﻿Species diversity, molecular phylogeny and ecological habits of Cyanosporus (Polyporales, Basidiomycota) with an emphasis on Chinese collections

﻿Abstract Cyanosporus is a genus widely distributed in Asia, Europe, North America, South America and Oceania. It grows on different angiosperm and gymnosperm trees and can cause brown rot of wood. Blue-tinted basidiomata of Cyanosporus makes it easy to distinguish from other genera, but the similar morphological characters make it difficult to identify species within the genus. Phylogeny and taxonomy of Cyanosporus were carried out based on worldwide samples with an emphasis on Chinese collections, and the species diversity of the genus is updated. Four new species, C.flavus, C.rigidus, C.subungulatus and C.tenuicontextus, are described based on the evidence of morphological characters, distribution areas, host trees and molecular phylogenetic analyses inferred from the internal transcribed spacer (ITS) regions, the large subunit of nuclear ribosomal RNA gene (nLSU), the small subunit of nuclear ribosomal RNA gene (nSSU), the small subunit of mitochondrial rRNA gene (mtSSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α gene (TEF). Our study expanded the number of Cyanosporus species to 35 around the world including 23 species from China. Detailed descriptions of the four new species and the geographical locations of the Cyanosporus species in China are provided.

Previously, species identification of the P. caesia complex was only based on morphological characters and host trees in China, and only two species were recorded from China before Dai (2012), viz., P. alni and P. caesia. Recently, taxonomic studies of P. caesia complex in China have been carried out, and some new species have been described based on both morphological characteristics and molecular data. Shen et al. (2019) carried out a comprehensive study on Postia and related genera, in which Cyanosporus was supported as an independent genus with 12 species were accepted in this genus. Liu et al. (2021a) studied the species diversity and molecular phylogeny of Cyanosporus and the number of Cyanosporus species was expanded to 31 around the world, including 19 species from China. These studies have greatly enriched the species of Cyanosporus in China. Currently, the morphological characteristics of the genus are as follows: basidiomata annual, pileate or resupinate to effused-reflexed, soft corky, corky to fragile. Pileal surface white to cream to greyish brown, usually with blue tint. Pore surface white to cream, frequently bluish; pores round to angular. Context white to cream, corky. Tubes cream, fragile. Hyphal system monomitic; generative hyphae clamped, IKI-, CB-. Cystidia usually absent, cystidioles occasionally present. Basidiospores narrow, allantoid to cylindrical, hyaline, usually slightly thickwalled, smooth, IKI-, weakly CB+.
Cyanosporus species usually have blue-tinted basidiomata, which makes it easy to recognize. Some specimens with blue-tinted basidiomata were collected during investigations into the diversity of polypores in China, and four undescribed species of Cyanosporus were discovered. To confirm the affinity of the undescribed species to Cyanosporus, phylogenetic analyses were carried out based on the combined datasets of ITS+TEF and ITS+nLSU+nSSU+mtSSU+RPB1+RPB2+TEF sequences. During the investigation and study of Cyanosporus, the information of host trees and distribution areas of species in the genus from China were also obtained (Table 1). Four new species are described and illustrated in the current study, and the geographical locations of the Cyanosporus species distributed in China are indicated on the map (Fig. 1).

Morphological studies
The examined specimens were deposited in the herbarium of the Institute of microbiology, Beijing Forestry University (BJFC), and some duplicates were deposited at the Institute of Applied Ecology, Chinese Academy of Sciences, China (IFP) and Southwest Forestry University (SWFC). Macro-morphological descriptions were based on the field notes and measurements of herbarium specimens. Special colour terms followed Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Cui et al. (2019) and Liu et al. (2021b). Sections were studied at a magnification up to × 1000 using a Nikon Eclipse 80i microscope and phase contrast  Miettinen et al. 2018 illumination (Nikon, Tokyo, Japan). Drawings were made with the aid of a drawing tube. Microscopic features, measurements and drawings were made from slide preparations stained with Cotton Blue and Melzer's reagent. Spores were measured from sections cut from the tubes. To present variation in the size of basidiospores, 5% of measurements were excluded from each end of the range and extreme values are given in parentheses.
