Taxonomy and Phylogeny of Polyporus Group Melanopus (Polyporales, Basidiomycota) from China

Melanopus is a morphological group of Polyporus which contains species with a black cuticle on the stipe. In this article, taxonomic and phylogenetic studies on Melanopus group were carried out on the basis of morphological characters and phylogenetic evidence of DNA sequences of multiple loci including the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (EF1-α), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), and β-tubulin gene sequences (β-tubulin). The phylogenetic result confirmed that the previously so-called Melanopus group is not a monophyletic assemblage, and species in this group distribute into two distinct clades: the Picipes clade and the Squamosus clade. Four new species of Picipes are described, and nine new combinations are proposed. A key to species of Picipes is provided.


Introduction
Melanopus Pat. was established by Patouillard [1] as a specific genus which including stipitate polypores with black stipes. Then it was accepted as a synonym of Polyporus P. Micheli ex Adans. [2]. Núñez and Ryvarden [3] treated genus Melanopus as an infrageneric group of Polyporus. They defined this group with following characters: basidiocarps coriaceous, tough when dry, context thin, stipe with a black cuticle, skeleto-binding hyphae mostly solid and narrow when mature, basidiospores medium size to large (6-12 × 2-4 μm). Eleven species of Polyporus were accepted as members of group Melanopus by Núñez and Ryvarden [3]. Among these species, P. mikawai Lloyd was transferred into Neofavolus Sotome & T. Hatt. as N. mikawai (Lloyd) Sotome & T. Hatt. based on morphological and phylogenetic analyses [4].
For RPB1 and RPB2, the PCR procedure was used as following: initial denaturation 94°C for 2 min, followed by 9 cycles at 94°C for 45 s, 60°C for 45 s (minus 1°C per cycle) and 72°C for 1min 30 s, then followed by 36 cycles at 94°C for 45 s, 53°C for 1 min and 72°C for 1min 30 s, finally with a final extension at 72°C for 10 min.
All PCR products were purified and sequenced in the BGI (Beijing Genomics Institute), China, with the same primers. All newly generated sequences were deposited at GenBank and listed in Table 1. Trametes conchifer (Schwein.) Pilát was selected as outgroup.
Phylogenetic analyses were done as in Zhao et al. [23][24][25]. All gene sequences were initially aligned separately using Clustal Omega [26] and then manually adjusted. One thousand partition homogeneity test (PHT) replicates of the combined dataset were tested by PAUP 4.0 beta 10 [27] to determine whether the partitions were homogeneous. The best-fit evolutionary model was selected by hierarchical likelihood ratio tests (hLRT) and Akaike information criterion (AIC) in MrModeltest 2.2 [28] after scoring 24 models of evolution by PAUP 4.0 beta 10. The best maximum likelihood (ML) phylogenetic bootstrap values (ML-BS) obtained from 1000 replicates were performed using RAxmlGUI [29], while the ML topology, maximum parsimony (MP) tree and bootstrap values (MP-BS) were performed using PAUP 4.0 beta 10. Bayesian phylogenetic inference and Bayesian posterior probabilities (BPPs) were performed with MrBayes v3.2 [30]. Four Markov chains were run for 5,000,000 generations until the split deviation frequency value <0.01, and sampled every 100th generation. All trees were viewed in FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).

Nomenclature Acts
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 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  31 Pi. tubaeformis      [31] and accepted by most succeeding researchers [3,4,7,8,32]. Morphologically, P. tuberaster was treated as a members of group Polyporus along with P. squamosus, however P. umbellatus was put into group Dendropolyporus [6].
Favolus clade and neofavolus clade are well supported as monophyletic lineages respectively (both 100/100/100 for Favolus and Neofavolus). These two clades are separately composed of species from genera Favolus and Neofavolus.

Taxonomy
Picipes Zmitr. et Kovalenko Basidiocarps annual, stipitate, of polyporoid morphotype; pilei fan-shaped to circular or infundibuliform, covered with hard cuticle, glabrous; corky to coriaceous when fresh and hard when dry; stipe usually covered with brownish to black cuticle when mature; pores round or angular; hyphal system dimitic with generative and skeleto-binding hyphae, uninflated; generative hyphae with clamp connections or simple septa; skeleto-binding hyphae strongly branched in trama; hyphae in cuticle bearing clamp connections or not, thick-walled with a wide lumen, usually unbranched; basidiospores oblong to cylindrical or fusiform, smooth, hyaline, less than 13 μm long and 5 μm wide; mainly growing on woods and causing a white rot, occasionally on ground or grass roots.  Fruitbody: Basidiocarps annual, centrally stipitate, solitary, coriaceous when fresh and tough when dry. Pilei infundibuliform, up to 5.5 cm wide and 2.5 mm thick. Pileal surface glabrous, reddish-brown to black in the center and becoming light ivory to pale-brown towards the edge in young specimens, black upon the whole pileus upon the old ones, with radially aligned stripes; margin straight when fresh and involute upon drying. Pore surface white when fresh, cream to buff upon drying; pores round to angular, 3-6 per mm; dissepiments thin, entire to slightly lacerate. Context white to buff, becoming woody hard upon drying, up to 1 mm thick. Tubes concolorous with pore surface, decurrent on the stipe, less than 1.5 mm thick. Stipe slender, bearing a black cuticle, wrinkled, 2.2 cm long and 5 mm in diam.
