Taxonomy and phylogeny of Sidera (Hymenochaetales, Basidiomycota): four new species and keys to species of the genus

Abstract Sidera is a polypore genus with white to cream or buff basidiomata, whose species in Hymenochaetales are poorly known. We study the phylogeny and diversity of Sidera based on our recent collections from tropic and subtropic Asian-Pacific regions. Phylogenetic analyses based on the internal transcribed spacer (ITS) and nuclear large subunit (nLSU) ribosomal RNA gene regions indicate that ten terminal lineages are well supported within Sidera. Based on morphological examination and phylogeny, four new species, viz. Sidera minutissima, S. parallela, S. srilankensis and S. tenuis are described, and a new combination, Sidera minutipora, is proposed. All these species are illustrated. Sidera minutissima is characterized by tiny basidiomata with bluish pores when fresh, generative hyphae dominating at the dissepiment edges, the presence of cystidioles, and allantoid basidiospores measuring 3.8–4.4 × 0.9–1.3 μm. Sidera parallela differs from other poroid species in the genus by having parallel tramal hyphae in combination with lunate basidiospores measuring 2.8–3.3 × 0.9–1.2 μm. Sidera srilankensis have generative and skeletal hyphae co-dominating at the dissepiment edges, and lunate basidiospores measuring 3.5–4 × 1–1.3 μm. Sidera tenuis is distinguished by small pores (8–10 per mm) and relatively long allantoid basidiospores measuring 4.2–5 × 0.8–1 μm. Sidera minutipora is characterized by buff to olivaceous buff basidiomata when dry, 5–7 pores per mm, rosette-like crystals rare, and allantoid basidiospores measuring 3.7–4.3 × 1–1.3 μm. An identification key to all accepted species is provided.

, and S. vesiculosa Rui Du & M. Zhou (Du et al. 2019). The genus is characterized by resupinate, white to cream or buff, mostly waxy basidiomata when fresh, 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 (Miettinen and Larsson 2011;Du et al. 2019). Species grow on decaying wood and cause a white-rot (Dai et al. 2007;Yuan and Dai 2008;Miettinen and Larsson 2011;Du et al. 2019).
In the phylogeny, current five Sidera species distributed in Europe, Asia, Pacific Ocean and South America were defined based on ITS and nLSU sequences. Sidera vesiculosa, S. lowei, S. vulgaris have distributions in Asian-Pacific regions. However, samples named as Sidera vulgaris from New Zealand and Australia were separated into two lineages (Miettinen and Larsson 2011;Du et al. 2019).
New specimens collected from the tropic and subtropic Asian-Pacific regions have been studied by morphological and DNA methods. As a result, four unknown Sidera species are found. Another species, originally described as Poria minutipora Rodway & Cleland from Australia, is proposed for transfer to Sidera, and the sample from Australia named as S. vulgaris by Miettinen and Larsson (2011) is also identified as the species. In addition, specimens or literatures and sequences of all ten accepted Sidera species are studied. Furthermore, an identification key to accepted species is provided.

Morphological studies
The studied specimens are deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions are based on field notes and dry herbarium specimens. Microscopic measurements and drawings were made from slide preparations of dried specimens stained with Cotton Blue and Melzer's reagent following Dai (2010). In presenting spore size variation, 5% of measurements were excluded from each end of the range and this value is given in parentheses. The following abbreviations were used: KOH = 2% potassium hydroxide, CB = Cotton Blue, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, 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). Special color terms follow Anonymous (1969) and Petersen (1996). Herbarium abbreviations follow Thiers (2018).

Molecular studies
A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract total genomic DNA from dried specimens following the manufacturer's instructions with some modifications Shen et al. 2019). ITS regions were amplified with primers ITS4 and ITS5 (White et al. 1990), and the nLSU with primers LR0R and LR7. The PCR procedure for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 sec, 54 °C for 45 sec and 72 °C for 1 min, and a final extension of 72 °C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 35 cycles at 94 °C for 1min, 50 °C for 1 min and 72 °C for 1.5 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.

