Resolution of phylogenetic position of Nigrofomitaceae within Hymenochaetales (Basidiomycota) and Nigrofomes sinomelanoporus sp. nov. (Nigrofomitaceae) from China

Abstract The family Nigrofomitaceae has been considered to be a member of Polyporales and a synonym of Polyporaceae for a long time. However, no molecular evidence supports this taxonomic opinion. For the first time, Nigrofomitaceae is included in a phylogenetic analysis, which shows that this family is separated from Polyporales and nested within Hymenochaetales as a distinct lineage from four well-known families, viz. Hymenochaetaceae, Neoantrodiellaceae, Oxyporaceae and Schizoporaceae. Therefore, Nigrofomitaceae is treated as the fifth family of Hymenochaetales. Nigrofomes melanoporus, the type species of Nigrofomitaceae, was considered to have a pantropical distribution. However, from both morphological and phylogenetic perspectives, the Chinese specimens labelled as N. melanoporus are found not to be conspecific with the specimens of N. melanoporus from Costa Rica, close to the type locality in Cuba. These Chinese specimens are thus described as a new species Nigrofomes sinomelanoporus. The species diversity of Nigrofomes in pantropical region is discussed.


Introduction
Polyporales, accommodating about 2000 species, is one of the largest orders of wood-inhabiting fungi within Agaricomycetes, Basidiomycota (Kirk et al. 2008). The taxonomy of members of Polyporales has been extensively studied, resulting in the emergence of an enormous number of new genera and species worldwide (e.g. Cao et al. 2012, Miettinen and Rajchenberg 2012, Zhou and Dai 2012, Dai et al. 2014, Spirin et al. 2016. With the aid of molecular phylogeny, Binder et al. (2005) for the first time recovered four clades in Polyporales, viz. core polyporoid clade, antrodia clade, phlebioid clade and residual polyporoid clade. Later, an additional four lineages, viz. the family Fragiliporiaceae, the genus Grifola Gray, gelatoporia clade (or cinereomyces clade) and tyromyces clade emerged (Tomšovský et al. 2010, Miettinen and Larsson 2011, Miettinen and Rajchenberg 2012. At the family level, 41 legitimate names are currently considered to belong to Polyporales (Binder et al. 2013. Of these members, several families, including Nigrofomitaceae, have not yet been included in any phylogenetic analysis.
Nigrofomitaceae was erected to accommodate the monotypic genus Nigrofomes Murrill (Jülich 1981). This genus was typified by Nigrofomes melanoporus (Mont.) Murrill that was originally described from Cuba, tropical America (Murrill 1904). Recently, Hattori and Sotome (2013) combined Trametes nigrivinea Corner typified by a specimen from Papua New Guinea to Nigrofomes as N. nigrivineus (Corner) T. Hatt. & Sotome, bringing the members in this genus to two. Nigrofomitaceae was long treated as a synonym of Polyporaceae (Kirk et al. 2008, Ryvarden 2015, although the phylogenetic position of either species of Nigrofomitaceae remains unclear.
In the present study, Costa Rican and Chinese specimens of Nigrofomes melanoporus are analysed from a phylogenetic perspective for the first time and the phylogenetic affinity of Nigrofomitaceae is clarified. Moreover, the Chinese specimens labelled as Nigrofomes melanoporus are found not to be conspecific with the Costa Rican specimens of N. melanoporus and are herein described as a new species.

