (Bull.) Šutara. Nigroboletus shares with Xerocomellus the medium-small size, dry subtomentose to velutinous pileus surface, wide, roundish to angular and radially arranged yellowish to olive-yellow pores, stipe without reticulate ornamentation and microscopically the trichodermic pileipellis with encrusting pigment, fertile stipe surface and the similar hymenophoral trama structure. Xerocomellus is morphologically delimited from Nigroboletus by the longer, ellipsoid-fusoid to subfusoid spores with more or less pronounced suprahilar depression, the lateral stipe stratum usually absent or, whenever present, very thin (up to 40 μm thick at most), the absence of congophilous plaques and the occurrence in North boreal and temperate habitats [47]. Apart from the comparative features listed above with respect to Xerocomellus s.l., the armeniacus clade further shares with Nigroboletus the presence of congophilous plaques on hyphal surface, whereas the differential characters also include the bright yellow-ochraceous to orange-yellow and unchangeable context in the stipe base and the dark blue-green reaction with FeSO 4 on pileus surface and in the stipe base context [47,48].

Taxonomic circumscription of N. roseonigrescens
Given the geographical range of the tropical belt and despite recent advances in mycological research, our present knowledge of tropical boletes is still scarce. As a result, even though boletes are thriving in pantropical habitats, only a few of them have been recognized and named to date.
The new taxon Nigroboletus roseonigrescens, originating from tropical south-eastern China, revealed to be morphologically and phylogenetically unique amongst Boletaceae and exhibits several clear-cut features. The diagnostic macromorphological features of N. roseonigrescens include the following combination: (1) Small to medium-small sized basidiomes, (2) pastel pink pileus, (3) very thin tubes, (4) yellow to pinkish-orange pruinose stipe, (5) pale cream to yellowish context, (6) white basal mycelium. Noteworthy is the color change of the tissues to blackish, which is likely to be addressed to the presence of abundant oleiferous hyphae and cystidia with dark brown, dextrinoid content spreading out after bruising or sectioning. Other distinctive features are: the shape and size of the spores as the small, broadly ellipsoid to subovoid outline without suprahilar depression is unlike most of Boletaceae and is conversely more reminiscent of the spore morphology found in Paxillaceae [49]; the presence of congophilous plaques on cystidial wall. Xerocomellus sp. (HKAS56311) from China might represent an additional species of Nigroboletus (Fig 1), but more material is required to elucidate the status of the taxon.
The new species seems to be fairly common and abundant but at present it is premature to determine the frequency and bio-geographical boundaries of Nigroboletus, even though a south-eastern Asian distribution in tropical and subtropical regions appears quite likely. We therefore expect N. roseonigrescens to be found at other localities in southern China and adjacent regions.