Two new forest pathogens in Phaeolus (Polyporales, Basidiomycota) on Chinese coniferous trees were confirmed by molecular phylogeny

Phaeolus schweinitzii (Fr.) Pat. was originally described in Europe and is considered a common forest pathogen on conifers in the Northern Hemisphere. Our molecular phylogeny based on samples from China, Europe, and North America confirms that P. schweinitzii is a species complex, including six taxa. P. schweinitzii sensu stricto has a distribution in Eurasia; the samples from Northeast and Southwest China are distantly related to P. schweinitzii sensu stricto, and two new species are described after morphological, phylogenetic, and geographical analyses. The species growing on Larix, Picea, and Pinus in Northeast China is described as Phaeolus asiae-orientalis. Another species mostly occurring on Pinus yunnanensis in Southwest China is Phaeolus yunnanensis. In addition, three taxa distributed in North America differ from P. schweinitzii sensu stricto. Phaeolus tabulaeformis (Berk.) Pat. is in Southeast North America, “P. schweinitzii-1” in Northeast North America, and “P. schweinitzii-2” in western North America.


Introduction
Phaeolus (Pat.) Pat. is a well-known polypore genus because of its type species; Phaeolus schweinitzii (Fr.) Pat. has conspicuous and colorful basidiocarps and is the major cause of butt rotting in many commercial timber species (Gilbertson and Ryvarden, 1987;Sinclair et al., 1987;Núñez and Ryvarden, 2001;Ryvarden and Melo, 2017). The genus is characterized by annual, pileate to stipitate, orange to brown basidiocarps with a fibrous to spongy context, a monomitic hyphal system with simple septate hyphae, the presence of gloeoplerous hyphae and cystidia, ellipsoid to cylindric, .
During investigations on brown-rot fungi in China, specimens morphologically similar to P. schweinitzii were collected from Northeast (NE) and Southwest (SW) China, but they are different from the European P. schweinitzii based on our preliminary phylogenetic study. Thus, it appears that P. schweinitzii is a species complex with more independent species that exist in the Northern Hemisphere. Then, more samples from North America and Europe were added for the multigene phylogeny, and six independent lineages were formed. After morphological examinations and phylogenetic and geographical analyses, six taxa are recognized in the P. schweinitzii complex; two taxa are confirmed as new members of Phaeolus, and they are pathogens on coniferous trees in NE and WS China. The present study aims to clarify the Chinese species of Phaeolus and outline the phylogeny of Phaeolus based on available data in the Northern Hemisphere.

Morphological studies
The examined specimens are deposited at the herbaria of the Institute of Microbiology, Beijing Forestry University (BJFC), and the Institute of Applied Ecology, Chinese Academy of Sciences (IFP). Macro-morphological descriptions were based on field notes and measurements of voucher herbarium specimens. Microscopic measurements and drawings were obtained from the slides prepared from voucher specimens and stained with Cotton Blue and Melzer's reagent following Wu et al. (2022) using a Nikon Eclipse 80i microscope. The following abbreviations were used in the description: CB = Cotton Blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, L = mean spore length (arithmetic average of spores), W = mean spore width (arithmetic average of spores), Q = variation in the L/W ratios between specimens studied, n (a/b) = number of spores (a) measured from a given number of specimens (b). In presenting spore size variation, mean ± SD (standard deviation at 95% confidence) was reported as the range; 5% of measurements were excluded from each end of the range, and the values are given in parentheses. Special color terms follow Anonymous (1969) and Petersen (1996). Herbarium abbreviations follow Thiers (2022).

DNA extraction and sequencing
The Rapid Plant Genome kit based on acetyl trimethylammonium bromide extraction (Aidlab Biotechnologies Co., Ltd, Beijing, China) was used to extract total genomic DNA from dried specimens and for polymerase chain reaction (PCR), according to the manufacturer's instructions with some modifications (Song and Cui, 2017;Xing et al., 2018). The ITS region was amplified with the primer pairs ITS5 and ITS4 (White et al., 1990). The nLSU region was amplified with primer pairs LR0R and LR7 (http:// www.biology.duke.edu/fungi/mycolab/primers.htm).
The PCR procedure was followed by Yuan et al. (2021). The PCR products were purified and sequenced at the Beijing Genomics Institute, China, with the same primers as in the original PCR amplifications.