In the text the following abbreviations were used: IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB + = cyanophilous, CB -= acyanophilous, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios between the specimens studied, n (a/b) = number of spores (a) measured from given number (b) of specimens.
The PCR cycling schedule for ITS, mtSSU and TEF included an initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 54 °C for ITS and mtSSU, 54-55 °C for TEF for 45 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min. The PCR cycling schedule for nLSU and nSSU included an initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 30 s, 50 °C for nLSU and 52 °C for nSSU for 1 min, 72 °C for 1.5 min, and a final extension at 72 °C for 10 min. The PCR procedure for RPB1 and RPB2 follow Justo and Hibbett (2011) with slight modifications: initial denaturation at 94 °C for 2 min, followed by 10 cycles at 94 °C for 40 s, 60 °C for 40 s and 72 °C for 2 min, then followed by 37 cycles at 94 °C for 45 s, 55 °C for 1.5 min and 72 °C for 2 min, and a final extension of 72 °C for 10 min. The PCR products were purified and sequenced at Beijing Genomics Institute (BGI), China, with the same primers. All newly generated sequences were deposited at GenBank (Table 1).
Most parsimonious phylogenies were inferred from the combined 2-gene dataset (ITS+TEF) and 7-gene dataset (ITS+nLSU+nSSU+mtSSU+RPB1+RPB2+TEF), and their congruences 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. Phylogenetic analyses approaches followed Liu et al. (2019) and Sun et al. (2020). In phylogenetic reconstruction, the sequences of Antrodia serpens (Fr.) Donk and A. tanakae (Murrill) Spirin & Miettinen obtained from GenBank were used as outgroups. Maximum parsimony analysis was applied to the combined multiple genes datasets, and the tree construction procedure was performed in PAUP* version 4.0b10. 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 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for each most Parsimonious Tree (MPT) generated. RAxmL v.7.2.8 was used to construct a maximum likelihood (ML) tree with a GTR+G+I model of site substitution including estimation of Gamma-distributed rate heterogeneity and a proportion of invariant sites (Stamatakis 2006). The branch support was evaluated with a bootstrapping method of 1000 replicates (Hillis and Bull 1993). The phylogenetic tree was visualized using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). MrModeltest 2.3 (Posada and Crandall 1998;Nylander 2004) was used to determine the best-fit evolution model for the combined multi-gene dataset for Bayesian inference (BI). Bayesian inference was calculated with MrBayes 3.1.2 with a general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Ronquist and Huelsenbeck 2003). Four Markov chains were run for 2 runs from random starting trees for 1.8 million generations (ITS+TEF), for 3.5 million generations (ITS+nLSU+nSSU+mtSSU+RPB1+RPB2+TEF) and trees were sampled every100 generations. The first one-fourth generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. Branches that received bootstrap support for maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP and ML) and 0.95 (BPP) were considered as significantly supported, respectively.

Phylogeny
The combined 2-gene (ITS+TEF) sequences dataset had an aligned length of 1015 characters, of which 502 characters were constant, 62 were variable and parsimony-uninformative, and 451 were parsimony-informative. MP analysis yielded 10 equally parsimonious trees (TL = 2396, CI = 0.379, RI = 0.735, RC = 0.279, HI = 0.621). The best model for the concatenate sequence dataset estimated and applied in the Bayesian inference was GTR+I+G with equal frequency of nucleotides. ML analysis resulted in a similar topology as MP and Bayesian analyses, and only the ML topology is shown in Fig. 2.
