Taxonomy and Phylogeny of the Fomitopsis pinicola Complex With Descriptions of Six New Species From East Asia

Fomitopsis pinicola is a common brown-rot fungal species found in northern hemisphere. It grows on many different gymnosperm and angiosperm trees. Recent studies show that it is a species complex; three species from North America and one species from Europe have been recognized in this complex. In the current study, six new species in the Fomitopsis pinicola complex were discovered from East Asia, based on morphological characters and phylogenetic analyses inferred from the sequence data of the internal transcribed spacer (ITS) regions, the second subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF). Detailed descriptions of the six new species are provided. Our results also indicates that species of the F. pinicola complex from East Asia usually have limited distribution areas and host specialization.


INTRODUCTION
Fomitopsis P. Karst. was established by Karsten and typified by F. pinicola (Sw.) P. Karst. (Karsten, 1881). It is characterized by a perennial or annual growth habit, is sessile to effused-reflexed, has tough to woody hard basidiocarps, has a white to tan or pinkish-colored pore surface with mostly small and regular pores, has a dimitic to trimitic hyphal system with clamped generative hyphae, and has a hyaline, thin-walled, smooth, and ellipsoid to subglobose basidiospores which are negative in Melzer's reagent; it causes a brown rot (Ryvarden and Johansen, 1980;Gilbertson and Ryvarden, 1986;Ryvarden and Gilbertson, 1993;Núñez and Ryvarden, 2001;Han et al., 2016).
Fomitopsis pinicola has been intensively studied because it has the function of dispelling wind-evil and dampness, and has anti-tumor (Dai et al., 2009;Sun et al., 2016), antifungal, antioxidant, immunomodulation, and neuroprotective activities (Guler et al., 2009;Bao et al., 2015;Sun et al., 2016;Guo and Wolf, 2018). Högberg et al. (1999) showed that all European populations of F. pinicola belong to one intersterility group. Ryvarden and Stokland (2008) described Fomitopsis ochracea Ryvarden & Stokland from Alberta on Populus tremuloides that was distinguished from F. pinicola by substrate preference, basidiospore morphology, and match flame test to the lacquered pilei surface. Subsequently, this species was proven to belong to the F. pinicola complex (Haight et al., 2019). Haight et al. (2016) suggested that F. pinicola is a species complex comprised of at least four well-supported phylogenetic species, three in North America (F. ochracea and two previously undescribed species) and one in Europe (F. pinicola). Haight et al. (2019) described two new species: F. mounceae Haight & Nakasone and F. schrenkii Haight & Nakasone from North America in the F. pinicola complex. Until now, four species have been recognized in the F. pinicola complex; F. mounceae, F. ochracea, and F. schrenkii from North America and F. pinicola from Europe.
In some cases, fungi species boundaries based on morphology misrepresents the number of existing species (Leavitt et al., 2011). Due to geographic isolation, lack of migration, and genetic drift there may be variation among populations, although this genetic variation is not always obvious (Haight et al., 2016). Cryptic species of species complexes are proving to be extremely common in higher fungi, particularly those with wide geographic distributions or host ranges (Haight et al., 2016); they share similar morphological characteristics and phylogenetic relationships to known species. More cryptic species could be discovered by combining evidence of morphological characters, molecular data, host trees, and distribution areas in species complexes (Liu et al., 2021).
In recent years, taxonomic and phylogenetic studies of Fomitopsis have been carried out in China and several new species have been described (Li et al., 2013;Han et al., 2014Han et al., , 2016Han and Cui, 2015;Liu et al., 2019), but none have been focused on the F. pinicola complex. Samples collected from China were still identified as F. pinicola complex in these studies. With more and more specimens collected from different areas of China, Vietnam, and of East Asia, six new species of the F. pinicola complex have been discovered based on morphological characters and phylogenetic analysis of ITS + RPB2 + TEF gene regions.

