All studied specimens are deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Morphological descriptions are based on field notes and microscopic examinations of voucher specimens. Special color terms are based on Anonymous (1969) and Petersen (1996). Microscopic structures refer to Spirin et al. (2013), Chen and Cui (2015), and Liu et al. (2022).

Acetyl trimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to obtain DNA templates from dried specimens and perform the polymerase chain reaction (PCR) according to the manufacturer’s instructions with some modifications (Chen and Dai, 2021; Liu et al., 2022). The primers of ITS, including nLSU, nSSU, mtSSU, TEF1, and RPB2, for amplifying the DNA regions are mentioned in Table 1. The PCR procedure for ITS, mtSSU, TEF1, and RPB2 follows Liu et al. (2022). All newly generated sequences have been submitted to GenBank and are listed in Table 2.

A total of 98 samples of Antrodia and related taxa were used for phylogenetic analysis in this study (Table 2). Oligoporus rennyi (Berk. & Broome) Donk and Postia lactea (Fr.) P. Karst. were selected as outgroups for phylogenetic analysis following Liu et al. (2022), based on the combined datasets of the internal transcribed spacer (ITS) region, the large subunit nuclear ribosomal RNA gene (nLSU), the small subunit nuclear ribosomal RNA gene (nSSU), the small subunit mitochondrial rRNA gene sequences (mtSSU), the translation elongation factor 1-α gene (TEF1), and the second subunit of RNA polymerase II (RPB2). Sequences were aligned with MAFFT v. 7 online¹ adjusting the direction of nucleotide sequences according to the first sequence (accurate enough for most cases), selecting the G-INS-i iterative refinement method (Katoh et al., 2017). The aligned sequences were deposited at TreeBase (submission ID 29874).²

The analyses of maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI) refer to Liu et al. (2022).

The concatenated dataset included 98 ITS sequences, 90 nLSU sequences, 67 nSSU sequences, 64 mtSSU sequences, 70 TEF1 sequences, and 66 RPB2 sequences representing 54 taxa. There are 4,608 characters in the dataset, including 3,044 were constant, 144 were variable and parsimony-uninformative, and 1,420 were parsimony-informative. MP analysis yields a tree (TL = 8,189, CI = 0.323, RI = 0.700, RC = 0.226, HI = 0.677). “GTR + I + G” was the best model for the BI analysis, lset nst = 6, rates = invgamma; prset statefreqpr = dirichlet (1,1,1,1). The average standard deviation of split frequencies in the BI analysis was 0.008957. Branches that received bootstrap support for MP (MP-BS), ML (ML-BS), and BI (BPP) greater than or equal to 50% (MP-BS and ML-BS) and 0.95 (BPP) are considered as significantly supported, respectively.

The current phylogeny placed all samples of Antrodia in a high supported clade (Figure 1). Two new species Antrodia aridula and A. variispora formed two well-supported lineages, respectively (100% MP 100% ML 1.00 BI and 99% MP 100% ML 1.00 BI). The two new species clustered with Antrodia macra (Sommerf.) Niemelä formed a well-supported subclade (96% MP 98% ML 1.00 BI).

Antrodia aridula Y.C. Dai, H.M. Zhou, Y.D. Wu & Shun Liu, sp. nov. Figures 2, 3.

MycoBank number: 846495.

Holotype: China. Inner Mongolia Autonomous Region, Alxa Left Banner, Helanshan Nature Reserve, elev. 2270 m, N 38.865282, E 105.899814, on fallen trunk of Picea crassifolia, 17 September 2022, Dai 24525 (BJFC).

Etymology: Aridula (Lat.): Referring to the species that prefer to the dry environment in Helan Mts. around the desert area of the Inner Mongolia Autonomous Region.

Basidiocarps: Annual, resupinate, tightly attached on wood, leathery when fresh, hard corky when dry, up to 5 cm long, 2 cm wide, and 0.5-mm thick at the center. Pore surface cream when fresh, becoming cream to buff when dry; sterile margin thinning out, white, up to 1 mm wide; pores angular to irregular, 2–3 per mm; dissepiments thin, lacerate. Subiculum cream, hard corky, paler contrast with tubes, up to 0.1-mm thick. Tubes concolorous with pores, hard corky, up to 0.4-mm long.

