The studied specimens are deposited in the herbaria of Beijing Forestry University (BJFC), Southwest Forestry University (SWFC), the Connecticut Agricultural Experiment Station Valley Laboratory (NHES), and Seoul National University Fungus Collection (SFC). Macro-morphological descriptions were based on field notes and voucher herbarium specimens. Microscopic measurements and drawings were made from slides prepared from voucher tissues and stained with Cotton Blue and Melzer’s reagent. The following abbreviations were used: KOH = 5% potassium hydroxide; CB = Cotton Blue; CB+ = cyanophilous in Cotton Blue; CB– = acyanophilous in Cotton Blue; IKI = Melzer’s reagent; IKI– = neither amyloid nor dextrinoid in Melzer’s reagent; L = mean basidiospore length (arithmetic average of basidiospores); W = mean basidiospore width (arithmetic average of basidiospores); Q = variation in the L/W ratios between specimens studied; n (a/b) = number of basidiospores (a) measured from the given number of specimens (b). In presenting basidiospore size variation, 5% of measurements were excluded from each end of the range and these values are given in parenthesis. Special color terms follow Anonymous (1969) and Petersen (1996).

A CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China), AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea), and FH plant DNA kit II (Demeter Biotech Co., Ltd., Beijing, China) were used to extract total genomic DNA from dried specimens and to perform the polymerase chain reaction (PCR) according to the manufacturer’s instructions with some modifications (Shen et al., 2019). The ITS region was amplified with primer pairs ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCCTCC GCT TAT TGA TAT GC) (White et al., 1990), and for nLSU, LR0R (ACC CGC TGA ACT TAA GC), and LR7 (TAC TAC CAC CAA GAT CT) (Vilgalys and Hester, 1990). The final PCR volume was 30 μl; each tube contained 1 μl of each primer, 1 μl extracted DNA, 12 μl ddH₂O, and 15 μl 2 × EasyTaq PCR Supermix (TransGen Biotech Co., Ltd., Beijing, China). PCRs were performed on S1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The PCR procedure for ITS was as follows: initial denaturation at 95°C for 3 min, followed by 34 cycles of denaturation at 94°C for 40 s, annealing at 54°C for 45 s, and extension at 72°C for 1 min, followed by the final extension at 72°C for 10 min. The PCR procedure for‘ nLSU was initial denaturation at 94°C for 1 min, followed by 34 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 1 min, and extension at 72°C for 1.5 min, followed by the final extension at 72°C for 10 min. The PCR products were purified and sequenced at the Beijing Genomics Institute (BGI), China, using PCR primers. All sequences analyzed in this study were deposited at GenBank and listed in Table 1.

Phylogenetic trees were constructed using ITS + nLSU rDNA sequences, and phylogenetic analyses were performed with the maximum likelihood (ML) and Bayesian inference (BI) methods. Sequences of the species and strains were primarily adopted from ITS-based and 28S-based tree topology, as described by González et al. (2021). New sequences generated in this study, along with reference sequences retrieved from GenBank (Table 1), were aligned by MAFFT version 7 (Katoh et al., 2019¹) using the “G-INS-i” strategy and manually adjusted in BioEdit (Hall, 1999). Unreliably aligned sections were removed before the analyses, and efforts were made to manually inspect and improve the alignment. The data matrix was edited in Mesquite version 3.70 (Maddison and Maddison, 2021). The sequence alignment was deposited at TreeBase (submission ID 29857). Sequences of Pseudofistulina radicata (Schwein.) Burds. obtained from GenBank were used as outgroups to root the trees in the ITS + nLSU analysis.

The research using ML was conducted using RAxML-HPC version 8.2.3 (Stamatakis, 2014) and RAxML-HPC through the CIPRES Science Gateway (Miller et al., 2009²). Statistical support values (BS) were obtained using non-parametric bootstrapping with 1,000 replicates.

jModelTest version 2.17 was used to determine the best-fit evolution model of the combined dataset for ML and BI (Darriba et al., 2012). Four unique partitions were established, and GTR + I + G was a selected substitution model for each partition. The BI was calculated with MrBayes version 3.2.6 (Ronquist et al., 2012) in two independent runs, each of which had four chains for 2 million generations and started from random trees. Trees were sampled every 100 generations. 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 FigTree version 1.4.4 (Rambaut, 2018). Branches that received bootstrap support (BS) for ML and BPPs ≥ 75% (BS) and 0.95 (BPP) were considered significantly supported, respectively.

The ITS + nLSU dataset contained sequences from 22 fungal specimens, representing eight species of Fistulina, Porodisculus pendulus, and P. radicata; it had an aligned length of 1,977 characters. The Bayesian analyses exported a nearly identical topology to the ML analyses with an average standard deviation of split frequencies = 0.007082. Therefore, only the ML tree is presented with the BS and BPP. The phylogeny (Figure 1) inferred from the ITS and nLSU sequences showed that the sequences of Fistulina americana sp. nov. and Fistulina orientalis sp. nov. nested in the Fistulina clade and formed two independent lineages; both new species are related to F. hepatica and F. subhepatica with strong support (98% BS, 1 BPP).

Fistulina americana Y.C. Dai, D.W. Li, and Meng Zhou, sp. nov. Figures 2, 3.

MycoBank: MB 846428.

Differs from other Fistulina species by ellipsoid basidiospores 4–4.8 × 3–3.3 μm, and growth on Quercus in North-East USA.

Type. USA, Massachusetts, Boston, Blackstone Square Park, 42°20′23.4″N, 71°04′24.8″W, on the stump of Quercus, 27.VII.2015, C.L. Zhao 147 (holotype, SWFC 000147; isotype, BJFC038583).

Etymology. Americana (Lat.): refers to North America, where the species was found.