Phylogenetic analyses
The phylogenetic tree was constructed using sequences obtained in this study and additional sequences downloaded from GenBank (Table 1). The sequences were aligned within MAFFT version 7 (Katoh et al., 2019) and ClustalX (Thompson et al., 1997), then manual proofreading was performed in BioEdit (Hall, 1999). The downloaded sequences were chosen to cover Laetiporaceae Jülich and related clades, including Fomitopsidaceae Jülich and Sparassidaceae Herter (Justo et al., 2017;Song and Cui, 2017;Song et al., 2018). Ambiguous regions were deleted, and gaps were manually adjusted to optimize alignment before phylogenetic analyses. Sparassis latifolia Y. C. Dai and Zheng Wang was used as an outgroup in the phylogeny of Phaeolus (Zhao et al., 2013; Figure 1). The data matrix was edited in Mesquite version 3.04 software. Phylogenetic analyses were performed with maximum parsimony (MP), maximum likelihood (ML), and Bayesian Inference (BI) based on ITS + nLSU aligned datasets.
MP analysis was applied to the dataset containing the ITS + nLSU sequences. The tree construction procedure was .
/fmicb. . performed using PAUP * version 4.0b10 (Swofford, 2002). All characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed, and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1,000 replicates (Felsenstein, 1985). Descriptive tree statistics: tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RCI), and homoplasy index (HI) were calculated for each maximum parsimonious tree generated. jModeltest v.2.17 (Darriba et al., 2012) was used to determine the best-fit evolution model of the combined dataset for ML and BI. Four unique partitions were established; GTR + I + G was the selected substitution model for each partition. RAxML version 8.2.12 (Stamatakis, 2014) was used for ML analysis with default parameters. Only the best maximum likelihood tree from all searches was kept.
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 10 million generations and started from random trees. Trees were sampled every 100 generations. The first 25% of sampled trees were discarded as burnin, whereas other trees were used to construct a 50% majority consensus tree and for calculating Bayesian posterior probabilities (BPPs).
Basidiocarps: Basidiocarps annual, pileate to laterally or centrally stipitate on substrate or ground from roots, soft and watery when fresh, and fragile and light in weight when dry. Pilei imbricate, sometimes developing a large number of imbricate, petaloid, or flabelliform and often confluent pilei, circular or semicircular to fan-shaped with irregularly lobed margin, projecting up to 15, 26 cm wide, up to 2 cm thick at the base or center; pileal surface cream, buff, orange when juvenile, pinkish buff, reddish brown to fuscous with age, concentrically zonate with various shades of cinnamon, brown, reddish brown to fuscous colors when fresh, become tomentose to hirsute, umber, rusty tawny to dark brown, indistinctly concentrically zonate when dry; margin acute, incurved when dry. Pore surface greenish yellow, citrine to sulfur yellow, become dark brown when bruised, rusty brown, bay, date brown to purplish chestnut when dry, sterile margin distinct, up to 3 mm wide; pores irregular and labyrinthine, 1-2 per mm; dissepiments thick, entire to lacerate. Context concolorous with a pileal surface, soft corky to fibrous, azonate, up to 10 mm thick. Tubes are pinkish buff to curry yellow, paler than the context, fragile when dry, decurrent, and up to 10 mm long. Stipe central or lateral, sometimes branched, concolorous with a pileal surface, and up to 2 cm long and 1 cm in diameter.
Context: Generative hyphae are hyaline to yellowish, thin-to slightly thick-walled with a wide lumen, occasionally branched, loosely interwoven, encrusted by crystals, and 4.2-15 µm in diameter; gloeoplerous hyphae are present, thin-walled, dark blue in CB.
Basidiocarps: Basidiocarps annual, pileate to laterally stipitate, always on wood, soft when fresh, corky to fragile, and light in weight when dry. Pilei applanate, semicircular to fan-shaped with lobed margin, projecting up to 9, 14 cm wide, and 13 mm thick at the base or center. Upper surface buff, fawn to vinaceous brown, concentrically zonate with various shades of buff, cinnamon, vinaceous brown to bay colors when fresh, tomentose to hirsute, indistinctly concentrically zonate when dry; margin acute or blunt, curved inwards when dry. Pore surface cream, greenish yellow to sulfur yellow, slightly glistening, become dark brown when bruised, cinnamon buff, cinnamon, rusty brown, bay to purplish chestnut when dry; sterile margin indistinct to almost lacking; pores irregular and labyrinthine, 0.5-1 per mm; dissepiments thin, entire in juvenile to lacerate to dentate with age. Context concolorous with a pileal surface, azonate, and corky when dry, up to 10 mm thick. Tubes are buff to orange-yellow, paler than the context, fragile when dry, decurrent, and up to 3 mm long. Stipe central or lateral, unbranched, concolorous with a pileal surface, up to 1 cm long and 1 cm in diameter.
Context: Generative hyphae are hyaline to yellowish, thin-to slightly thick-walled with a wide lumen, occasionally branched, .
Phaeolus asiae-orientalis and P. yunnanensis are common species in both natural and planted coniferous forests in China; they were previously considered P. schweinitzii, a forest pathogen in the Chinese forests (Dai et al., 2007;Dai, 2012). The present results demonstrate that the three pathogenetic species of Phaeolus exist in China: viz. P. asiae-orientalis mostly on Larix gmelinii, L. olgensis, Picea jezoensis, and Pinus koraiensis in Northeast China, Phaeolus yunnanensis usually on Pinus yunnanensis in Southwest China, and P. schweinitzii on Larix in Northwest China. P. asiae-orientalis is the cause of butt rot in natural coniferous forests in Northeast China, while P. yunnanensis causes butt rot in planted coniferous forests in Southwest China.
Phaeolus schweinitzii was also reported in New Zealand and Australia on species of Araucariaceae and Myrtaceae (Cunningham, 1965;Buchanan and Ryvarden, 2000) and in the tropical pacific areas of Hawaii Island on Acacia (the Bega, 1979). The taxon differs from P. schweinitzii sensu stricto and will be published by another team (personal communication).
Phaeolus amazonicus M.A. De Jesus & Ryvarden was described from Brazil, and it differs from the members of the genus in the Northern Hemisphere by its dimitic hyphal structure (Jesus and Ryvarden, 2010). Phaeolus manihotis (Heim, 1931) was described from Madagascar. It is different from our two new species by the presence of cuticle or crust at the upper surface and the absence of cystidia (Heim, 1931). Phaeolus subbulbipes (Henn.) O. Fidalgo & M. Fidalgo was also originally described from Brazil, and it differs from our new species by its smaller and globose basidiospores measuring 3.5-4.1 µm (Fidalgo and Fidalgo, 1957).

Data availability statement
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