Taxa Sampling and Morphological Study
The examined specimens were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC, Beijing, P. R. China). Morphological descriptions and abbreviations used in this study follow Han et al. (2016) and Liu et al. (2019).

DNA Extraction and Molecular Analyses
The procedures for DNA extraction and polymerase chain reaction (PCR) used in this study were the same as described by Chen et al. (2017) and Song and Cui (2017). The primer pairs ITS5 and ITS4 for ITS regions, fRPB2-f5F and bRPB2-7.1R for the RPB2 gene, and EF1-983 F and EF1-1567R for the TEF gene used in this study are the same as in previous studies (White et al., 1990;Rehner, 2001;Matheny, 2005).
The PCR cycling schedules for different DNA sequences of ITS, RPB2, and TEF genes used in this study followed those used in Zhu et al. (2019) and Sun et al. (2020) with some modifications. The PCR procedure for ITS was the initial denaturation at 95 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 40 s, annealing at 54 • C for 45 s, and extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. The PCR procedure for RPB2 was the initial denaturation at 94 • C for 2 min, followed by 37 cycles of denaturation at 94 • C for 45 s, annealing at 56 • C for 90 s, and extension at 72 • C for 2 min, and a final extension at 72 • C for 10 min. The PCR procedure for TEF was the initial denaturation at 95 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 40 s, annealing at 54-57 • C for 45 s and extension at 72 • C for 1 min, and a final extension at 72 • C for 10 min. The PCR products were purified and sequenced at the Beijing Genomics Institute (BGI), China, with the same primers. All newly generated sequences were deposited in GenBank (Table 1). Additional sequences for phylogenetic analyses were downloaded from GenBank ( Table 1). All sequences were aligned in MAFFT 7 (Katoh and Standley, 2013) 1 and manually adjusted in BioEdit (Hall, 1999). Alignments were spliced in Mesquite (Maddison and Maddison, 2017). The missing sequences were coded as "N, " ambiguous nucleotides were coded as "N" followed Chen et al. (2017). The final concatenated sequence alignment was deposited in TreeBase 2 (submission ID: 27439).
Phylogenetic analyses approaches used in this study followed Han et al. (2016) and Cui et al. (2019). The congruences of the 3-gene (ITS, RPB2 and TEF) were evaluated with the incongruence length difference (ILD) test (Farris et al., 1994) implemented in PAUP * 4.0b10 (Swofford, 2002), under heuristic search and 1000 homogeneity replicates. The sequences of Daedalea quercina (L.) Pers obtained from GenBank were used as outgroups for the phylogeny of the Fomitopsis pinicola complex, and sequences of Laetiporus zonatus B.K. Cui & J. Song were used as outgroups for the phylogeny of the Fomitopsis pinicola complex and related taxa. A maximum parsimony (MP) analysis was performed in PAUP * version 4.0b10 (Swofford, 2002). A maximum likelihood (ML) analysis was performed in RAxmL v.7.2.8 with a GTR + G + I model (Stamatakis, 2006). Bayesian inference (BI) was calculated by MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites determined by MrModeltest 2.3 (Posada and Crandall, 1998;Nylander, 2004). Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein, 1985). The branch support was evaluated with a bootstrapping method of 1000 replicates (Hillis and Bull, 1993). Branches that received bootstrap supports for the MP and ML, greater than or equal to 75% and Bayesian posterior probabilities (BPP) greater than or equal to 0.95, were considered

Molecular Phylogeny
The combined 3-gene (ITS, RPB2, TEF) dataset to infer the phylogeny of species in the Fomitopsis pinicola complex included sequences from 52 fungal samples representing 11 taxa. The dataset had an aligned length of 1750 characters including gaps (553 characters for ITS, 641 characters for RPB2, 556 characters for TEF), of which 1403 characters were constant, 32 were variable and parsimony-uninformative, and 315 were parsimonyinformative. Maximum parsimony analysis yielded 12 equally parsimonious trees (TL = 470, CI = 0.785, RI = 0.872, RC = 0.684, HI = 0.215). The best model for the combined ITS + RPB2 + TEF sequences dataset estimated and applied in the Bayesian analysis was GTR + I + G with equal frequency of nucleotides. Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, and only the MP tree inferred from the combined three-gene dataset is shown in Figure 1.
