Two new species of Antrodia (Polyporales, Basidiomycota) in western China

Two new species of Antrodia, A. aridula and A. variispora, are described from western China. Phylogeny based on a six-gene dataset (ITS + nLSU + nSSU + mtSSU + TEF1 + RPB2) demonstrates that samples of the two species form two independent lineages within the clade of Antrodia s.s. and are different in morphology from the existing species of Antrodia. Antrodia aridula is characterized by its annual and resupinate basidiocarps with angular to irregular pores of 2–3 mm each and oblong ellipsoid to cylindrical basidiospores measuring 9–12 × 4.2–5.3 μm, growing on gymnosperm wood in a dry environment. Antrodia variispora is characterized by its annual and resupinate basidiocarps with sinuous or dentate pores with a size of 1–1.5 mm each and oblong ellipsoid, fusiform, pyriform, or cylindrical basidiospores measuring 11.5–16 × 4.5–5.5 μm, growing on the wood of Picea. The differences between the new species and morphologically similar species are discussed in this article.


Introduction
Antrodia P. Karst. is one of the major groups of fungi causing brown rot, mostly on gymnosperm wood in the Northern Hemisphere (Gilbertson and Ryvarden, 1986;Núñez and Ryvarden, 2001;Ryvarden and Melo, 2017;Wu et al., 2022), and traditionally, the members of the genus are characterized by annual to perennial, leathery, mostly light colored, resupinate to effused-reflexed or distinctly pileate basidiocarps; a dimitic hyphal structure or a few species with a monomitic hyphal system; generative hyphae with clamp connections; skeletal hyphae negative in Melzer's reagent; some species with amyloid skeletal hyphae; and hyaline, thin-walled basidiospores which are negative in Melzer's reagent and cotton blue (Spirin et al., 2013;Liu et al., 2022). Recent studies demonstrated that this genus is polyphyletic including 12 small and monophyletic genera (Ortiz-Santana et al., 2013;Spirin et al., 2013;Runnel et al., 2019;Liu et al., 2022), and Antrodia s. str. Has been delimited as the species grouped around Antrodia serpens (Fr.) P. Karst. within the antrodia clade of Polyporales (Hibbett and Donoghue, 2001;Spirin et al., 2013).
During an investigation on polypores in western China, five resupinate, cream to buff specimens were collected from Qinghai Province and Inner Mongolia Autonomous Region, western China. Macromorphology and the ecology of brown rot on gymnosperm wood showed that they belong to Antrodia s.l., and further morphological examination and phylogenetic analysis indicated that they represent two undescribed species of Antrodia s. str. Thus, we describe them as two new species in the present article.

Morphological studies
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).

DNA extraction, amplification, and sequencing
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.

Phylogenetic analysis
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 online 1 adjusting the direction of nucleotide sequences 1 https://mafft.cbrc.jp/alignment/server/ 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).

FIGURE 2
Basidiocarps of Antrodia aridula (holotype, Dai 24525). 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.
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.  Basidiocarp of Antrodia variispora (holotype, Dai 23995). Scale bar: 2 cm. 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.
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.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.