Global Diversity and Taxonomy of Sidera (Hymenochaetales, Basidiomycota): Four New Species and Keys to Species of the Genus

The genus Sidera is a polypore genus with resupinate, white to cream or buff fresh basidioma, poroid or hydnoid hymenophore, a monomitic or dimitic hyphal system with generative hyphae bearing clamp connections, the presence of rosette-like crystals and allantoid to lunate basidiospores. We study the phylogeny and diversity of Sidera herein by using both morphological and molecular methods. Phylogenetic analyses are based on the ITS dataset, the combined 2-locus dataset (5.8S + nLSU) and 7-locus dataset (ITS + nLSU + RPB1 + RPB2 + TEF1 + mtSSU + nSSU) of 15 taxa of Sidera all over the world. Among them, four species are new to science and described and illustrated in this paper, viz. S. inflata, S. malaysiana, S. punctata and S. roseo-bubalina. In addition, three taxa were treated as Sidera vulgaris sensu lato. An identification key of the 14 accepted species of Sidera worldwide is provided.

During investigations on the diversity of polypores in tropical Asia, four resupinate polypore specimens were collected from China and Malaysia. They were characterized by a monomitic or dimitic hyphal system with generative hyphae bearing clamp connections, the presence of rosette-like crystals and allantoid to lunate basidiospores. These morphological characteristics demonstrated that these specimens may represent species of Sidera. To confirm their affinities, phylogenetic analyses were carried out based on the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), the translation elongation factor 1-α gene (TEF1), the small subunit mitochondrial rRNA gene sequences (mtSSU) and the small subunit (nSSU) of nuclear ribosomal RNA gene. As a result, these specimens were found to represent four new terminal lineages in the Sidera clade. In addition, the specimens or literature and sequences of all 14 currently accepted taxa of Sidera were studied, with their morphological characteristics summarized in Table 1. Furthermore, an identification key of accepted species is provided. Table 1. The main characteristics of Sidera species. Pore and basidiospore sizes partly from Du et al. (2020) [2], Rajchenberg (1987) [4], Du et al. (2019) [7], Niemelä and Dai (1997) [8] and Niemelä (2005) [9].

Morphological Studies
Morphological descriptions were based on field notes and dry herbarium specimens. Microscopic measurements and drawings were made from slide preparations of dry tissues stained with Cotton Blue and Melzer's reagent following Dai (2010) [10]. Pores were measured by subjectively choosing the straightest line of pores possible and measuring how many fit per mm. The following abbreviations were used: KOH = 5% potassium hydroxide; CB = Cotton Blue; CB-= acyanophilous in Cotton Blue; IKI = Melzer's reagent; IKI-= neither amyloid nor dextrinoid in Melzer's reagent; 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 specimens studied; n (a/b) = number of spores (a) measured from given number of specimens (b). In presenting spore size variation, 5% of measurements were excluded from each end of the range, and this value is given in parentheses. Special color terms follow Anonymous (1969) and Petersen (1996) [11,12]. Herbarium abbreviations follow Thiers (2018) [13]. The studied specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC).
The PCR procedure for ITS and mtSSU was as follows: initial denaturation at 95 • C for 3 min, followed by 34 cycles at 94 • C for 40 s, 54 • C for ITS and 55 • C for mtSSU for 45 s and 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR procedure for nLSU, nSSU and TEF1 was as follows: initial denaturation at 94 • C for 1 min, followed by 34 cycles at 94 • C for 30 s, 50 • C for nLSU and 59 • C for TEF1 for 1 min and 72 • C for 1.5 min, and a final extension of 72 • C for 10 min. The PCR procedure for RPB1 and RPB2 was as follows: 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 in the Beijing Genomics Institute, China, with the same primers used in the PCR reactions.
Sequences were also analyzed using maximum likelihood (ML) with RAxML-HPC through the CIPRES Science Gateway ( [24]; http://www.phylo.org (accessed on 24 March 2021)). Statistical support values (BS) were obtained using nonparametric bootstrapping with 1000 replicates. The optimal substitution models for the combined dataset were determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.2 [25] after scoring 24 models of evolution by PAUP* version 4.0 beta 10 [26]. The selected model applied in the Bayesian phylogenetic inference (BI) analyses and maximum likelihood (ML) analyses was the model GTR + I + G.
The BI analysis was performed with MrBayes 3.2.5 [27]. Four Markov chains were run for two runs from random starting trees for 3 million generations (ITS, 5.8S + nLSU and ITS + nLSU + RPB1 + RPB2 + TEF1 + mtSSU + nSSU) until the split deviation frequency value reached <0.01, and trees were sampled every 1000 generation. The first 25% of the sampled trees were discarded as burn-in, and the remaining ones were used to reconstruct a majority rule consensus tree and calculate Bayesian posterior probabilities (BPP) of the clades.

