Basidioradulum mayi and B. tasmanicum spp. nov. (Hymenochaetales, Basidiomycota) from both sides of Bass Strait, Australia

Basidioradulum was morphologically considered to be a synonym of Xylodon. Here, its independence within Hymenochaetales is confirmed from a phylogenetic perspective. Basidioradulum radula, the generic type, is widely distributed in Northern Hemisphere. Two Southern Hemisphere species close to B. radula are newly described as B. mayi and B. tasmanicum, respectively, from Victoria and Tasmania, Australia. Basidioradulum mayi differs from B. radula by lack of cystidia. Moreover, the hymenial surface of B. radula is normally much more strongly hydnoid than that of B. mayi. Basidioradulum tasmanicum is distinct from B. radula and B. mayi by having capitate cystidia, ellipsoid to subglobose basidiospores, and crystal-covered hyphae. Although morphologically distinct, the two new species isolated by Bass Strait have an almost identical ITS region, and could not be differentiated by nLSU- and ITS-based phylogenetic analyses. This case reminds us that basing phylogeny simply on the ITS as a barcode region may underestimate fungal species diversity.

The phylogeny inferred from the nLSU region did not classify families well within Hymenochaetales (Fig. 1), but did separate the fully supported Basidioradulum lineage from other species belonging to Schizoporaceae, including Xylodon quercinus (Pers.) Gray, the generic type of Xylodon. The Basidioradulum lineage was divided into two clades. The clade of B. radula with five Northern Hemisphere specimens received a moderate BS value of 68% and a high BPP value of 0.97. The Southern Hemisphere clade with three Australian specimens was strongly supported (99% BS, 1 BPP).
The midpoint-rooted tree inferred from the ITS region indicates that all 13 collections of Basidioradulum formed two fully supported clades (Fig. 2). One clade including eight collections originating from the Northern Hemisphere represented B. radula, while the other clade had five Southern Hemisphere collections.
The pairwise distance matrix for ITS sequences of Basidioradulum (Table 2) shows that the 13 collections were separated into two groups that are congruent with the two clades in the ITS-based phylogeny (Fig. 2). The distances among Group 1 including five Southern Hemisphere collections and among Group 2 including eight Northern Hemisphere collections were, respectively, 0.2% and 0.9%, while the distance between the two groups was 3.4%.
All the above molecular evidence supported the Southern Hemisphere specimens as distinct from Basidioradulum radula. Moreover, further morphological examinations indicated that among the Southern Hemisphere specimens the two Victorian specimens were extremely different to the three Tasmanian specimens. Therefore, two new species of Basidioradulum are described below. Basidiocarps annual, effused, adnate, hard and cracked when dry. Hymenial surface smooth, odontoid to hydnoid, the teeth conical or irregular, up to 1-2 mm long and 0.5-1 mm wide, becoming gradually shorter towards margin, whitish-cream to light-ochraceous. Margin slightly fibrillose, paler or concolorous with fertile area. Subiculum homogeneous, whitish-cream, up to 0.8 mm thick.

