Species Diversity and Molecular Phylogeny of Cyanosporus (Polyporales, Basidiomycota)

Cyanosporus is a cosmopolitan brown-rot fungal genus, recognizable by blue-tinted basidiocarps. Species in this genus were usually treated as belonging to the Postia caesia complex, however, recent phylogenetic analyses showed that this complex represents an independent genus. During further studies on Cyanosporus, five new species were discovered based on morphological features and molecular data. Phylogenetic analyses of Cyanosporus were conducted using 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); illustrated descriptions of the new species are provided. In addition, fifteen species previously belonging to the Postia caesia complex are transferred to Cyanosporus and proposed as new combinations.


INTRODUCTION
Boletus caesius Schrad. was described based on material from Germany (Schrader, 1794), and this name was subsequently sanctioned by Fries (1821), who considered B. coeruleus Schumach. a synonym of Polyporus caesius (Schrad.) Fr. In 1881 Karsten transferred Boletus caesius to Postia Fr. as Postia caesia (Schrad.) P. Karst. (Karsten, 1881). Murrill (1907) transferred this species to Tyromyces P. Karst. as T. caesius (Schrad.) Murrill and later McGinty (1909) proposed a new monotypic genus Cyanosporus McGinty for Polyporus caesius, based on its cyanophilous basidiospores, but Cyanosporus caesius was not widely used in subsequent studies (Donk, 1960;Jahn, 1963;Lowe, 1975), while Tyromyces caesius was commonly used. Later, Postia caesia was widely used (Papp, 2014). David (1974David ( , 1980 described another two species: P. luteocaesia (A. David) Jülich and P. subcaesia (A. David) Jülich from Europe besides Postia caesia (Schrad.) P. Karst. Jahn (1979) noted that David's P. subcaesia included many forms and introduced P. subcaesia "f. minor, " which was described as P. alni Niemelä & Vampola by Niemelä et al. (2001). Subsequently, Pieri and Rivoire (2005) introduced the fifth European species, P. mediterraneocaesia M. Pieri & B. Rivoire in the Postia caesia complex. Papp (2014) provided a detailed nomenclatural review on the Postia caesia complex and proposed the subgenus Postia subg. Cyanosporus (McGinty) V. Papp for this complex which included five species viz., P. alni, P. caesia, P. luteocaesia, P. mediterraneocaesia and P. subcaesia. Miettinen et al. (2018) focused on the taxonomy of the Postia caesia complex based on morphological features and molecular evidence and increased the species number of the Postia caesia complex from 10 to 24. In their study, they only focused on the species concept of the Postia caesia complex, the taxonomic status of this complex among Postia and related genera are not mentioned. Shen et al. (2019) carried out a comprehensive study on Postia and related genera and confirmed that the genus Cyanosporus is an independent genus rather than subgenus which containing 12 accepted species including seven new species of the Postia caesia complex. Furthermore, phylogenetic analyses showed that Cyanosporus belongs to the antrodia clade (Shen et al., 2019). Morphologically, Cyanosporus differs from other related genera by its bluish basidiocarps, usually narrow allantoid and thinto slightly thick-walled basidiospores (McGinty, 1909;Shen et al., 2019). In the current study, the phylogenetic analysis of Cyanosporus was carried out based on the combined sequence dataset of ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF rRNA gene regions. Combining morphological characters and molecular evidence, thirty-one species belonging to the Postia caesia complex are now recognized in Cyanosporus, including five new species and fifteen new combinations.

Taxa Sampling and Morphological Study
The examined specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC) with some duplicates at the Institute of Applied Ecology, Chinese Academy of Sciences (IFP). Morphological descriptions and abbreviations used in this study following Liu et al. (2019); Sun et al. (2020).