Hyphal structure: Hyphal system dimitic; generative hyphae with clamp connections; skeletal hyphae IKI–, CB–; tissue unchanged in KOH.

Subiculum: Generative hyphae hyaline, thin-to slightly thick-walled, rarely branched, 2–3 μm in diam; skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen to subsolid, rarely branched, 2–5 μm in diam.

Tubes: Generative hyphae frequent, hyaline, thin-to slightly thick-walled, rarely branched, 2–3 μm in diam; skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen to subsolid, rarely branched, interwoven, 3–4 μm in diam. Cystidia absent; cystidioles present, fusoid, thin-walled, 21–26 × 5–7 μm. Basidia clavate, with a basal clamp connection and four sterigmata, 23–30 × 9–10 μm; basidioles in shape similar to basidia, but smaller.

Spores: Basidiospores oblong ellipsoid to cylindrical, gently arcuate and tapering toward the apiculus, hyaline, thin-walled, smooth, sometimes within a few small guttules, IKI–, CB–, (8.5–)9–12(−12.8) × (4–)4.2–5.3(−5.6) μm, L = 10.31 μm, W = 4.7 μm, Q = 2.14–2.22 (n = 90/3).

Additional specimens examined (paratypes): China. Inner Mongolia Autonomous Region, Alxa Left Banner, Helanshan Nature Reserve, elev. 2270 m, N 38.865282, E 105.899814, on fallen trunk of Picea crassifolia, 17 September 2022, Dai 24526 (BJFC), Dai 24527 (BJFC); Helanshan Forest Park, elev. 2070 m, N 38.971847, E 105.905048, on fallen branch of Pinus tubaeliformis, 18 September 2022, Dai 24530 (BJFC).

Notes: Antrodia aridula is characterized by its annual and resupinate basidiocarps with angular to irregular pores of 2–3 per mm, a dimitic hyphal structure, the presence of fusoid cystidioles, basidiospores oblong ellipsoid to cylindrical, gently arcuate and tapering toward the apiculus, 9–12 × 4.2–5.3 μm, and growing on gymnosperm wood in a dry environment of West China. Antrodia aridula and Cartilosoma ramentaceum (Berk. & Broome) Teixeira (≡ Antrodia ramentacea (Berk. & Broome)) Donk have similar pores and growing on the wood of Pinus, but the latter differs from the former by gelatinous basidiocarps, smaller basidiospores (6.2–8.1 × 2.1–2.7 μm vs. 9–12 × 4.2–5.3 μm) and wider geographical distribution (Niemelä, 2005). Cartilosoma is different from Antrodia by completely resupinate and soft basidiocarps when fresh, and gelatinous hymenophore (Liu et al., 2022). Antrodia aridula is similar to A. macra (Sommerf.) Niemelä in macromorphology, and both species are closely related (Figure 1), but the latter species has smaller basidiospores (7.4–11 × 3–4.3 μm vs. 9–12 × 4.2–5.3 μm, Niemelä, 2005), and growing on Salix and Populus only (Ryvarden and Melo, 2017). Antrodia aridula is also closely related to Antrodia variispora (Figure 1), but the latter species has bigger and variable basidiospores (9–12 × 4.2–5.3 μm vs. 11.5–16 × 4.5–5.5 μm).

Ecologically, Antrodia aridula grows on fresh fallen gymnosperm trunks and branches in a dry environment, indicating a pioneer decayer in the coniferous forests of western China.

Antrodia variispora Y.C. Dai, H.M. Zhou, Y.D. Wu & Shun Liu, sp. nov. Figures 4, 5.

MycoBank number: 846496.

Holotype: China. Qinghai Province, Nangqian County, Baizha Forest Park, elev. 4090 m, N 31.855040, E 96.467402, on stump of Picea likiangensis var. balfouriana, 7 August 2022, Dai 23995 (BJFC).

Etymology: Variispora (Lat.): Referring to the species having variable basidiospores.