Materials and methods
Morphological examination. The studied specimens were originally deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC) in China and the private herbarium of Josef Vlasák (JV) in the Czech Republic. In addition, the duplicates of all these specimens have been preserved at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP) in China.
Macroscopic characters of the specimens were observed by the naked eye and also with the aid of a stereomicroscope. The microscopic procedure followed Kan et al. (2016).
Specimen sections were mounted in Cotton Blue (CB), Melzer's reagent (IKI) and 5% potassium hydroxide (KOH) and examined using a Nikon Eclipse 80i microscope at magnification up to 1000×. Measurements were taken in CB. The basidiospore size variation was presented by placing 5% of measurements from each end of the range in parentheses. Special colour terms followed Petersen (1996). Drawings were made with the aid of a drawing tube. The following abbreviations are used in the text: L = mean basidiospore length (arithmetic average of all measured basidiospores), W = mean basidiospore width (arithmetic average of all measured basidiospores), Q = variation in the L/W ratios between the specimens studied and n = number of basidiospores measured from a given number of specimens.
Molecular sequencing. Crude DNA was extracted from 0.02 to 0.2 g of dry basidiocarps of Costa Rican specimens using CTAB/NaCl followed by repeated extractions with chloroform and isopropanol precipitation. After purification and dilution, the DNA was used as a template for subsequent PCR amplifications. The primer pairs LR0R and LR7 (Vilgalys and Hester 1990) and ITS5 and ITS4 (White et al. 1990) were, respectively, selected for amplifying nLSU and ITS regions. The PCR procedure was as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles for nLSU region or 30 cycles for ITS region at 94 °C for 5 s, 55 °C for 15 s and 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR products were sequenced with the same primers in PCR amplifications in the Genomics Laboratory of Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice, on an ABI 3730xl DNA analyser, using BigDye Terminator 3.1 kit.
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd, Beijing) was used to extract DNA from Chinese specimens according to the manufacturer's instructions. The DNA was directly used as a template for PCR amplifications of the nLSU and ITS regions using the same primers as above. The PCR procedure was as follows for the nLSU region: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min and a final extension at 72 °C for 10 min, while for the ITS region: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 54 °C for 45 s and 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR products were sequenced with the same primers as those used for PCR at the Beijing Genomics Institute, China.
Phylogenetic analysis. The nLSU dataset, exploring the phylogenetic position of Nigrofomes, included sequences from species in Hymenochaetales and Polyporales as the ingroup and those in Thelephorales as the outgroup. To clarify the phylogenetic relationship between specimens of Nigrofomes from Costa Rica and China, the ITS dataset with Oxyporus populinus (Schumach.) Donk as an outgroup taxon focused on taxa closely related to Nigrofomes according to the topology inferred from the nLSU dataset. These two datasets were aligned using MAFFT 7.110 (Katoh and Standley 2013) with the g-ini-i option (Katoh et al. 2005). The resulting alignments were deposited in TreeBASE (http://www.treebase.org; accession number S21400). GTR + I + G and HKY + I + G were estimated as the best-fit evolutionary models for the resulting alignments from nLSU and ITS datasets, respectively, using jMod-elTest (Guindon andGascuel 2003, Posada 2008). Following the corresponding models, the two alignments were subjected to phylogenetic analyses by maximum likelihood (ML) and Bayesian Inference (BI) methods. ML analysis was performed using raxmlGUI 1.2 (Silvestro andMichalak 2012, Stamatakis 2006) and bootstrap (BS) replicates were evaluated under the auto FC option (Pattengale et al. 2010). BI analysis was conducted using MrBayes 3.2 (Ronquist et al. 2012). Two independent runs, each including four chains of 10 million generations and starting from random trees, were employed. Trees were sampled every 1000 th generation. The first 25% of sampled trees was removed and the remaining trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs). Chain convergence was judged using Tracer 1.5 (http://tree.bio.ed.ac.uk/ software/tracer/). For each alignment, the ML and BI methods generated nearly congruent topologies and, thus, only the topologies generated from the ML method are presented along with the BS values and BPPs, respectively, above 50% and 0.8 simultaneously at the nodes.

Results
Three nLSU and six ITS sequences were newly generated for this study and deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank; Figs 1, 2). The nLSU dataset, being composed of 147 sequences, resulted in an alignment of 956 characters. The BS search stopped after 400 replicates in the ML analysis, while all chains converged in the BI analysis, which was indicated by the effective sample sizes (ESSs) of all parameters above 1500 and the potential scale reduction factors (PSRFs) close to 1.000. In the nLSU-based phylogeny, Hymenochaetales (100% in ML and 1 in BI) and Polyporales (59% in ML and 0.98 in BI) were well separated; one Costa Rican and two Chinese specimens of Nigrofomes separated from each other (although not strongly supported in BI) but formed a distinct clade (100% in ML and 1 in BI) from widely accepted families within Hymenochaetales, viz. Hymenochaetaceae, Schizoporaceae, Oxyporaceae and Neoantrodiellaceae and also from other genera and species with uncertain phylogenetic position at the family level (Fig. 1).
The alignment, resulting from the ITS dataset of 24 sequences, comprised 1011 characters. After 250 replicates, the BS search stopped, while chain convergence was evidenced by the ESSs of all parameters above 5500 and the PSRFs equal to 1.000. The ITS-based phylogeny, focusing on Nigrofomes and related taxa within Hymenochaetales, shows that four Chinese and three Costa Rican specimens are clustered together but separated as two independent lineages, all with full statistical supports corresponding to their geographic origins (Fig. 2).   Description. Basidiocarps perennial, effused-reflexed, pileate, solitary, without odour or taste when fresh, woody hard. Pilei triquetrous or applanate, fan-shaped to semicircular, projecting up to 7 cm long, 15 cm wide and 4 cm thick at base. Pileal surface dark brown to black, rimose with age, glabrous to tuberculate, distinctly concentrically zonate and sulcate with a distinct crust; margin sharp, black. Pore surface mouse-grey to vinaceous grey, glancing; sterile margin vinaceous brown, up to 5 mm wide; pores angular, 7-9 per mm; dissepiments thin, entire to slightly lacerated. Context vinaceous grey, woody hard, distinctly concentrically zonate, upside integrating with a distinct crust on the pileal surface, up to 1 cm thick. Tubes greyish brown to vinaceous grey, the fresh layer dark grey to black, woody hard, up to 3 cm long.
Regarding the hyphal system of N. melanoporus, Ryvarden (2015) recognised it as "probably dimitic" and Lowe (1966) as "dimitic". They both mentioned the so-called skeletal hyphae are sometimes septate. According to the authors' observations, there are two kinds of hyphae present in N. melanoporus and N. sinomelanoporus and one of them is frequently septate, whereas the other rarely or occasionally septate. When describing this kind of hyphal system, we prefer "pseudodimitic" to "dimitic" because genuine skeletal hyphae are generally defined as aseptate.