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); Song and Cui (2017). The primer pairs ITS5 and ITS4 for ITS regions, LR0R and LR7 for nLSU regions, NS1 and NS4 for nSSU regions, MS1 and MS2 for mtSSU regions, RPB1-Af and RPB1-Cr for RPB1 gene, fRPB2-f5F and bRPB2-7.1R for RPB2 gene, EF1-983 F and EF1-1567R for TEF gene used in this study are the same as previous studies (White et al., 1990;Rehner, 2001;Matheny et al., 2002;Matheny, 2005).
The PCR cycling schedules for different DNA sequences of ITS, nLSU, nSSU, mtSSU, RPB1, RPB2, and TEF genes used in this study followed those used in Liu et al. (2019); Shen et al. (2019), Zhu et al. (2019); Sun et al. (2020) with some modifications. 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" following Chen et al. (2017). The final concatenated sequence alignment was deposited in TreeBase ( 2 submission ID: 27274).

Molecular Phylogeny
The ITS + TEF sequences dataset had an aligned length of 1166 characters, of which 646 characters were constant, 68 were variable and parsimony-uninformative, and 452 were parsimonyinformative. MP analysis yielded 4 equally parsimonious trees (TL = 1983, CI = 0.417, RI = 0.726, RC = 0.303, HI = 0.583). 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. ML analysis resulted in a similar topology as MP and Bayesian analyses, and only the ML topology is shown in Figure 1.
The combined three-gene (ITS + nLSU + TEF) sequences dataset had an aligned length of 2475 characters, of which 1752   characters were constant, 134 were variable and parsimonyuninformative, and 589 were parsimony-informative. MP analysis yielded 8 equally parsimonious trees (TL = 2222, CI = 0.473, RI = 0.770, RC = 0.365, HI = 0.527). 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 Figure 2.
The combined seven-gene (ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF) sequences dataset had an aligned length of 5855 characters, of which 4194 characters were constant, 300 were variable and parsimony-uninformative, and 1361 were parsimony-informative. MP analysis yielded 10 equally parsimonious trees (TL = 3951, CI = 0.585, RI = 0.806, RC = 0.471, HI = 0.415). 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 Figure 3.
The phylogenetic trees (Figures 1-3) inferred from ITS + TEF, ITS + nLSU + TEF and ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF gene sequences were obtained from 99 fungal samples representing 45 taxa of Cyanosporus and its related genera in the antrodia clade. Seventy-five samples representing thirty-one taxa of Cyanosporus MycoBank: MB 838417 Differs from other Cyanosporus species by its tomentose, cream to buff pileal surface when fresh becoming cream to pinkish buff when dry and a white to cream pore surface when fresh becoming straw yellow to buff when dry, and round to angular small pores (5-8 per mm).
Basidiocarps. -Annual, pileate, solitary, soft and watery when fresh, becoming soft corky to fragile upon drying. Pileus shellshaped, projecting up to 2.5 cm, 3.5 cm wide and 1.5 cm thick at base. Pileal surface white to cream when fresh, finely tomentose, becoming cream to pinkish buff upon drying; margin acute. Pore surface white to cream when fresh, becoming straw yellow to buff when dry; sterile margin narrow to almost lacking; pores round to angular, 5-8 per mm; dissepiments thin, entire to lacerate. Context white, corky, up to 1.2 cm thick. Tubes cream, fragile, up to 5 mm long. FIGURE 1 | Maximum likelihood tree illustrating the phylogeny of Cyanosporus and its related genera in the antrodia clade based on sequences dataset of ITS + TEF. Branches are labeled with maximum likelihood bootstrap higher than 50%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.90 respectively. Bold names = New species.
Notes. -In the phylogenetic trees (Figures 1-3), two specimens of Cyanosporus bubalinus formed a highly supported lineage (Figures 1-3), closely related to C. coeruleivirens Cyanosporus and its related genera in the antrodia clade based on the combined sequences dataset of ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF. Branches are labeled with maximum likelihood bootstrap higher than 50%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.90 respectively. Bold names = New species.
