Molecular Phylogeny and Global Diversity of the Genus Haploporus (Polyporales, Basidiomycota)

Phylogeny and taxonomy of the genus Haploporus were carried out based on a larger number of samples covering a wider geographic range including East Asia, South Asia, Europe, and America, and the species diversity of the genus is updated. Four species, Haploporus bicolor, H. longisporus, H. punctatus and H. srilankensis, are described as new species based on morphology and molecular phylogenetic analyses inferred from the internal transcribed spacer (ITS), the large subunit nuclear ribosomal RNA gene (nLSU), and the small subunit mitochondrial rRNA gene (mtSSU). Haploporus bicolor is characterized by the distinctly different colors between the pore surface and the tubes, small pores measuring 5–7 per mm, and narrow basidiospores measuring 10.5–11.9 × 4.5–5 µm; H. longisporus differs from other species in the genus by its large pores measuring 2–3 per mm, hyphae at dissepiment edge with simple septum, and the long basidiospores (up to 22 µm); H. punctatus is distinguished by its cushion-shaped basidiocarps, wide fusiform cystidioles with a simple septum at the tips, the absence of dendrohyphidia and the cylindrical to slightly allantoid basidiospores measuring 9–10.8 × 3.8–5 µm; H. srilankensis is characterized by its perennial habit, small pores measuring 4–5 per mm, dextrinoid skeletal hyphae, the presence of cystidioles and dendrohyphidia. An identification key to accepted species of Haploporus is provided.

The aim of the study is to explore the phylogeny and species diversity of Haploporus based on samples from Asia, Europe and America. The taxonomy and phylogeny are updated, and four new species, respectively, from China, Ecuador, and Sri Lanka, were confirmed to be members of the Haploporus. In this paper, we describe and illustrate those new species.

Morphological Studies
Specimens examined were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC) and the private herbarium of Josef Vlasák (JV) which will forward specimens to the National Museum of Czech Republic in Prague (PRM). Macro-morphological descriptions were based on field notes. Sections of basidiocarps were studied microscopically according to Zhou and Cui [12] at magnifications 1000× using a Nikon Eclipse 80i microscope with phase contrast illumination. Drawings were made with the aid of a drawing tube. Microscopic features, measurements, and drawings were made from sections stained with Cotton Blue and Melzer's reagent. Basidiospores were measured from sections cut from the tubes. To present basidiospores size variation, the 5% of measurements excluded from each end of the range are given in parentheses. Basidiospore spine lengths were not included in the measurements. Abbreviations include IKI = Melzer's reagent, IKI+ = amyloid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, L = mean spore length (arithmetic average of all spores), W = mean basidiospores width (arithmetic average of all basidiospores), Q = the L/W ratio, and n = number of basidiospores measured from given number of specimens. Special color terms follow Petersen [13]. Herbarium abbreviations follow Thiers [14].

DNA Extraction and Sequencing
A cetyl trimethylammonium bromide (CTAB)rapid plant genome extraction kit (Aidlab Biotechnologies, Beijing, China) was used to obtain PCR products from dried specimens, according to the manufacturer's instructions with some modifications [15,16]. The internal transcribed spacer (ITS) region was amplified with primer pair ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGATAT GC) [17]. The large subunit nuclear ribosomal RNA gene (nLSU) region was amplified with primer pair LR0R (ACC CGC TGA ACT TAA GC) and LR7 (TAC TAC CAC CAA GAT CT) ( http://www.biology.duke.edu/fungi/mycolab/primers.htm). The small subunit mitochondrial rRNA gene (mtSSU) region was amplified with primer pair MS1 (CAG CAG TCA AGA ATA TTA GTC AAT G) and MS2 (GCG GAT TAT CGA ATT AAA TAA C) [17]. The PCR procedure for ITS and mtSSU was as follows: initial denaturation at 95 • C for 3 min, followed by 34 cycles at 94 • C for 40 s, 54 • C for ITS and 55 • C for mtSSU for 45 s and 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR procedure for nLSU was as follows: 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 of 72 • C for 10 min [5]. The PCR products were purified with a Gel Extraction and PCR Purification Combo Kit (Spin-column) in Beijing Genomics Institute, Beijing, P.R. China. The purified products were then sequenced on an ABI-3730-XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers as in the original PCR amplifications.