Basidiospores: Basidiospores cylindrical, thin-walled, colorless, smooth, usually bearing one to three guttules, IKI-, CB-, (5.8-)6.6-7. Etymology: subtropicus (Lat.): referring to the geographic distribution in subtropical regions. Fruitbody: Basidiocarps annual, laterally stipitate, gregarious, coriaceous when fresh and tough when dry. Pilei fan-shaped to semicircular, up to 4.8 cm wide and 2.5 mm thick. Pileal surface glabrous, black towards the base and becoming reddish-brown to orange-brown towards the edge when fresh, frequently becoming black to chestnut upon the whole pileus, sometimes brown-beige or pastel-yellow towards the edge when dry; margin straight when fresh and straight or slightly involute upon drying. Pore surface white when fresh, white to brown-beige when dry; pores angular to subcircular, 8-9 per mm when young and becoming 5-7 per mm when aged; dissepiments thin, entire to slightly lacerate. Context white to buff and woody hard upon drying, up to 2 mm thick. Tubes white when fresh and white to brown-beige upon drying, less than 1 mm thick, decurrent on one side of the stipe. Stipe very short or forming a flattened base, bearing a black cuticle, up to 5 mm long and 5 mm in diam. Hyphal structure: Hyphal system dimitic; generative hyphae bearing clamp connections, colorless, thin-walled; skeleto-binding hyphae colorless, thick-walled, with arboriform branches and tapering ends, IKI-, CB+; tissue unchanged in KOH.
Fruitbody: Basidiocarps annual, centrally to laterally stipitate, solitary, coriaceous when fresh and tough when dry. Pilei irregularly semicircular or ellipsoidal, with shallow central depression, up to 7.8 cm wide and 2 mm thick. Pileal surface glabrous, reddish-brown to chestnut in the center or towards the stipe and changing to signal-orange to clay-brown towards the edge when dry in young specimens, becoming reddish-brown to chestnut upon whole surface in mature ones, with radially aligned stripes; margin involute upon drying. Pore surface buff to festucine when dry, shining; pores round to angular, 4-6 per mm; dissepiments thin, entire to lacerate. Context white to buff, becoming woody hard upon drying, up to 1 mm thick. Tubes concolorous with pore surface, less than 1.5 mm thick, sometimes decurrent on one side of stipe. Stipe bearing a terra-brown to black cuticle, up to 1.2 cm long and 3.5 mm in diam. Hyphal structure: Hyphal system dimitic; generative hyphae bearing clamp connections, colorless, thin-walled; skeleto-binding hyphae colorless, thick-walled, with arboriform branches and tapering ends, IKI-, CB+; tissue unchanged in KOH.
Etymology: tibeticus (Lat.): referring to the locality of the type specimens in Tibet. Fruitbody: Basidiocarps annual, centrally to laterally stipitate, solitary or scattered, coriaceous when fresh and tough when dry. Pilei irregular fan-shaped or semicircular, usually shallow towards the stipe, up to 10.5 cm wide and 1 mm thick. Pileal surface glabrous, orangebrown to brown when fresh, becoming orange-brown to reddish-brown or blackish-brown upon drying, more or less radially wrinkled when dry; margin straight or slightly involute when fresh and involute upon drying. Pore surface white when fresh, becoming buff to yelloworange when dry; pores angular to subcircular, 6-9 per mm; dissepiments thin, entire to slightly lacerate. Context white when fresh, becoming woody hard upon drying, up to 0.5 mm thick. Tubes concolorous with pore surface, less than 0.9 mm thick, decurrent. Stipe bearing a black cuticle, wrinkled, up to 4.5 cm long and 9 mm in diam.