Basidiocarps. -Annual, pileate, soft corky, without odor or taste when fresh, becoming corky to fragile upon drying. Pileus flabelliform to semicircular, projecting up to 5.2 cm, 9.5 cm wide and 1.5 cm thick at base. Pileal surface ash gray to light grayish brown with bluish gray zone when fresh, becoming grayish to grayish brown when dry, distinctly hirsute; margin acute. Pore surface cream when fresh, becoming straw yellow to olivaceous buff when dry; sterile margin narrow to almost lacking; pores angular, 5-7 per mm; dissepiments thin, entire. Context white, soft corky, up to 9 mm thick. Tubes cream, fragile, up to 7 mm long.
Basidiocarps. -Annual, effused-reflexed to pileate, solitary, soft and watery, without odor or taste when fresh, becoming soft corky to fragile upon drying. Pileus flabelliform to semicircular, projecting up to 2.4 cm, 4.2 cm wide and 0.7 cm thick at base. Pileal surface buff to olivaceous buff when fresh, finely tomentose, becoming smooth, pale mouse gray to buff yellow when dry; margin acute. Pore surface white to cream when fresh, becoming cream to buff yellow when dry; sterile margin narrow to almost lacking; pores angular, 4-6 per mm; dissepiments thin, entire to lacerate. Context white, soft corky, up to 4 mm thick. Tubes cream, fragile, up to 3 mm long.
-Cyanosporus caesius is also found in Tasmania, Australia. It has pileate or effused reflexed basidiocarps with a tomentose pileal surface and similar pores to C. nothofagicola, but it differs from the later by its plumbeous to bluish gray or grayish brown pileal surface .
Additional specimen ( (Figures 4G-H, 8) MycoBank: MB 838420 Differs from other Cyanosporus species by its cream to pinkish buff pileal surface and white to smoke gray pore surface when fresh, buff to buff yellow pileal surface and buff to olivaceous buff pore surface when dry.
Type Basidiocarps. -Annual, pileate, soft and watery, without odor or taste when fresh, becoming corky to woody hard upon drying. Pileus flabelliform to semicircular, projecting up to 3.2 cm, 6.5 cm wide and 1.3 cm thick at base. Pileal surface cream to pinkish buff when fresh, velutinate, becoming rugose, buff to buff yellow when dry; margin acute. Pore surface white to smoke gray when fresh, becoming buff to olivaceous buff when dry; sterile margin narrow to almost lacking; pores round, 6-9 per mm; dissepiments thin, entire. Context cream to buff, corky, up to 5 mm thick. Tubes pale mouse gray to cream, fragile, up to 7 mm long.
Basidiocarps. -Annual, effused-reflexed to pileate, soft corky, without odor or taste when fresh, becoming corky to fragile upon drying. Pileus flabelliform, projecting up to 1.5 cm, 1.8 cm wide and 0.4 cm thick at base. Pileal surface buff to olivaceous buff when fresh, finely tomentose, becoming cream to olivaceous buff when dry; margin acute. Pore surface white to cream when fresh, becoming buff yellow to pinkish buff when dry; sterile margin narrow to almost lacking; pores angular, 5-7 per mm; dissepiments thin, entire to lacerate. Context cream, soft corky, up to 2 mm thick. Tubes pinkish buff, fragile, up to 2 mm long.
Additional specimen ( Notes. -Postia arbuti was described from the United States . It is characterized by conchate, pendant to effused-reflexed basidiocarps with almost glabrous to matt pileal surfaces. It usually grows on Arbutus menziesii and is widely distributed in temperate areas of North America (North-West). In our study, the phylogenetic analysis (Figures 1-3 Notes. -Postia auricoma was described by Miettinen et al. (2018) and Cyanosporus mongolicus was described by Shen et al. (2019). Phylogenetically, the two species formed a highly supported lineage (Figures 1-3) and as the morphological characters of C. mongolicus fit well with Postia auricoma, we treat C. mongolicus as a synonym of P. auricoma, and P. auricoma is transferred to Cyanosporus as a new combination. Notes. -Postia bifaria was described by Miettinen et al. (2018). The morphological characters of the two specimens from China fit well with Postia bifaria and phylogenetically they clustered together within the genus Cyanosporus. Based on morphological characters and phylogenetic analysis, we transferred Postia bifaria to Cyanosporus as a new combination. For a detailed description of Postia bifaria, see Miettinen et al. (2018).