Phylogenetic Analyses
ITS is the most important locus for barcoding fungi, especially at the generic and species levels. However, the genetic variation inside Sidera is significant and, for example, the alignment of the whole ITS region is difficult among Sidera polypores [1]. Therefore, we used the most stable and conservative portion of ITS (5.8S) and LSU to analyse the phylogenetic relationship of Sidera species (Figure 1). The concatenated 5.8S + nLSU dataset contained sequences from 46 fungal specimens representing 15 Sidera taxa (three are treated as S. vulgaris sensu lato; ( Table 2). Sequences of Exidia candia and Exidiopsis calcea were used as the outgroup [1,12,23]. The best model for the 5.8S + nLSU dataset estimated and applied in the Bayesian analysis was GTR + I+G. The ML analysis resulted in the best tree ( Figure 1). BI analyses resulted in almost identical tree topologies compared to the ML analysis, with an average standard deviation of split frequencies of 0.009930 (BI). Additionally, only the ML tree is presented along with the support values from the BI analyses. Our new species and their related species are nested in Group A and Group B. Thus, we used the whole ITS region to analyse the phylogenetic relationship of new Sidera species with their closely related species (Figure 2).  The ITS dataset contained sequences from ten fungal specimens representing eight Sidera taxa (four new species and another four Sidera taxa). The sequence of Sidera lunata was used as the outgroup. The best model for the ITS dataset estimated and applied in the Bayesian analysis was GTR + I + G. The ML analysis resulted in the best tree ( Figure 2). BI analyses resulted in almost identical tree topologies compared to the ML analysis, with an average standard deviation of split frequencies of 0.001361 (BI). Thus, only the ML tree is presented along with the support values from the BI analyses.
The concatenated ITS + nLSU + RPB1 + RPB2 + TEF1 + mtSSU + nSSU dataset contained sequences from 46 fungal specimens representing 15 Sidera taxa (three were treated as S. vulgaris sensu lato; Table 2). Sequences of Exidia candia and Exidiopsis calcea were used as the outgroup [1,12,23]. The best model for the ITS + nLSU + RPB1 + RPB2 + TEF1 + mtSSU + nSSU dataset estimated and applied in the Bayesian analysis was GTR + I+G. The ML analysis resulted in the best tree ( Figure 3). BI analyses resulted in almost identical tree topologies compared to the ML analysis, with an average standard deviation of split frequencies of 0.009821 (BI). Additionally, only the ML tree is presented along with the support values from the BI analyses.
The phylogenetic trees (Figures 2 and 3) revealed four new and independent lineages represented by our specimens, indicating that they are phylogenetically distinct from the species currently known in the genus. In addition, another three taxa were treated as S. vulgaris sensu lato, although they formed four independent lineages too.

1.
Sidera Basidioma-Annual, resupinate, soft corky when fresh and dry, up to 4 cm long, 1.5 cm wide and less than 1 mm thick at center; pore surface white to buff and shiny when fresh, becoming cream to buff yellow and shiny when dry; sterile margin distinct, white, cottony, thinning out; pores angular, 9-10 per mm; dissepiments thin, lacerate; subiculum very thin to almost absent; tubes concolorous with the poroid surface, less than 1 mm long.
Notes-Sidera inflata was found in China, and the species is characterized by annual, resupinate basidioma with a white to buff fresh pore surface, which becomes cream to buff-yellow upon drying; angular pores (9-10 per mm); a dimitic hyphal system; skeletal hyphae at dissepiment edge bearing abundant fine thorn-like crystals; skeletal hyphae in all structures obviously swelling in KOH; and allantoid basidiospores measuring 3-3.3 × 0.9-1.1 µm. Morphologically, S. inflata can be distinguished from other species in Sidera by its skeletal hyphae at the dissepiment edge, which are usually covered by abundant fine thorn-like crystals. Although S. inflata clustered together with the S. vulgaris sensu lato (Dai 21057 and Dai 22151) with a moderate support (87% BS, 0.94 BPP; Figure 3), the latter taxon had perennial basidioma, and its skeletal hyphae were unchanged in KOH.  Basidioma-Annual, resupinate, very difficult to separate from substrate, soft corky when fresh and dry, up to 4 cm long, 2 cm wide and less than 1 mm thick at center; pore surface white to cream when fresh and dry; sterile margin indistinct; pores round to angular, 9-11 per mm; dissepiments thin, entire; subiculum very thin to almost absent; tubes white, less than 1 mm long.
Notes-Sidera malaysiana was found in Malaysia, and the species is characterized by annual, resupinate basidioma with a white to cream pore surface, round to angular pores (9-11 per mm), a dimitic hyphal system, skeletal hyphae in all structures become slightly swollen in KOH, subicular skeletal hyphae bearing rosette-like crystals, irregular crystals and fine thorn-like crystals and lunate basidiospores measuring 2.9-3.2 × 1-1.2 µm. Morphologically, S. malaysiana can be distinguished from other species in Sidera by its subicular skeletal hyphae, which are usually covered by abundant irregular crystals and fine thorn-like crystals. S. malaysiana is closely related to S. srilankensis in our phylogeny (100% BS, 1.00 BPP; Figure 2), but S. malaysiana is different from S. srilankensis due to its smaller pores (9-11 per mm vs. 6-8 per mm, [2]) and skeletal hyphae in all structures becoming slightly swollen in KOH, while skeletal hyphae are unchanged in KOH in S. srilankensis. S. malaysiana resembles S. parallela due to its white fresh pore surface. However, S. malaysiana differs from S. parallela due to its smaller pores (9-11 per mm vs. 6-8 per mm, [2]). In addition, tramal hyphae are parallel in S. parallela, while they are interwoven in S. malaysiana. Additionally, they are phylogenetically distant (Figure 2).  Basidioma-Annual, resupinate, soft corky when fresh and dry, up to 13 cm long, 4 cm wide and less than 1 mm thick at center; pore surface white to cream when fresh, becoming cinnamon buff to white when dry; sterile margin distinct, white, cottony, thinning out; pores round, 8-9 per mm; dissepiments thin, entire; subiculum very thin to almost absent; tubes darker than the poroid surface, less than 1 mm long.
Notes-Sidera punctata was discovered in China, and the species is characterized by annual, resupinate basidioma with a white to cream fresh pore surface which becomes cinnamon-buff to white upon drying, round pores (8-9 per mm), a monomitic hyphal system, and allantoid to lunate basidiospores measuring 3.8-4.8 × 1-1.3 µm. Phylogenetically, S. punctata is close to S. vesiculosa (100% BS, 1.00 BPP; Figure 2) and it is similar to S. vesiculosa by annual, resupinate basidioma and some generative hyphae with swollen tips, but S. punctata has a cinnamon-buff to white dry pore surface, while the pore surface is cream upon drying in S. vesiculosa. Above all, S. punctata can be distinguished from other species in Sidera by its rosette-like crystals are more abundant in subiculum than in tubes, while rosette-like crystals are more abundant in tubes than in subiculum in other members of the genus. Basidioma-Annual, resupinate, soft corky when dry, up to 7 cm long, 3 cm wide, and less than 1 mm thick at center; pore surface pinkish buff to yellowish brown when dry; sterile margin distinct, white, cottony, thinning out; pores round, 6-7 per mm; dissepiments thin, entire to lacerate; subiculum very thin to almost absent; tubes concolorous with the poroid surface, less than 1 mm long.