Basidiomata. Annual, lateral stipitate, fleshy, and readily exuding a reddish blood-like sap when squeezed or bruised when fresh, hard corky when dry. Pileus dimidiate to fan-shaped, projecting up to 6 cm, 5 cm wide, and 8 mm thick at the base when dry. Pileal surface pinkish brown to reddish brown, slimy, radially striate when fresh, becoming cinnamon to vinaceous gray, irregularly zonate when dry; margin blunt, concolorous with pileal surface or paler than pileal surface. Pore surface white to flesh-pink when fresh, become brownish when bruised, fulvous to umber when dry, bruised part become black when dry; sterile margin almost absent; pores round, 7–8 per mm, consisting of individual, crowed but easily separable tubes; dissepiments thin, usually entire, slightly pruinose. Context pale mouse gray and corky when dry, up to 5 mm thick. Tubes peach, paler than pore surface, slightly rigid when dry, up to 3 mm long. Stipe concolorous with pileal surface when fresh and dry, up to 13 mm long and 10 mm in diameter.

Hyphal structure. Hyphal system monomitic; generative hyphae with clamp connections and simple septa, IKI–, CB– to slightly CB+, become swollen in KOH.

Context. Generative hyphae hyaline, thin-walled, occasionally branched, interwoven, some collapsed, 7–11 μm in diameter, some inflated up to 22 μm in diameter; gloeoplerous hyphae present.

Tubes. Generative hyphae hyaline, thin- to slightly thick-walled, rarely branched, gelatinous, parallel along the tubes, 5–7 μm in diameter. Basidia clavate with four sterigmata and a basal clamp connection, 19–25 × 4.5–7 μm; basidioles in shape similar to basidia, but slightly smaller. Cystidial elements present at dissepimental edges, hyaline, thin-walled, smooth, with an oily substance, 74–87 × 7–9 μm.

Spores. Basidiospores ellipsoid, hyaline, thick-walled, smooth, with a big guttule, IKI–, CB+, (3.8–)4–4.8(–5) × (2.8–)3–3.3(–3.5) μm, L = 4.18 μm, W = 3.06 μm, Q = 1.37 (n = 30/1).

Additional specimens examined. USA. Connecticut, Avon, Avon Old Farm School, on the fallen trunk of Quercus palustris, 24.IX.2022, DL-22-192 (NHES, Dupl. BJFC); East Hampton, Hurd State Park, on the stump of Quercus, 4.IX.2022, DL-22-189 (NHES, dupl. in BJFC and JV); West Harford, 6 Reservoir, on stump of Quercus, 10.IX.2021, DL-21-209 (NHES, dupl. in BJFC and JV).

Fistulina orientalis Y.C. Dai, D.P. Bao, and Y.W. Lim, sp. nov. Figures 4, 5.

MycoBank: MB 846430.

Differs from other Fistulina species by its small pores of 11–12 per mm, small basidiospores of 3–4 × 2.7–3 μm, and growth on Castanopsis in East Asia.

Type. China, Anhui Province, Huangshan County, Huangshan Forest Park, Diaoqiao, on the living tree of Castanopsis eyrei, alt. 500 m, 30°08′35.44″ N, 118°06′45.59″ E, 7.IX.2018, R.H. Yang 217 (holotype, BJFC038584).

Etymology. Orientalis (Lat.): refers to East Asia where the species was found.

Basidiomata. Annual, lateral stipitate, fleshy and readily exuding a reddish blood-like sap when squeezed or bruised when fresh, woody hard to bone hard when dry. Pileus dimidiate to fan-shaped, projecting up to 4 cm, 5 cm wide, and 6 mm thick at the base when dry. Pileal surface salmon to scarlet, slimy, and faintly radially furrowed when fresh, becoming black to blackish blue and irregularly zonate upon drying; margin acute, concolorous with pileal surface. Pore surface flesh-pink when fresh, become brown when bruised, clay buff when dry, bruised part become black when dry; sterile margin almost absent; pores round, 11–12 per mm, consisting of individual, crowed but easily separable tubes; dissepiments thick, entire, pruinose. Context cream when fresh, dark gray and bone hard when dry, up to 4 mm thick. Tubes concolorous with pore surface, rigid when dry, and up to 2 mm long. Stipe concolorous with pileal surface when fresh, become dark gray when dry, up to 25 mm long and 5 mm in diameter.

Hyphal structure. Hyphal system monomitic; generative hyphae with clamp connections and simple septa, IKI–, CB–, become swollen in KOH.

Context. Generative hyphae hyaline to pale brownish, thin-walled, occasionally branched, interwoven, some collapsed, 6–10 μm in diameter, gloeoplerous hyphae present.

Tubes. Generative hyphae hyaline, thin- to slightly thick-walled, rarely branched, gelatinous, parallel along the tubes, 3–6 μm in diameter. Basidia clavate with four sterigmata and a basal clamp connection, 19–22 × 5–7 μm; basidioles in shape similar to basidia, but slightly smaller. Cystidial elements present at dissepimental edges, hyaline, smooth, thin-walled, with an oily substance, 57–85 × 5–7 μm.

Spores. Basidiospores ovoid to subglobose, hyaline, thick-walled, smooth, IKI–, CB+, (2.9–)3–4(–4.1) × (2.6–)2.7–3(–3.2) μm, L = 3.36 μm, W = 2.93 μm, Q = 1.15 (n = 30/1).

Additional specimen examined. Korea. Jeju Island, Seogwipo-si Gosali Forest Road, on dead root of living Castanopsis sieboldii, alt. 300 m, 33°31′63.86″ N, 126°59′76.26″ E, 18.V.2021, H.T. Jang (SFC20210518-01).