Phylogenetic analyses
Phylogenetic analyses were applied to ITS+nLSU sequences. Sequences generated in this study were aligned with additional sequences downloaded from GenBank (Table 1) using Clustal X ( Thompson et al. 1997) and manually adjusted in BioEdit (Hall 1999). Prior to phylogenetic analysis, ambiguous sequences at the start and the end were deleted and gaps were manually adjusted to optimize the alignment. Sequence alignment was deposited at TreeBase (submission ID 26119). Phylogenetic analysis was done as in Li et al. (2014) and Zhu et al. (2019). Sequences of Exidia candia Lloyd and Exidiopsis calcea (Pers.) K. Wells outside Hymenochaetales were used as outgroup referred to Miettinen and Larsson (2011) and Yuan et al. (2016), because some species related to Sidera in Polyporales, like Skeletocutis species, were added in phylogenetic analysis.
Maximum parsimony analysis (MP) was performed in 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 1000 random sequence additions. Max-trees were set to 1000, branches of zero length were collapsed and all parsimonious trees were saved. Clade robustness (BP) was assessed using a bootstrap analysis with 1000 replicates (Felsenstein 1985).
The optimal substitution models for the combined dataset were determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.2 (Nylander 2004) after scoring 24 models of evolution by PAUP* version 4.0 beta 10 (Swofford 2002). 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 (Ronquist et al. 2012). Four Markov chains were run for 5 million generations and trees were sampled every 1000 generations. 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. The ML analysis was conducted on RAxmlGUI 1.31 (Michalak 2012), and all parameters used default settings. Statistical support values (BS) were obtained using non-parametric bootstrapping with 1000 replicates. The best fit maximum likelihood tree from all searches was kept. Branches that received bootstrap support values for MP and ML greater than or equal to 70% and BPP greater than or equal to 0.95 were considered as significantly supported.

Phylogenetic analyses
The combined ITS+nLSU dataset included sequences from 37 specimens representing 32 species (Table 1). The specimen Dai 19587 was not included because of its lack of ITS sequence, but it has an nLSU sequence with 100% identity to Dai 19529. The dataset had an aligned length of 1718 characters, of which 909 are constant, 148 are variable but parsimony-uninformative, and 661 are parsimony-informative. BI analyses resulted in a best tree (Figure 1), where the ESSs of all parameters were superior to 1000 and the PSRFs were close to 1.0. MP and ML analyses produced consensus trees similar to BI analysis, and only the BI tree is presented along with support values from MP and ML analyses. Our newly generated sequences formed five robustly supported lineages within the Sidera clade, which we interpret as four new species and support for one new combination.

Taxonomy
Sidera minutipora (   center; pore surface cream to buff when fresh, become buff to olivaceous buff when dry; sterile margin distinct, fimbriate, thinning out; pores round, 5-7 per mm; dissepiments thin, lacerate; subiculum very thin to almost absent; tubes darker than the poroid surface, up to 1 mm long.
Specimen examined. Australia. Tasmania Etymology. Minutissima (Lat.), refers to the species having small basidiomata. Description. Basidiomata: Annual, resupinate, soft when fresh, soft corky to fragile when dry, up to 5 cm long, 3 cm wide, and approximately 1 mm thick at center; pore surface bluish to more or less turquoise when fresh, becoming cream to buff yellow when dry; sterile margin distinct, fimbriate, thinning out; pores round, 7-9 per mm; dissepiments thin, entire; subiculum very thin to almost absent; tubes concolorous with pore surface, up to 1 mm long.
Description. Basidiomata: Annual, resupinate, soft corky when fresh, soft corky when dry, up to 11 cm long, 4 cm wide, and approximately 1.5 mm thick at center; pore surface white when fresh, becoming cream to buff yellow upon drying; sterile margin distinct, fimbriate, thinning out; pores round, 6-8 per mm; dissepiments thick, entire; subiculum very thin to almost absent; tubes concolorous with pore surface, up to 1.5 mm long.