Discussion
In this study, the phylogenetic position of Basidioradulum was reevaluated by sampling more collections of this genus in the nLSU-based phylogeny (Fig. 1). Although the resolution at the family level was poor, Basidioradulum was clearly separated from Xylodon quercinus, the generic type of Xylodon, as in previous studies [3][4][5] . Therefore, we reject the transfer of B. radula, the generic type of Basidioradulum to Xylodon proposed by Ţura et al. 2 , and treat Basidioradulum as a distinct genus from Xylodon. Because comprehensive phylogenetic sampling of taxa of the order Hymenochaetales is lacking, we consider Basidioradulum to be a genus incertae sedis at the family rank within Hymenochaetales.
Basidioradulum radula as defined morphologically is considered to be widely distributed in Northern Hemisphere 2 . The sequences of B. radula from Chinese, Korean and American collections analyzed in this study also confirm its wide distribution. Meanwhile, the two Southern Hemisphere relatives of B. radula are distinct, and newly described as B. mayi and B. tasmanicum. These two species cannot be differentiated by molecular evidence on the basis of ITS region, which is used as the fungal barcode (Fig. 2, Table 2). The situation where B. mayi and B. tasmanicum have distinct morphological characters but cannot be delimited by DNA data indicates  www.nature.com/scientificreports www.nature.com/scientificreports/ that these two species isolated by Bass Strait may be undergoing an ongoing allopatric speciation event 9 . However, more evidence, such as mating test between these two species and multi locus-based phylogeny, is needed to further confirm this event. Nevertheless, the current case of Basidioradulum does stimulate consideration of the multitude of species concepts and recognition criteria as applied to fungi 10,11 . Although the molecular phylogenetic method normally has been considered to be a powerful means to discover new fungal lineages 12,13 , it is concluded from the current case that if we only consider differences in the ITS region, fungal diversity may be underestimated. The speciation of fungi needs time, and during the process of speciation the divergence of phenotype and genotype could happen at differentiated paces. Therefore, neither the genotype nor phenotype alone is suitable to be used as evidence to describe species. Instead, polyphasic evidence from morphology, phylogeny, ecology and so on, if available, should be used together to delimit fungal species.
conclusion Two wood-inhabiting basidiomycetous species Basidioradulum mayi and B. tasmanicum are newly described from both sides of Bass Strait, Australia. These two species have almost identical fungal barcoding ITS sequences but distinct morphological characters. The hymenial surfaces of basidiocarps were observed under a stereomicroscope. Special color terms followed Petersen 14 . Microscopic characters were examined using a Nikon Eclipse 80i microscope at magnification up to 1000×. Specimen sections were stained in Cotton Blue (CB), Melzer's reagent (IKI) and 5% potassium hydroxide. All measurements were taken from CB-stained sections. The basidiospore size variation was presented by putting 5% of measurements from each end of the range in parentheses. Drawings were made with the aid of a drawing tube. Photos of basidiospores were taken using a Nikon Digital Sight DS-U3 camera. The following abbreviations are used in the text: L = mean basidiospore length (arithmetic average of all basidiospores), W = mean basidiospore width (arithmetic average of all basidiospores), Q = variation in the L/W ratios between the specimens studied, and n = number of basidiospores measured from given number of specimens.
Crude DNA was extracted from basidiocarps of dry specimens using FH Plant DNA Kit (Beijing Demeter Biotech Co., Ltd., Beijing, China), and then directly used as template for subsequent PCR amplifications. The nLSU and ITS regions were amplified and sequenced using primer pairs LR0R and LR7 15 , and ITS1F 16 and ITS4 17 , respectively. The PCR procedure was as follows: for nLSU region initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 47.2 °C for 1 min and 72 °C for 1.5 min, and a final extension at 72 °C for 10 min, while for ITS region initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 57.2 °C for 45 s www.nature.com/scientificreports www.nature.com/scientificreports/ and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR products were sequenced at the Beijing Genomics Institute, Beijing, China. The newly generated sequences were deposited in GenBank (https://www. ncbi.nlm.nih.gov/genbank/; Table 1).
The nLSU dataset was used to explore the phylogenetic position of Basidioradulum, and included sequences from all main lineages in Hymenochaetales and two species in Polyporales as ingroup taxa and Amaurodon viridis (Alb. & Schwein.) J. Schröt. in Thelephorales as an outgroup taxon. The ITS dataset was used to differentiate the phylogenetic relationships among specimens of Basidioradulum. The datasets were aligned using MAFFT 7.110 18 under the g-ini-i option 19 , and the resulting alignments, after manual inspection, were deposited in TreeBASE (http://www.treebase.org; accession number 24505). jModelTest 20,21 was used to estimate the best-fit evolutionary model of the two alignments. Maximum likelihood (ML) and Bayesian inference (BI) methods were utilized for phylogenetic analyses of the two alignments following the evolutionary models. The ML method was performed using raxmlGUI 1.2 22,23 with the calculation of bootstrap (BS) replicates under the auto FC option 24 . The BI method was carried out using MrBayes 3.2 25 with two independent runs, each including four chains of 10 million generations and starting from random trees. The first 25% of the sampled trees every 1000th generation was removed, and the other 75% trees were remained for constructing a 50% majority consensus tree and calculating Bayesian posterior probabilities (BPPs). Tracer 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) was used to judge whether chains converged.