Discussion
For the first time, Nigrofomitaceae was phylogenetically evidenced to separate from Polyporales and belong to Hymenochaetales (Fig. 1). Like Polyporales, Hymenochaetales is also an order mainly being composed of wood-inhabiting fungi. Four families, viz. Hymenochaetaceae, Schizoporaceae, Oxyporaceae and Neoantrodiellaceae were nested within Hymenochaetales in previous studies (Larsson et al. 2006, Zmitrovich and Malysheva 2014, Ariyawansa et al. 2015. However, the phylogenetic frame of this fungal order is not well resolved, which is indicated by the ambiguous phylogenetic position of many members of Hymenochaetales at the family level ( fig. 1; Larsson et al. 2006, Miettinen and Larsson 2011, Ariyawansa et al. 2015. Even regarding the four accepted families, their circumscriptions are uncertain. For example, Coltricia Gray and Coltriciella Murrill, two genera morphologically and phylogenetically belonging to Hymenochaetaceae (Dai 2010, Ariyawansa et al. 2015, were excluded from Hymenochaetaceae according to the phylogenetic analysis inferred from nLSU and 5.8S regions (Larsson et al. 2006). Schizoporaceae has never yet been evidenced as monophyletic (Larsson et al. 2006). Oxyporaceae and Neoantrodiellaceae, two recently erected families, respectively based on one and four genera (Zmitrovich andMalysheva 2014, Ariyawansa et al. 2015), have not yet been fully explored. The current nLSU-based phylogeny did not resolve the circumscriptions of these four families similar to previous studies mentioned above, but did support the fact that Nigrofomitaceae, represented by the type genus Nigrofomes, occupied a distinct lineage outside these four well-known families and thus was considered to be the fifth family in Hymenochaetales (Fig. 1). In future, more comprehensive phylogenetic studies including many more representative samples and employing more loci will improve our understanding of the taxonomy of Hymenochaetales, which may result in the emergence of more taxonomic units at the family level.
Nigrofomes melanoporus was considered to have a pantropical distribution (Ryvarden 2015). However, after more careful morphological examination, the Chinese specimens previously labelled as N. melanoporus show distinct characters from the tropical American specimens. In the nLSU-based phylogeny (Fig. 1), the Chinese and Costa Rican specimens were separated as two lineages, but the clade of the Chinese specimens did not receive reliable support, whereas the phylogeny inferred from the ITS dataset including more samples of Nigrofomes strongly supported the Chinese specimens as an independent lineage (Fig. 2). Therefore, these Chinese specimens were newly described as Nigrofomes sinomelanoporus distinct from N. melanoporus from both morphological and phylogenetic perspectives. Hattori and Sotome (2013) distinguished Nigrofomes nigrivineus from N. melanoporus by the presence of clamp connections in the contextual generative hyphae of the former species. Only a single specimen of N. nigrivineus is known and no molecular sequence was provided (Hattori and Sotome 2013), which makes the position of this species ambiguous. However, both N. sinomelanoporus and N. nigrivineus indicate that the species diversity of Nigrofomes in pantropical regions could be higher than previously supposed.