The combined 7-gene (ITS+nLSU+nSSU+mtSSU+RPB1+RPB2+TEF) sequences dataset had an aligned length of 5634 characters, of which 3843 characters were constant, 247 were variable and parsimony-uninformative, and 1544 were parsimony-informative. MP analysis yielded 23 equally parsimonious trees (TL = 5756, CI = 0.468, RI = 0.752, RC = 0.352, HI = 0.532). The best model for the concatenate sequence dataset estimated and applied in the Bayesian inference was GTR+I+G with equal frequency of nucleotides. ML analysis resulted in a similar topology as MP and Bayesian analyses, and only the ML topology is shown in Fig. 3.
The phylogenetic trees inferred from ITS+TEF and ITS+nLSU+nSSU+mtSSU+R PB1+RPB2+TEF gene sequences were all obtained from 106 fungal samples represent-  ing 65 taxa of Cyanosporus and its related genera within the antrodia clade. 74 samples representing 35 taxa of Cyanosporus clustered together and separated from species of Postia and other related genera. As for Cyanosporus, the sequences used in phylogenetic analyses include 28 holotype specimen sequences, one isotype specimen sequence and one neotype specimen sequence (Table 1) Diagnosis. Cyanosporus flavus is characterised by flabelliform to semicircular and hirsute pileus with ash grey to light vinaceous grey pileal surface when fresh, buff to lemon-chrome pore surface when dry, and allantoid and slightly curved basidiospores (4.6-5.2 × 0.8-1.3 μm).
Etymology. Flavus (Lat.): referring to its lemon-chrome pore surface when dry. Fruiting body. Basidiomata annual, pileate, soft and watery, without odour or taste when fresh, becoming corky to fragile and light in weight upon drying. Pileus flabelliform to semicircular, projecting up to 3.2 cm, 5.7 cm wide and 0.9 cm thick at base. Pileal surface ash-grey to light vinaceous grey when fresh, becoming pale mouse-grey to mousegrey when dry, hirsute; margin acute to slightly obtuse, white with a little blue tint when fresh, olivaceous buff to greyish brown when dry. Pore surface white to cream when fresh, becoming buff to lemon-chrome when dry; sterile margin narrow to almost lacking; pores angular, 5-7 per mm; dissepiments thin, entire to lacerate. Context white to cream, soft corky, up to 6 mm thick. Tubes pale mouse-grey to ash-grey, fragile, up to 4 mm long.
Spores. Basidiospores slim allantoid, slightly curved, hyaline, thin-to slightly thick-walled, smooth, IKI-, CB-, 4.6-5.  Diagnosis. Cyanosporus rigidus is characterised by corky, hard corky to rigid basidiomata with a buff yellow to clay-buff and tomentose pileal surface when fresh, becom-   Etymology. Rigidus (Lat.): referring to the rigid basidiomata. Fruiting body. Basidiomata annual, pileate, corky, without odour or taste when fresh, becoming hard corky to rigid upon drying. Pileus flabelliform, projecting up to 1.6 cm, 3.8 cm wide and 0.6 cm thick at base. Pileal surface tomentose, buff yellow to clay-buff, when fresh, becoming smooth, rugose, olivaceous buff to greyish brown when dry; margin obtuse. Pore surface white to cream when fresh, becoming buff-yellow to pinkish buff when dry; sterile margin narrow to almost lacking; pores angular, 5-8 per mm; dissepiments thin, entire to lacerate. Context cream to buff, hard corky, up to 4 mm thick. Tubes cream to pinkish buff, brittle, up to 5 mm long.
Type of rot. Brown rot. Diagnosis. Cyanosporus subungulatus is characterised by shell-shaped pileus with a pale mouse-grey to ash-grey pileal surface when fresh, dark-grey to mouse-grey when dry, allantoid to cylindrical and slightly curved basidiospores (4.5-5.2 × 1. Fruiting body. Basidiomata annual, pileate, soft corky, without odour or taste when fresh, becoming corky to fragile and light in weight upon drying. Pileus shell-shaped, projecting up to 1.7 cm, 2.8 cm wide and 1.2 cm thick at base. Pileal surface velutinate, pale mouse-grey to ash-grey when fresh, becoming smooth, rugose, dark-grey to mouse-grey when dry; margin obtuse. Pore surface white to cream when fresh, becoming cream to pinkish buff when dry; sterile margin narrow to almost lacking; pores round, 4-6 per mm; dissepiments thin, entire to lacerate. Context white to cream, soft corky, up to 5 mm thick. Tubes pale mouse-grey to ash-grey, fragile, up to 6 mm long. Hyphal structure. Hyphal system monomitic; generative hyphae with clamp connections, IKI-, CB-; hyphae unchanged in KOH.