Phylogenetic Analysis
The sequences generated in this study were aligned with additional sequences downloaded from GenBank (Table 1) using ClustalX [18] and manually adjusted in BioEdit [19]. The sequence quality was checked following Nilsson et al. [20]. Perenniporia hainaniana B.K. Cui and C.L. Zhao and P. medulla-panis (Jacq.) Donk were used as outgroups, following Shen et al. [5]. Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps were manually adjusted to optimize the alignment. Se-quence alignment was deposited at TreeBase (http://purl.org/phylo/treebase; submission ID 27556). Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian inference (BI) were employed to perform phylogenetic analysis of the three aligned datasets. The three phylogenetic analysis algorithms generated nearly identical topologies for each dataset, thus only the topology from the MP analysis is presented along with statistical values from the MP, ML and BI algorithms. Most parsimonious phylogenies were inferred from the ITS + nLSU + mtSSU, and their combinability was evaluated with the incongruence length difference (ILD) test [23] implemented in PAUP* 4.0b10 [24], under a heuristic search and 1000 homogeneity replicates giving a P value of 1.000, much greater than 0.01, which means there is no discrepancy among the two loci in reconstructing phylogenetic trees. Phylogenetic analysis approaches followed [22]. The tree construction procedure was performed in PAUP* version 4.0b10 [24]. All characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [25]. Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each maximum parsimonious tree (MPT) generated.
jModeltest v.2.17 [26] was used to determine the best-fit evolution model of the combined dataset for Maximum likelihood (ML) and Bayesian inference (BI). Four unique partitions were established, GTR + I + G was the selected substitution model for each partition. RaxmlGUI 1.2 [27,28] was used for ML analysis. All parameters in the ML analysis used default settings. Statistical support values were obtained using non-parametric bootstrapping with 1000 replicates. The Bayesian inference (BI) was conducted with MrBayes 3.2.6 [29] in two independent runs, each of which had four chains for 10 million generations and started from random trees. Trees were sampled every 1000th generation. The first 25% of sampled trees were discarded as burn-in, whereas other trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).
Phylogenetic trees were visualized using Treeview [30]. Branches that received bootstrap support for Maximum likelihood (BS), Maximum parsimony (BP) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (BS/BP) and 0.95 (BPP) were considered as significantly supported, respectively.

