Three new species of Fomitiporella (Hymenochaetales, Basidiomycota) based on the evidence from morphology and DNA sequence data

Abstract Fomitiporella austroasiana, F. mangrovei and F. vietnamensis are described and illustrated as new species based on morphological characters and molecular evidence. They have annual to perennial, mostly resupinate basidiomata with grayish fresh pores, an indistinct subiculum, lack any kind of setae, have brownish, thick-walled basidiospores, and cause a white rot. The distinctive morphological characters of the new species and their related species are discussed. Phylogenies based on the nuclear ribosomal large subunit (28S) and the nuclear ribosomal ITS region show that these three new species form three distinct lineages in the Fomitiporella clade. A key to known species of Fomitiporella is given.

As a continuation of the revision of Fomitiporella Murrill, phylogenetic inferences based on 28S and ITS DNA sequences revealed three new species. The taxonomic affinity and the evolutionary relationships among the new species and relates species are outlined.

Morphological studies
Specimens studied are deposited in the herbarium of Beijing Forestry University (BJFC) and will be forwarded to the National Museum Prague of Czech Republic (PRM). The sections were prepared in 5% potassium hydroxide (KOH), Melzer's reagent (IKI) and Cotton Blue (CB). The following abbreviations were used: KOH = 5% potassium hydroxide, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, CB = Cotton Blue, CB+ = cyanophilous, CB(+) = cyanophilic after 12 hours stained with Cotton Blue, CB-= acyanophilous, L = mean spore length (arithmetic average of the spores), W = mean spore width (arithmetic average of the spores), Q = variation in the ratios of L/W between specimens studied and n = number of spores measured from new specimens. The microscopic procedure follows He and Li (2013) and the special color terms follow Petersen (1996). Sections were studied at magnifications up to 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 illustrations were made from slide preparations stained with Cotton Blue. Spores were measured from sections cut from the tubes.

Molecular study and phylogenetic analysis
A CTAB-based rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing) was used to obtain genomic DNA from dried specimens. The primer pair ITS4 and ITS5 was used for amplification of the ITS region (White et al. 1990), while the primer pair LR0R and LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm) was used for providing the D1-D4 regions of the 28S (https://unite.ut.ee/primers.php). The PCR procedure for ITS amplification was as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles at 94 °C for 40 s, 54 °C for 45 s and 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for 28S was as follows: initial denaturation at 94 °C for 1 min, followed by 35 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. The PCR products were purified and sequenced at the Beijing Genomics Institute, China, with the same primers.
Reference ITS and 28S sequences from various species of Fomitiporella, available from GenBank (Benson et al. 2017), were compiled and complemented with sequences generated for this study. Additionally, we also used sequences from Ji et al. (2017) ( Table 1). Phellinus laevigatus (P. Karst.) Bourdot & Galzin and P. populicola Niemelä were selected as the outgroup representatives both in the ITS dataset and 28S dataset (Wagner and Fischer 2002). The sequences were aligned using ClustalX 1.83 (Chenna et al. 2003) and alignments were curated manually in BioEdit 7.0.5.3 (Hall 1999). Prior to phylogenetic analyses, ambiguous regions at the start and the end were deleted. The sequence alignment was deposited at TreeBase (submission ID 22036; www.treebase. org). Phylogenetic analyses were carried out as described previously (Ji et al. 2017).
Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) analyses were performed for the two 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 ML, MP and BI algorithms (Bootstrap support < 50 % and Bayesian posterior probabilities < 0.9 are not shown) at the nodes. MP analyses were performed using PAUP* 4.0b10 (Swofford 2002) with gaps in the alignments treated as missing data. Trees were generated using 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch-swapping algorithm with all characters given equal weight. Branch supports (BS) for all parsimony analyses were estimated by performing 1,000 bootstrap replicates (Felsenstein 1985) with a heuristic search of 10 randomaddition replicates for each bootstrap replicate. Sequences were also analyzed using MLwith RAxML-HPC2 on Abe through the Cipres Science Gateway (www.phylo. org). BI was calculated with MrBayes3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). The ITS region was divided into three partitions, ITS1, 5.8S and ITS2, for the Bayesian analysis. MrModeltest2.3 (Posada andCrandall 1998, Nylander 2004) was used to determine the best-fit evolution model for each dataset. Trees were visualized in TreeView 1.6.6 (Page 1996).

Results
Fifty-six 28S rDNA sequences, including eight sequences generated in this study (GenBank accession numbers MG657320-MG657327) and forty-six ITS rDNA sequences, including six sequences generated in this study (GenBank accession numbers MG657328-MG657333) were used to infer the phylogenetic trees. Sequence information is provided in Table 1. The 28S dataset had an aligned length of 898 characters, of  (Ji et al. 2017). However, each clade has its unique characters distinct from other Fomitiporella species. We therefore describe them as new species.  Basidiomata perennial, resupinate, hard corky and without odor or taste when fresh, woody hard when dry, up to 12 cm long, 5 cm wide and 12 mm thick at center.  Pore surface ash-gray to grayish brown when fresh, grayish brown to olivaceous, more or less shiny and uncracked on drying; margin yellowish-brown, less than 1 mm wide, thinning out; pores circular, 8-10 per mm; dissepiments thick, entire; tubes woody hard, concolorous with pores, each layer up to 2 mm deep, white mycelial strands present in old tubes. Subiculum very thin to almost lacking.
Additional specimens examined ( Etymology. Mangrovei (Lat.): referring to the species growing in mangrove. Basidiomata annual, resupinate, inseparable, without odor or taste when fresh, woody hard on drying, up to 30 cm long, 7 cm wide and 5 mm thick at center. Pore surface ash-gray to bluish gray when fresh, becomes pale clay-buff to pale brown and uncracked when dry; pores angular, 3-5 per mm; dissepiments thin, more or less entire to slightly lacerate; tubes woody hard, dark brown, up to 5 mm long. Subiculum very thin to almost lacking.
Additional specimen examined (
The phylogenetic analyses based on 28S or the ITS dataset produced trees with near-identical topologies, and each of the three new species formed a distinct, wellsupported clade.
An identification key to the accepted species of Fomitiporella is provided as follows: Key to species of Fomitiporella