The combined three-gene (ITS, RPB2, TEF) dataset infer the phylogeny of species in the Fomitopsis pinicola complex and the related group included sequences from 74 fungal samples 3 http://tree.bio.ed.ac.uk/software/figtree/ representing 21 taxa. The dataset had an aligned length of 1848 characters including gaps (641 characters for ITS, 641 characters for RPB2, 566 characters for TEF), of which 1063 characters were constant, 41 were variable and parsimony-uninformative, and 744 were parsimony-informative. MP analysis yielded 10 equally parsimonious trees (TL = 2428, CI = 0.521, RI = 0.762, RC = 0.397, HI = 0.479). The best model for the concatenate sequence dataset estimated and applied in the Bayesian inference was GTR + I + G with an equal frequency of nucleotides. Bayesian analysis and ML analysis resulted in a similar topology as the MP analysis, and only the MP tree inferred from the combined three-gene sequences dataset is shown in Figure 2.
Basidiocarps. -Annual to perennial, pileate, sessile, solitary, hard corky, without odor or taste when fresh, woody hard and light in weight upon drying. Pilei semicircular to ungulate, projecting up to 6.5 cm long, 8.5 cm wide, 2.5 cm thick at base. Pileal surface cream to pinkish buff when fresh, becoming honey-yellow to grayish brown when dry, glabrous, small nodules appear near the base, rough, azonate; margin cream, slightly paler than pileal surface, obtuse. Pore surface cream to pinkish buff when fresh, becoming buff to curry-yellow when dry; sterile margin distinct, white to cream when fresh, becoming olivaceous buff to clay-buff when dry, up to 10 mm wide; pores round to angular, 2-4 per mm; dissepiments slightly thick to thick, entire. Context cream to straw-yellow, woody hard, up to 1.5 cm thick. Tubes concolorous with pore surface, woody hard, up to 1 cm long.
MycoBank: MB 838909 Fomitopsis hengduanensis is characterized by laccate pileus with pale dark gray to reddish brown surface at base and cream to flesh-pink toward the margin when fresh, oblong-ellipsoid to ellipsoid basidiospores (5.2-6 × 3.2-3.6 µm) and is distributed in high altitude areas of the Hengduan Mountains.
Etymology. -Hengduanensis (Lat.), refers to the species distributed in the area of Hengduan Mountains.
Basidiocarps. -Annual to perennial, pileate, sessile, solitary, hard corky, without odor or taste when fresh, woody hard and light in weight upon drying. Pilei applanate, semicircular to ungulate, projecting up to 7.5 cm long, 9 cm wide, 3 cm thick at base. Pileal surface laccate, colors varied but usually pale dark gray to reddish brown at base and cream to flesh-pink toward the margin when fresh, curry-yellow, mouse-gray to reddish brown at base and buff to clay-buff toward the margin when dry, glabrous, sulcate, concentrically zonate; margin acute to obtuse. Pore surface white to cream when fresh, becoming buff to straw-yellow when dry; sterile margin distinct, cream, up to 4 mm wide; pores round to angular, 6-8 per mm; dissepiments thick, entire. Context cream to straw-yellow, woody hard, up to 1.4 cm thick. Tubes concolorous with pore surface, woody hard, up to 0.5 cm long.
MycoBank: MB 838910 Fomitopsis kesiyae is characterized by its buff yellow to orange-yellow buff pileal surface when fresh, reddish brown to yellowish brown when dry, and grows on Pinus kesiya and is distributed in tropical areas of Vietnam.