Molecular Phylogeny
The ITS-based phylogeny included ITS sequences from 46 fungal collections representing 23 species. The dataset had an aligned length of 720 characters, of which 333 characters are constant, 63 are variable and parsimony-uninformative, and 324 are parsimonyinformative. MP analysis yielded a tree (TL = 945, CI = 0. 624, RI = 0.872, RC = 0.544, HI = 0.376). The best model for the ITS sequences dataset estimated and applied in the BI was GTR+I+G. BI resulted in a similar topology with an average standard deviation of split frequencies = 0.006018 to MP analysis, and thus only the MP tree is provided. Both BT values (≥50%) and BPPs (≥0.90) are shown at the nodes ( Figure 1).
The combined three gene (ITS + nLSU + mtSSU) sequences dataset from 46 fungal specimens representing 23 taxa did not show any conflicts in tree topology for the reciprocal bootstrap trees, which allowed us to combine them (p > 0.01). The dataset had an aligned length of 2706 characters, of which 1792 characters are constant, 220 are variable and parsimony-uninformative, and 694 are parsimony-informative. Maximum parsimony analysis yielded a tree (TL = 1929, CI = 0.627, RI = 0.847, RC = 0.531, HI = 0.373). The best model for the combined ITS + nLSU + mtSSU dataset estimated and applied in the Bayesian analysis was GTR + I + G. Bayesian analysis resulted in a similar topology with an average standard deviation of split frequencies = 0.005181 to MP analysis, and thus only the MP tree is provided. Both BT values (≥50%) and BPPs (≥0.90) are shown at the nodes ( Figure 2).
In both ITS + nLSU + mtSSU-and ITS-based phylogenies (Figures 1 and 2), four new well-supported lineages were identified. Among them are two well-supported terminal clades and two isolated branches. Four specimens from Sri Lanka formed a well-supported clade (98% MP 100% ML and 1.00 BI), named as Haploporus srilankensis, sister to H.  Figure 1). The combined three gene (ITS + nLSU + mtSSU) sequences dataset from 46 fungal specimens representing 23 taxa did not show any conflicts in tree topology for the reciprocal bootstrap trees, which allowed us to combine them (p > 0.01). The dataset had an aligned length of 2706 characters, of which 1792 characters are constant, 220 are variable and parsimony-uninformative, and 694 are parsimony-informative. Maximum parsimony analysis yielded a tree (TL = 1929, CI = 0.627, RI = 0.847, RC = 0.531, HI = 0.373). The best model for the combined ITS + nLSU + mtSSU dataset estimated and applied in the Bayesian analysis was GTR + I + G. Bayesian analysis resulted in a similar topology with an average standard deviation of split frequencies = 0.005181 to MP analysis, and thus only the MP tree is provided. Both BT values (≥50%) and BPPs (≥0.90) are shown at the nodes ( Figure 2). Etymology-Bicolor (Lat.): referring to the species having different colors between the pore surface and the tubes.
Fruiting body-Basidiocarp annual, resupinate, difficult to separate from the substrate, soft corky and white to cream when fresh, become buff when bruised, corky when dry, up to 5 cm long, 2 cm wide and 0.7 mm thick at the center. Pore surface cream to buff with peach tint when dry; sterile margin distinct, white, up to 1 mm; pores round to angular, 5-7 per mm; dissepiments thick, entire. Subiculum clay buff, corky, up to 0.4 mm thick. Tubes clay buff, corky, up to 0.3 mm long. In both ITS + nLSU + mtSSU-and ITS-based phylogenies (Figures 1 and 2   Hyphal structure-Hyphal system dimitic; generative hyphae bearing clamp connections, hyaline, thin-walled; skeletal hyphae dominant, thick-walled, frequently branched, IKI-, CB+; tissues unchanging in KOH.
Fruiting body-Basidiocarp annual, resupinate, difficult to separate from the substrate, corky when dry, up to 10 cm long, 1.5 cm wide and 2 mm thick at the center. Pore surface cream to pale buff when dry; sterile margin indistinct; pores round to angular, 2-3 per mm; dissepiments thick, entire. Subiculum olivaceous buff, corky, up to 1 mm thick. Tubes olivaceous buff, corky, 1 mm long.

MycoBank: MB 838452
Diagnosis-Differs from other Haploporus species by its cushion-shaped basidiocarps, dextrinoid skeletal hyphae and basidiospores, wide fusiform cystidioles with a simple septum at the tips, the absence of dendrohyphidia and the cylindrical to slightly allantoid basidiospores measuring 9-10.8 × 3.8-5 µm.
Fruiting body-Basidiocarps annual, resupinate, difficult to separate from the substrate, corky when dry, cushion-shaped, distinctly thickened in center and receding at margin, up to 3 cm long, 3 cm wide and 4 mm thick at center. Pore surface pale buff when dry; sterile margin distinct, cream, up to 2 mm; pores round to angular, 3-5 per mm; dissepiments thick, entire. Subiculum olivaceous buff to clay buff, corky, up to 0.5 mm thick. Tubes buff, corky, up to 3.5 mm long.
All the species of Haploporus with molecular evidence and close to our new species are discussed above. Furthermore, two species of Haploporus, H. brasiliensis and H. pileatus, were described from Brazil based on morphology only [7]. Haploporus brasiliensis distinctly differs from our four new species by its obviously smaller basidiospores and the absence of cystidioles and dendrohyphidia [7]. Haploporus pileatus is distinguished from all the species in this genus by its distinct pileate basidiocarps [7].
Currently, 23 species are accepted in Haploporus, and their main morphological characteristics are listed in Table 2. A key to accepted species of Haploporus is provided as follows.