Two New Species of Sidera (Hymenochaetales, Basidiomycota) from Southwest China

Two new wood-inhabiting fungi, Sidera salmonea sp. Nov. and S. tibetica sp. Nov. in the order Hymenochaetales from southwest China, are described and illustrated based on molecular and morphological evidence. They were found on gymnosperm wood that is rotten and charred. The characteristics of S. salmonea include annual, resupinate basidioma, salmon pores with distinctly white margins, angular pores (7–9 per mm), a dimitic hyphal system, and lunate basidiospores that are 3–3.5 × 0.9–1.1 μm. The characteristics of S. tibetica include annual, resupinate basidioma with a white to cream fresh pore surface that becomes cream to honey-yellow and shiny when dry, round pores (7–8 per mm), a dimitic hyphal system, and lunate basidiospores that measure 2.9–3.1 × 1–1.1 μm. A phylogenetic analysis based on the combined 2-locus dataset (5.8S + nuclear large subunit RNA (nLSU)) shows that the two species are members of the genus Sidera, and they are morphologically compared with related species, respectively. This paper provides a key to the identification of 16 accepted species of Sidera that are found throughout the world.


Introduction
Sidera Miettinen &K.H. Larss. is a small genus in the Rickenellaceae Vizzini (Hymenochaetales), which is distributed throughout the world. The genus was typified by S. lenis (P. Karst.) Miettinen [1]. The name is derived from sidus (star in Latin) and refers to the star-like or rosulate crystals [1]. Thus, the presence of rosette-like crystals is the most striking morphological characteristic of this genus. Its characteristics include resupinate, white to cream or buff fresh basidioma, poroid or hydnoid hymenophores, a monomitic or dimitic hyphal system with generative hyphae that bear clamp connections, the presence of rosette-like crystals, and allantoid to lunate basidiospores [1]. Species in this genus cause a white rot. A total of 14 species to date have been accepted in the genus that is found throughout the world [2].
Six resupinate polypore specimens were collected from the Eastern Himalayas of southwest China during studies on wood-inhabiting fungi, and their morphology corresponded to the concept of Sidera. A phylogenetic analysis based on the 5.8S and nuclear large subunit RNA (nLSU) rDNA sequences was conducted to confirm their affinity. Both morphological and molecular evidence demonstrated that these six specimens represent two undescribed species of Sidera. Thus, they are described in this paper. In addition, the specimens, literature, and the sequences of all 14 currently accepted taxa of Sidera were studied, and their morphological characters are summarized in Table 1. Furthermore, this paper provides an identification key to the accepted species.

Site Description
The type and paratype specimens were collected from eastern Tibet, in southwestern China, ca. E 92 • 09 , N 26 • 52 , alt. 3000-3800 m. The vegetation is typical of boreal natural forests and the dominant trees are Abies georgei, Picea linzhiensis and Pinus armandii and Pinus yunnanensis, etc.

Morphological Studies
Macro-morphological descriptions were based on voucher specimens and field notes. Microscopic structures were prepared from slide preparations of dried tissues stained with Cotton Blue and Melzer's reagent, as described by Dai [3]. The following abbreviations are used: CB = Cotton Blue; CB-= acyanophilous in Cotton Blue; IKI = Melzer's reagent; IKI-= neither amyloid nor dextrinoid in Melzer's reagent; KOH = 5% potassium hydroxide; n (a/b) = the number of spores (a) measured from a given number of specimens (b); L = mean spore length (arithmetic average of basidiospores); W = mean spore width (arithmetic average of basidiospores); and Q = variation in the L/W ratios between the specimens studied. When the variation in spore size is shown, 5% of the measurements were excluded from each end of the range, and these values are shown in parentheses.
Special color terms follow [4] and then herbarium abbreviations [5]. The voucher specimens for the present study are been deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC), Beijing, China.

DNA Extraction, PCR, and Sequencing
Total genomic DNA was extracted from dried specimens using a CTAB Rapid Plant Genome Extraction Kit (Aidlab Biotechnologies Company, Ltd., Beijing, China) according to the manufacturer's instructions with some modifications [6]. The ITS regions were amplified with primers ITS4 and ITS5 [7]. The nLSU regions were amplified with primers LR0R and LR7 (http://www.biology.duke.edu/fungi/mycolab/primers.htm, accessed on 7 March 2022).
The polymerase chain reaction (PCR) procedure for the ITS was as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 40 s, 58 • C for 45 s, 72 • C for 1 min, and a final extension of 72 • C for 10 min. The PCR procedure for the nLSU was as follows: initial denaturation at 94 • C for 1 min, followed by 35 cycles at 94 • C for 30 s, 48 • C for 1 min, and 72 • C for 1.5 min, and a final extension of 72 • C for 10 min [8]. Aliquots of PCR products were examined on 2% agarose gels stained with GelStar Nucleic Acid Gel Stain (Lonza Rockland, Inc., Rockland, YN, USA) and examined under UV light. The sequencing of the PCR products was conducted by the Beijing Genomics Institute, Beijing, China, with the same primers used in the PCR reactions. Species were identified by sequence comparison with accessions in the NCBI databases using the BLAST program.