Type Basidiocarps. -Annual, pileate, sessile, hard corky, without odor or taste when fresh, woody hard and light in weight upon drying. Pilei applanate, semicircular to sectorial, projecting up to 4.7 cm long, 6.5 cm wide, 4 cm thick at base. Pileal surface laccate, buff yellow to orange-yellow buff when fresh, becoming reddish brown to yellowish brown when dry, glabrous, sulcate, azonate; margin cream, distinctly paler than the pileal surface, obtuse. Pore surface white to cream when fresh, olivaceous buff to cinnamon-buff when dry; sterile margin distinct, buff to honeyyellow, up to 3 mm wide; pores round to angular, 6-8 per mm, dissepiments thick, entire. Context cream to straw-yellow, corky, up to 1.2 cm thick. Tubes concolorous with pore surface, hard corky, up to 1 cm long.
MycoBank: MB 838911 Fomitopsis massoniana is characterized by its effused-reflexed to pileate basidiocarps, applanate to triquetrous or irregular pilei with buff-yellow to apricot-orange pileal surface when fresh, buff to grayish brown when dry, a white to cream pore surface when fresh, cream to buff when dry, and grows on Pinus massoniana.
Basidiocarps. -Annual, effused-reflexed to pileate, sessile, hard corky, without odor or taste when fresh, woody hard and light in weight upon drying. Pilei applanate to triquetrous or irregular, projecting up to 4 cm long, 4.2 cm wide, 1.5 cm thick at base. Pileal surface laccate, buff-yellow to apricot-orange when fresh, becoming buff to grayish brown when dry, glabrous, sulcate, azonate; margin white to cream, obtuse. Pore surface white to cream when fresh, turning cream to buff when dry; sterile margin distinct, cream, up to 4 mm wide; pores round, 5-7 per mm, dissepiments thick, entire. Context cream to straw-yellow, woody hard, up to 0.8 cm thick. Tubes concolorous with pore surface, woody hard, up to 0.4 cm long.
MycoBank: MB 838912 Fomitopsis subpinicola is characterized by its apricot-orange, scarlet to fuscous pileal surface when fresh, reddish brown to dark brown when dry, occasionally septated skeletal hyphae and is distributed in Northeast China.
Basidiocarps. -Annual, pileate, sessile, hard corky, without odor or taste when fresh, woody hard and light in weight upon drying. Pilei applanate, circular to sectorial, projecting up to 7.5 cm long, 8.5 cm wide, 4.5 cm thick at base. Pileal surface laccate, apricot-orange, scarlet to fuscous when fresh, becoming reddish brown to dark brown upon drying, glabrous, sulcate, azonate; margin white to cream, distinctly paler than the pileal surface, obtuse. Pore surface white to cream when fresh, turning buff yellow to buff when dry; sterile margin distinct, white to cream, up to 6 mm wide; pores round, 6-8 per mm, dissepiments thick, entire. Context cream to straw-yellow, woody hard, up to 1.2 cm thick. Tubes concolorous with pore surface, woody hard, up to 0.5 cm long.
MycoBank: MB 838913 Fomitopsis tianshanensis is characterized by its effusedreflexed to pileate basidiocarps with soft corky texture when fresh, large pores (1-3 per mm) and long tubes (up to 2.5 cm), Etymology. -Tianshanensis (Lat.), refers to the species located at the Tianshan regions.
Basidiocarps. -Annual to perennial, effused-reflexed to pileate, sessile, soft corky, without odor or taste when fresh, hard corky and light in weight upon drying. Pilei applanate, semicircular to ungulate, projecting up to 11 cm long, 20 cm wide, 7 cm thick at base. Pileal surface dark bluish gray to yellowish brown when fresh, becoming fawn to deep olive when dry, slightly velutinate, small nodules appear near the base, rough, azonate; margin cream to cinnamon, obtuse to acute. Pore surface cream to pinkish buff when fresh, becoming faint yellow to light pink when dry; sterile margin distinct, cream to buff, up to 3 mm wide; pores mostly round, occasionally angular, 1-3 per mm, dissepiments thick, entire. Context cream to buff, corky, up to 3.5 cm thick. Tubes concolorous with pore surface, hard corky, up to 2.5 cm long.