Discussion
Previously five species of Sidera, viz. S. lenis, S. lowei, S. lunata, S. vesiculosa and S. vulgaris, were described or transferred to the genus. In this paper, Sidera minutissima, S. parallela, S. srilankensis and S. tenuis are described as new to science. In addition, Sidera minutipora is proposed as a new combination based on Poria minutipora. All these species with resupinate, white to cream or buff, bluish to more or less turquoise basidiomata when fresh, a dimitic hyphal system with generative hyphae bearing clamp connections, the presence of rosette-like crystals and allantoid to lunate basidiospores fit well in Sidera. Besides, they formed distinct lineages within the Sidera clade inferred from ITS and nLSU datasets (Figure 1). Eight names were listed as synonyms of S. lenis (Index Fungorum and Mycobank): Poria lunulispora Pilát (type from Siberia), P. chakasskensis Pilát (type from Siberia), P. earlei Murrill (type from Jamaica), P. tenuipora Murrill (type from Jamaica), P. montana Murrill (type from Jamaica), P. consimilis Rick (type from Brazil), P. subvulgaris Rick (type from Brazil) and P. minutipora (type from Tasmania). Buchanan and Ryvarden (1993) indicated that the holotype of P. minutipora was not found, but an isotype PDD 7115 labelled part of type collection was studied. This comprised fragments of two species, Diplomitoporus lenis (P. Karst.) Gilb. & Ryvarden (=Sidera lenis) and , and the portion of the isotype conforming to D. lenis was selected as lectotype for P. minutipora.
Three taxa were treated as 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, and they most probably represent a single species of S. vulgaris which was originally described from Sweden (Niemelä and Dai 1997).
In our phylogeny Gates FF257 clustered with Cui 16720 with high support within the Sidera clade (Figure 1), and both samples were collected from Tasmania, Australia. The sample Gates FF257 was named as S. vulgaris by Miettinen and Larsson (2011), but S. vulgaris was originally described from Europe and is different from the Australian specimens by having shorter basidiospores (2.9-3.6 × 0.9-1.4 μm according to Niemelä and Dai 1997, vs. 3.7-4.3 × 1-1.3 μm in Cui 16720). According to the protologue of Poria minutipora pores are 7 per mm and the only microscopic characteristic mentioned is that hyphae are 2-3 μm wide (Rodway and Cleland 1929). We did not study the type but our specimen Cui 16720 fits well with the description. Cunningham (1965) treated P. minutipora as a synonym of P. lenis (P. Karst.) Sacc. (=Sidera lenis), and indicated that spores were 2.5-4 × 1-1.5 μm. Sidera tenuis is also described from Tasmania in the present paper, but differs from S. minutipora by smaller pores (8-10 per mm) and longer basidiospores (4.2-5 × 0.8-1 μm). A European ITS sequence of Skeletocutis vulgaris (Fr.) Niemelä & Y.C. Dai (ex. CBS 465.50 GenBank: MH856711.1) is close to Skeletocutis species and far from Sidera species in the phylogeny. Ryvarden 37198 from New Zealand also named as Sidera vulgaris by Miettinen and Larsson (2011) clustered with Sidera lenis from Finland with high support, but we didn't examine their morphology, thus we keep their name.
Poria chakasskensis and P. lunulispora were described from Siberia (Pilát 1933(Pilát , 1935 and both types were studied by Pouzar (1989, 1991). The type of P. chakasskensis has basidiospores measuring 5.5-8.5 × 2-2.4 μm and represents Ceriporia purpurea (Fr.) Donk (Kotlaba and Pouzar 1991). Poria lunulispora was collected on wood of Pinus, and is true S. lenis (Kotlaba and Pouzar 1989, as Diplomitoporus lenis). Poria consimilis and P. subvulgaris were described from Brazil (Rick 1937). Rajchenberg (1987) studied the types and considered them as synonyms of S. lenis. Sidera lenis is a perennial species, and its basidiospores are more than 1.5 μm wide. Our newly described species have annual basidiomata and basidiospores are less than 1.5 μm wide. Poria earlei, P. montana and P. tenuipora were described from Jamaica (Murrill 1920a, b). Types of these species were studied by Niemelä and Dai (1997). They found that all types are sterile, but also that P. earlei and P. montana are conspecific and have perennial basidiomata, and that P. tenuipora has skeletal hyphae that are 3-4 μm in diam. Our new species are all annual and skeletal hyphae are 2-3 μm in diam.
Morphologically Sidera minutipora resembles S. srilankensis by sharing similar size of pores and basidiospores. However, the former species has allantoid basidiospores, and its skeletal hyphae become swollen in KOH while basidiospores are lunate and skeletal hyphae are unchanged in KOH in S. srilankensis.
Sidera parallela can be distinguished from other species by its parallel tramal hyphae. Sidera srilankensis resembles S. parallela by sharing pore size and lunate basidiospores, but in addition to the parallel tramal hyphae S. parallela also has smaller basidiospores measuring 2.8-3.3 × 0.9-1.2 μm.
Sidera tenuis has the smallest pores of all species in the genus (8-10 per mm) and also the narrowest basidiospores (0.8-1 μm).
In this paper four new species and a new combination of Sidera are described from tropic and subtropic Asian-Pacific regions. Although the type species, Sidera lenis, has a distribution in boreal forests, the majority of species are so far found in tropical and subtropical regions.