DISCUSSION
The genus Cyanosporus, usually with blue-tinted basidiocarps, is easy to recognize, but identification to species level is difficult as morphological features are quite similar among the species. The main morphological characters of the species in Cyanosporus are provided in Table 2.
In our current phylogenetic analyses, the genus Cyanosporus is supported as an independent genus; 31 species grouped together and formed a highly supported lineage (100% BS, 100% MP, 1.00 BPP; Figures 1-3), and were distant from other genera of Postia sensu lato. The current study also confirmed that Cyanosporus belongs to the antrodia clade phylogenetically and clusters with other brown-rot fungal genera, such as Amaropostia B.K. Cui
The nomenclatural and taxonomic history of the Postia caesia complex was critically reviewed by Papp (2014), the generic name of the P. caesia complex have been changed many times, Papp proposed Cyanosporus as a subgenus of Postia to contain the P. caesia complex. Miettinen et al. (2018) revised the species concept of the Postia caesia complex based on morphology and two gene markers (ITS and TEF) and discussed that TEF sequences are more reliable for molecular identification of the P. caesia complex than ITS sequences. They also indicated that host tree is important for species identification of the P. caesia complex; their study raised the species number of the Postia caesia complex from 10 to 24, but their study did not focus on the taxonomic status for the P. caesia complex of Postia sensu lato.
Previously, species identification of the Postia caesia complex was only based on morphological characters and host trees in China. Samples grown on angiosperm woods were usually identified as Postia alni Niemelä & Vampola and those on gymnosperm woods as Postia caesia; only two species were recorded from China before Dai (2012). Shen et al. (2019) carried out a comprehensive phylogenetic and taxonomic study of Postia and related genera based on morphological characters and the combined seven-gene (ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF) sequences, Cyanosporus was confirmed as an independent genus of Postia sensu lato and 12 species belonging to the P. caesia complex were recognized in Cyanosporus, which was distant from Postia s. s.
In the current study, the novel species were supported by phylogenetic analyses based on ITS + TEF sequences, ITS + nLSU + TEF sequences and ITS + nLSU + nSSU + mtSSU + RPB1 + RPB2 + TEF sequences, respectively, but for the ITS sequences, several species have very similar base pairs and could not be separated by ITS sequences. The suitable DNA barcoding gene is the TEF gene for species identification of Cyanosporus, this result is consistent with a previous study by Miettinen et al. (2018). Our study expanded the number of Cyanosporus species to 31 around the world including 19 species from China. Our study indicated that more cryptic species could be discovered by combined evidence of morphological characters, molecular data, host trees and distribution areas in species complexes. However, several species of the Postia caesia complex, such as, P. africana (Ryvarden) V. Papp, P. amyloidea (Corner) V. Papp, P. atrostrigosa (Cooke) Rajchenb. and P. caesioflava (Pat.) V. Papp, are not included in the current phylogenetic analysis due to the lack of DNA sequences. Although the morphological characters of these species fit well with Cyanosporus, we did not transfer them to Cyanosporus in the current study. A fully resolved phylogeny for Cyanosporus and its related genera requires evolutionary information from more samples and more conserved gene markers.
In addition, some species with yellow basidiocarps, such as C. auricoma, C. caesioflava, and C. luteocaesius, and some species with white to cream colored basidiocarps, such as C. bubalinus and C. nothofagicola, are grouped together within Cyanosporus; this expanded the concept of the genus to include taxa without blue-tinted basidiocarps. More novel species might be discovered from different regions in future studies.

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 below: http://purl.org/phylo/ treebase, 27274.