Picipes subtropicus was found in subtropical areas of China. It can be identified by a continuous variation in pore size, bright pileal surface color, short black stipe-like base and medium cylindrical basidiospores (5.1-6.2 × 2.2-2.7 μm). In phylogenetic analysis (Fig 1), it did not cluster with any other species in our study set. Polyporus dictyopus also has chestnut upper surface when aged, in addition, its pore size and pore surface are similar to Pi. subtropicus; but P. dictyopus has longer and thicker stipe (up to 3 cm long and 1 cm thick), larger basidiospores (7-8.5 × 2.5-4 μm) and pantropical distribution [3]. Picipes badius share similar basidiocarps and pore size with P. subtropicus; but it differs in its larger basidiospores (7.5-9.5 × 3-3.5 μm), simple-septate generative hyphae and absence of cystidioles [3]. Picipes baishanzuensis was also found in subtropical areas of China, but its infundibuliform pilei, slender stipe and lager basidiospores (6.6-7.9 × 2.5-3.1 μm) are quite different from Pi. subtropicus.
Picipes admirabilis was was initially collected on wood of apple trees in northeastern United States [38]. Lloyd considered that Pi. admirabilis is a variety of P. varius or belongs to group Melanopus for its black stipe [39,40]. Núñez (Fig 1). Morphologically, Pi. admirabilis has a long (up to 8 cm) and pale buff to black stipe, firm-corky basidiocarps and uninflated hyphae [3]. Based on our specimen collected from northeast of China, the balck stem, corky basidiocarps, uninflated hyphae and strongly branched skeleto-binding hyphae in trama are more similar to species of Picipes.
Picipes is showed to be a monophyletic group based on the 8-gen-squences data analysis, and sixteen species are included in this clade (Fig 1). Among these species, Pi. badius was previously put into Royoporus A.B. De by De [43] for its simple septa. But our analysis strongly supported Pi. badius as a member of Picipes. Corner [44] considered that Pi. badius, P. dictyopus and Pi. melanopus were conspecific, but our analysis showed that they are three different species. Zmitrovich & Kovalenko [9] considered that Picipes only includes the species with small pores (more than 5 per mm), cylindrical basidiospores and lignicolous habit. According to our study with more samples, we find several species with large pores, oblong basidiospores and terrestrial habit are also members of Picipes.
Polyporus umbellatus is a particular species that merely grows on the ground from a sclerotium close to stumps of hardwoods, and characterized by numerous stipitate pilei arising from a common, strongly branched stipe [3]. It was morphologically treated as a member of Polyporus group Dendropolyporus [3]. Phylogenetically, P. umbellatus was reported as a distinctive species that could not cluster with any other species [7,9]. Zmitrovich & Kovalenko [9] transferred P. umbellatus into genus Cladomeris Quél. But in our analysis, P. umbellatus strongly clusters with P. tuberaster and P. hapalopus in the core polyporus clade (Fig 1). Morphologically, basidiomata of the three species are fleshy when fresh and brittle upon drying [3,45]. Besides, all of them usually have inflated skeleton-binding hyphae up to 17 μm wide, generative hyphae dominant to almost monomitic in trama, cylindrical basidiospores and light-colored stipes. Thus, the core polyporus clade is treated as a natural group of Polyporus.
Species in the squamosus clade together with several species of Datronia Donk were transferred into Cerioporus [9]. Zmitrovich & Kovalenko [9] considered that Cerioporus spp. have inflated skeleto-binding hyphae, but the skeleto-binding hyphae of P. guianensis and P. leprieurii are uninflated and up to 5 μm wide [3]. So we prefer to maintain their previously taxonomic names here.
Previous phylogenetic analyses showed that favolus clade and neofavolus clade did not gather together and favolus clade has closer relationships with P. tuberaster [4,8]. But our phylogenetic result (Fig 1) shows that Favolus spp. closely related to Neofavolus spp. rather than other Polyporus spp. Seelan et al. [46] estimated that the ancestral state for Neofavolus is angular pores. They transferred Lentinus suavissimus Fr., a gilled species with sub-poroid lamellae, into genus Neofavolus. Zmitrovich & Kovalenko [9] also treated Lentinus suavissimus as a member of Neofavolus and named it N. suavissimus (Fr.) Zmitr. et Kovalenko, but this name is illegitimate because of its earlier homonym N. suavissimus (Fr.) J.S. Seelan.
Our phylogenetic analysis based on multiple gene sequences data of ITS, nLSU, EF1-α, mtSSU, β-tubulin, RPB1, RPB2 and nSSU suggested that species of group Melanopus distribute into two different clades: picipes clade and squamosus clade. This conclusion verified the view that Melanopus group is not a monophyletic assemblage of dark-stiped Polyporus species, and whether having black cuticle on stipe or not, is not a sufficient feature to define the natural Melanopus group. In our study, sixteen species including four new species of Picipes are recognized. A key to species of Picipes is provided.