Phylogenetic Analyses
Although ITS is an important marker used as a barcode for fungal species, it can be difficult to align ITS sequences for many groups of fungi, including Sidera [1,2]. Therefore, we used the most stable and conservative portion of ITS (5.8S) and the partial LSU (a fragment of about 1400 bp of the LSU) to analyze the phylogenetic relationship of the Sidera species. Phylogenetic analyses were performed with the Maximum Parsimony (MP), Maximum Likelihood (ML), and Bayesian Inference (BI) methods. Sequences of the species and strains were primarily adopted from 5.8S-based and 28S-based tree topologies as described by Liu et al. [2]. New sequences generated in this study, along with reference sequences retrieved from GenBank (Table 2), were aligned by MAFFT 7 ([9]; http://mafft. cbrc.jp/alignment/server/, accessed on 7 March 2022) using the "G-INS-i" strategy and manually adjusted in BioEdit [10]. Unreliably aligned sections were removed before the analyses, and efforts were made to manually inspect and improve the alignment. The data matrix was edited in Mesquite v3.70 [11]. Sequences of Skvortzovia furfurella (Bres.) Bononi & Hjortstam and S. furfuracea (Bres.) G. Gruhn & Hallenberg obtained from GenBank were used as outgroups to root the trees in 5.8S + nLSU analysis.
Maximum Parsimony analysis was applied to the 5.8S + nLSU dataset sequences. The approaches to phylogenetic analysis utilized those conducted by Chen and Cui [12], and the tree was constructed using PAUP* version 4.0 beta 10 [13]. All the characters were equally weighted, and gaps were treated as missing data. Trees were inferred using the heuristic search option with tree bisection and reconnection (TBR) branch swapping, and 1000 random sequence addition maxtrees were set to 5000. Branches of zero length were collapsed, and all the parsimonious trees were saved. Clade robustness was assessed using a bootstrap (BT) analysis with 1000 replicates [14]. Descriptive tree statistics, including the Consistency Index (CI), Homoplasy Index (HI), Rescaled Consistency index (RC), Retention Index (RI), and tree length (TL), were calculated for each Maximum Parsimonious Tree (MPT) generated. The research using ML was conducted using RaxML-HPC v. 8.2.3 [15] and RaxML-HPC through the CIPRES Science Gateway ( [16]; http://www.phylo.org, accessed on 7 March 2022). Statistical support values (BS) were obtained using nonparametric bootstrapping with 1000 replicates. The BI analysis was performed with MrBayes 3.2.7a [17]. Four Markov chains were run for two runs from random starting trees for 1 million generations until the split deviation frequency value <0.01, and the trees were sampled at every 1000th 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 the Bayesian Posterior Probabilities (BPP) of the clades.
A total of 24 models of evolution were scored using PAUP* version 4.0 beta 10 [13]. Optimal substitution models for the combined dataset were then determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.3 [18,19]. The model GTR + I + G was selected for use in the Maximum Likelihood (ML) and Bayesian Inference (BI) analyses.
Branches that received bootstrap support for Maximum Likelihood (BS), Maximum Parsimony (BP), and Bayesian Posterior Probabilities (BPP) >70% (BS), 70% (BP), and 0.95 (BPP) were considered to be significantly supported. In addition, the ML analysis resulted in the best tree, and only the ML tree is shown along with the support values from the MP and BI analyses. FigTree v1.4.4 [20] was used to visualize the resulting tree.

Phylogenetic Analyses
The concatenated 5.8S + nLSU dataset contained sequences from 27 fungal specimens that represented 14 taxa of Sidera (two are treated as S. vulgaris sensu lato) ( Table 2).
The dataset had an aligned length of 1514 characters, with 1334 characters that were constant, 57 that were variable but parsimony-uninformative, and 123 that were parsimonyinformative. The MP analysis yielded three equally parsimonious trees (TL = 341, CI = 0.607, RI = 0.790, RC = 0.479, and HI = 0.393). In addition, the average standard deviation of the split frequencies was 0.007886 (BI).
Basidioma-Annual, resupinate, soft corky when fresh and dry, up to 10 cm long, 6 cm wide, and 12 mm thick at the center; pore surface locally verruculose, salmon (6A4) and slightly shiny when fresh and dry, uncracked when fresh or occasionally cracked when dry; sterile margin distinct, fimbriate, white; pores angular, 7-9 per mm; dissepiments thin to thick, entire; subiculum white, cottony and up to 5 mm thick; tubes white to salmon (6A4), up to 7 mm long.
Basidiospores-Lunate, smooth, thin-walled, hyaline, occasionally with one or more guttules, IKI-, CB-, 3-3. Basidioma-Annual, resupinate, soft corky when fresh and dry, 4 cm wide, up to 10 cm long, and <0.8 mm thick at the center; pore surface white to cream when fresh, becoming cream to honey yellow and shiny when dry; sterile margin indistinct, cottony, white, thinning out; pores round, 7-8 per mm; dissepiments thin, entire to slightly lacerate; subiculum white, up to 0.1 mm thick; tubes concolorous with a poroid surface, up to 0.7 mm long.
Species in the genus Sidera are lignicolous and cause a white rot. They are found in North and South America, Eurasia, Africa, Australia, and New Zealand, in boreal, temperate, and tropical climates [1]. Two new species were collected from Tibet. Tibet has the most important virgin forests in southwest China, and such forests (typical boreal natural forests) provide favorable environments for wood-decaying fungi. Numerous new species have been found in Tibet [6,[22][23][24][25][26][27][28][29], and this paper confirms that the diversity of fungi is very rich in the montane forests of the Eastern Himalayas.  Data Availability Statement: The sequence alignment was deposited at TreeBase (submission ID 29499; http://purl.org/phylo/treebase/phylows/study/TB2:S29499?x-access-code=770f9dfc79ea7 f489fbf2fc56e55ec7e&format=html accessed on 7 March 2022).