Basidiocarp: Annual, resupinate, tightly attached to wood, leathery when fresh, hard corky to rigid when dry, up to 16 cm long, 6 cm wide, and 1.3-mm thick at the center. Pore surface cream when fresh, becoming cinnamon-buff when dry; sterile margin very narrow to almost lacking; pores sinuous or dentate, (0.5–)1–1.5(−2) per mm; dissepiments thin, lacerate. Subiculum cream, hard corky, paler contrast with tubes, up to 0.3-mm thick. Tubes concolorous with pores, rigid, up to 1-mm long.

Hyphal structure: Hyphal system dimitic; generative hyphae with clamp connections; skeletal hyphae IKI–, CB–; tissue unchanged in KOH.

Subiculum: Generative hyphae infrequent, hyaline, thin-to slightly thick-walled, rarely branched, 2–4 μm in diam; skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen to subsolid, rarely branched, interwoven, 2.5–4 μm in diam.

Tubes: Generative hyphae frequent, hyaline, thin-to slightly thick-walled, frequently branched, 2.5–4 μm in diam; skeletal hyphae dominant, hyaline, thick-walled with a narrow lumen to subsolid, occasionally branched, interwoven, 3–5 μm in diam. Cystidia absent; cystidioles present, fusoid, thin-walled, 22–41 × 4.5–5 μm. Basidia clavate to pyriform, with a basal clamp connection and four sterigmata, 24–35 × 7–10 μm; basidioles in shape similar to basidia, but smaller.

Spores: Basidiospores variable, oblong ellipsoid, fusiform, pyriform or cylindrical, gently arcuate and tapering toward the apiculus, hyaline, thin-walled, smooth, usually within a few small guttules, IKI–, CB–, (11–)11.5–16(−18.5) × 4.5–5.5(−5.8) μm, L = 13.47 μm, W = 5.12 μm, Q = 2.63 (n = 30/1).

Notes: Antrodia variispora is characterized annual and resupinate basidiocarps with sinuous or dentate pores of 1–1.5 per mm, a dimitic hyphal structure, the presence of fusoid cystidioles, basidiospores variable, oblong ellipsoid, fusiform, pyriform or cylindrical, 11.5–16 × 4.5–5.5 μm, and growing on the wood of Picea in West China. Antrodia variispora is similar to Adustoporia sinuosa (Fr.) Audet (≡ Antrodia sinuosa (Fr.) P. Karst.) in macromorphology, but the latter species has distinctly smaller basidia (11–15 × 4–5 μm vs. 24–35 × 7–10 μm, Ryvarden and Gilbertson, 1993) and smaller basidiospores (4.9–6 × 1.4–1.8 μm vs. 11.5–16 × 4.5–5.5 μm, Niemelä, 2005). Adustoporia differs from Antrodia s. str. by pale brown pore surface when fresh, smaller basidia (15–22 × 4–5 μm), smaller basidiospores (4–6 × 1–2 μm, Audet, 2017b). Antrodia variispora and Dentiporus albidoides (A. David & Dequatre) Audet (≡ Antrodia albidoides A. David & Dequatre) have similar pore and basidiospore dimensions (Ryvarden and Melo, 2017), but the latter species has skeletocystidia, uniformed cylindrical basidiospores, and growing on angiosperm wood in Mediterranean area (Ryvarden and Melo, 2017). In addition, phylogenetically both species are distantly related (Figure 1). The genus Dentiporus differs from Antrodia s. str by often resupinate basidiocarps usually with rose pink tinted pore surface and round to irpicoid pores (Audet, 2017a). Antrodia variispora is closely related to A. macra (Figure 1), but A. macra has regularly cylindrical to oblong ellipsoid basidiospores which are smaller than those in Antrodia variispora (7.4–11 × 3–4.3 μm vs. 11.5–16 × 4.5–5.5 μm, Niemelä, 2005).

Ecologically, Antrodia variispora grows on a large stump of Picea likiangensis var. balfouriana in a virgin forest, and we tried to find more samples in a similar environment of the same forest, and unfortunately, we did not find the second sample. Thus, Antrodia variispora seems to be a rare species in old growth forests.