Etymology. Tenuicontextus (Lat.): referring to the species having thin context. Fruiting body. Basidiomata annual, pileate, soft corky, without odour or taste when fresh, becoming corky to fragile and light in weight upon drying. Pileus flabelliform, projecting up to 1.3 cm, 3.2 cm wide and 0.5 cm thick at base. Pileal surface velutinate, cream to pinkish buff with a little blue tint when fresh, becoming glabrous, light vinaceous grey to pale mouse-grey when dry; margin acute. Pore surface white to cream when fresh, becoming pinkish buff to buff when dry; sterile margin narrow to almost lacking; pores round, 6-8 per mm; dissepiments thin, entire to lacerate. Context cream to buff, soft corky, up to 0.8 mm thick. Tubes pale mouse-grey to buff, fragile, up to 4.3 mm long.

Discussion
In the current phylogenetic analyses based on the combined datasets of ITS+TEF and ITS+nLSU+mtSSU+nSSU+RPB1+RPB2+TEF sequences, species of Cyanosporus formed a highly supported lineage, distant from Postia and other brown-rot fungal genera (Figs 2, 3) and consistent with previous studies (Shen et al. 2019;Liu et al. 2021a). Based on morphological characters and phylogenetic analyses, 35 species are accepted in Cyanosporus around the world, including four new species from China, viz., C. flavus, C. rigidus, C. subungulatus and C. tenuicontextus. The main ecological habits of the species in Cyanosporus with an emphasis on distribution areas and host trees are provided in Table 2.
Phylogenetically, Cyanosporus rigidus form a separate lineage different from other species in the genus. Morphologically, C. submicroporus share similar pores and basidiospores with C. rigidus, but C. submicroporus differs by having cream to pinkish buff pileal surface and white to smoke grey pore surface when fresh, buff to buff-yellow pileal surface and buff to olivaceous buff pore surface when dry. Cyanosporus auricomus and C. luteocaesius resemble C. rigidus in morphology by producing yellow-colored basidiomata, but C. auricomus differs from C. rigidus by having a hirsute pileal surface and larger basidiospores (4.4-5.6 × 1.5-1.8 μm; Miettinen et al. 2018); C. luteocaesius differs from C. rigidus by having larger pores (3-5 per mm) and basidiospores (4.3-6.1 × 1.5-1.9 μm; Miettinen et al. 2018). to the complex and diverse ecological environment and diverse host trees in this region, which provide a rich substrate for the growth of Cyanosporus species. The geographical locations of the Cyanosporus species distributed in China are indicated on the map (Fig. 1).
In summary, we performed a comprehensive study on the species diversity and phylogeny of Cyanosporus with an emphasis on Chinese collections. So far, 35 species are accepted in the Cyanosporus around the world, including 23 species from China. Currently, Cyanosporus is characterized by an annual growth habit, resupinate to effused-reflexed or pileate, soft corky, corky, fragile to hard corky basidiomata, velutinate to hirsute or glabrous pileal surface with blue-tinted, white to cream or yellow-colored, white to cream pore surface with round to angular pores, a monomitic hyphal system with clamped generative hyphae, and hyaline, thin-to slightly thick-walled, smooth, narrow, allantoid to cylindrical basidiospores that are usually weakly cyanophilous; it grows on different angiosperm and gymnosperm trees, causes a brown rot of wood and has a distribution in Asia, Europe, North America, Argentina in South America and Australia in Oceania (McGinty 1909;Shen et al. 2019;Liu et al. 2021a).