5.
Sidera vulgaris sensu lato Previously, the ten species of Sidera, viz. S. lenis, S. lowei, S. lunata, S. minutipora, S. minutissima, S. parallela, S. srilankensis, S. tenuis, S. vesiculosa and S. vulgaris were described or transferred to the genus. In this paper, S. inflata, S. malaysiana, S. punctata and S. roseobubalina were described as new to science, and they have resupinate, white to cream or buff fresh basidiocarps; a monomitic or dimitic hyphal system with generative hyphae bearing clamp connections; the presence of rosette-like crystals; and allantoid to lunate basidiospores. These characteristics fit well with the generic concept of Sidera. Thus far, 14 species are accepted in Sidera, and a key of accepted species is provided below.

Discussion
As is well known, the genus Sidera is challenging for distinguishing species morphologically, and two species, S. lenis and S. vulgaris, were recognized before phylogenetical analyses.
Poria earlei, P. montana and P. tenuipora were described from the Caribbean (Jamaica) (Murrill [31,32]). The type of specimens of three species were studied by Niemelä and Dai [8]. They found that P. earlei and P. montana are conspecific, and the species is perennial, resupinate and has small pores (7-9 per mm). Sidera punctata and S. vesiculosa have similar pores (8-9 per mm in S. punctata vs. 7-9 per mm in S. vesiculosa [7]) to P. tenuipora as well, but the former two species have a monomitic hyphal system, while P. tenuipora has a dimitic hyphal system. S. vesiculosa differs from S. punctata due to the presence of vesicular cells of swollen hyphae in the subiculum.
Du et al. [2] summarized three synonyms of Sidera vulgaris (Index Fungorum and MycoBank): Boletus papyraceus Schrank, B. proteus Bolton and B. cellulosus O.F. Müll, and all of them were originally described from Europe. In addition, the specimen Ryvarden 37198 from New Zealand was named S. vulgaris by Miettinen and Larsson too [1]. In the present paper, we found three taxa with similar morphologies to S. vulgaris, but we did not study the types of the above-mentioned taxa, and no sequence data are available for them. Although our three taxa formed three distinct lineages in our phylogenies (Figure 3), we refrained from describing these as new, and treated them as S. vulgaris sensu lato in this paper ( Table 2). The description of these species is the subject of a forthcoming paper.
The sequence of OTU1581 is from GenBank. We failed to obtain specimens of the taxa, but it formed a distinct lineage within the Sidera clade, so we treated OTU1581 as Sidera sp. temporarily here.
Polypores are an extensively studied group of Basidiomycota, and more than 1500 species have been recorded in the world [36][37][38][39][40][41][42]. Molecular phylogenies have demonstrated that more new taxa exist in the world [43][44][45][46][47], and more crypto species will be confirmed after molecular analyses of some traditional species in sensu lato. Thus, in order to understand the diversity, phylogeny and evolution of the fungi, future taxonomic and phylogenetic work should be based on both molecular and morphological characteristics.  Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/; https://www.mycobank.org/page/Simple%20 names%20search; http://purl.org/phylo/treebase, submission ID 27909, 27910 and 27911.