Type of rot.

DISCUSSION
Based on the phylogenetic analyses, 10 species of the Fomitopsis pinicola complex grouped together (Figures 1, 2), including six new species from East Asia: F. abieticola, F. hengduanensis, F. kesiyae, F. massoniana, F. subpinicola, and F. tianshanensis. The main morphological characters of species in the F. pinicola complex are provided in Table 2.
Morphologically, Fomitopsis massoniana is similar to F. kesiyae; both species have an annual growth habit and a similar colored pileal surface when fresh. However, F. kesiyae differs in having smaller basidiospores (4.8-5.3 × 3-3.5 µm), is distributed in Vietnam, and grows on Pinus kesiya. Fomitopsis hengduanensis has similar sized pores (6-8 per mm), but compared to F. massoniana, F. hengduanensis has larger sized basidiocarps, a laccate pileal surface with pale dark gray to reddish brown at base and cream to flesh-pink toward the margin when fresh, and smaller sized basidiospores (5.2-6 × 3.2-3.6 µm). Phylogenetically, these two species are distinct from each other (Figures 1, 2). Fomitopsis massoniana is closely related to F. subpinicola, they share a cream to buff pore surface and have similar sized pores, but F. subpinicola has smaller basidiospores (4.3-5.5 × 2.7-3.3 µm).
Fomitopsis subpinicola can be easily separated from F. pinicola by its apricot-orange, scarlet to fuscous pileal surface when fresh, reddish brown to dark brown when dry, smaller basidiospores (4.3-5.5 × 2.7-3.3 µm) and is located in the Northeast of China. Phylogenetically, F. subpinicola is closely related to F. massoniana. Morphologically, F. subpinicola is similar to F. massonian, which has an annual growth habit and white to cream pore surface when fresh. But F. massoniana differs by its effused-reflexed to pileate basidiocarps, lager basidiospores (6.2-7.3 × 3.3-4 µm) and grows on the Pinus massoniana tree. Fomitopsis hengduanensis and F. kesiyae have similar sized pores (6-8 per mm), but F. hengduanensis has larger sized basidia (16.6-34.5 × 5.4-10.2 µm), F. kesiyae has a buff yellow to orangeyellow buff pileal surface when fresh, reddish brown to yellowish brown when dry, and grows on the Pinus kesiya tree.
Previously, Fomitopsis pinicola was used as a broad species concept, specimens from Europe, North America, and East Asia were all identified as F. pinicola based on morphological characters (Gilbertson and Ryvarden, 1986;Ryvarden and Gilbertson, 1993;Núñez and Ryvarden, 2001;Dai, 2012;Ryvarden and Melo, 2014). Recent phylogenetic analyses indicated that F. pinicola is a species complex and represent  (Ryvarden and Melo, 2014). In North America, F. mounceae and F. ochracea is distributed in Canada and the northern United States, and they grow on different gymnosperm or angiosperm wood (Ryvarden and Stokland, 2008;Haight et al., 2019); F. schrenkii is distributed in western and southwestern regions of the United States and mostly grows on different gymnosperm wood, rarely on angiosperm wood (Haight et al., 2019). Among the woodrotting fungi, some other polypore genera also have limited distribution areas and host specializations, such as Bondarzewia Singer , Heterobasidion Bref. (Chen et al., 2015;Yuan et al., 2020), Laetiporus Murrill (Song and Cui, 2017), and Sanghuangporus Sheng H. Wu, L.W. Zhou & Y.C. Dai (Zhu et al., 2019).
Key to Accepted Species of Fomitopsis pinicola Complex

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in an online repository. The name of the repository and accession number can be found below: https://treebase.org/treebaseweb/search/study/summary.html?id=27994&x-access-code=ab24 95717aaf081f0557e6680c381710&agreement=ok, submission ID: 27994.