﻿Taxonomy and phylogeny of Sidera (Hymenochaetales, Rickenella clade) from China and North America revealing two new species

﻿Abstract Sidera, belonging to the Rickenella clade of Hymenochaetales, is a worldwide genus with mostly poroid hymenophore of wood-inhabiting fungi. Two new species in the genus, Sideraamericana and S.borealis, are described and illustrated from China and North America based on morphological and molecular evidence. They were mainly found growing on rotten wood of Abies, Picea and Pinus. S.americana is characterized by annual, resupinate basidiomata with silk sheen when dry, round pores (9–11 per mm), a dimitic hyphal system, and allantoid basidiospores measuring 3.5–4.2 × 1 μm. S.borealis is characterized by annual, resupinate basidiomata with cream to pinkish buff dry pore surface, angular pores (6–7 per mm), a dimitic hyphal system, and allantoid basidiospores measuring 3.9–4.1 × 1–1.1 μm. Phylogenetic analysis based on a combined 2-locus dataset [ITS1-5.8S-ITS2 (ITS) + nuclear large subunit RNA (nLSU)] shows that the two species are members of Sidera, and they are compared with morphologically similar and phylogenetically related species, respectively. An identification key to 18 accepted species of Sidera in worldwide is provided.

Larss.] is a member of Rickenella clade in their phylogenetic analysis of Hymenochaetales. Miettinen and Larsson (2011) Miettinen], because these four species formed a monophyletic group and didn't group together with any other species or genera within Rickenella clade in their phylogenetic analysis of 5.8S + nLSU. Yu et al. (2021) studied the taxonomic positions of the genera Resinicium Parmasto and Skvortzovia Bononi & Hjortstam, which belong to Rickenella clade. With the much richer sampling available to us, the phylogenetic analyses also prompted us to study the taxonomic position of Sidera within the Rickenella clade.
Sidera, a genus with mostly poroid hymenophore of wood-inhabiting fungi distributed in most continents except Africa (Miettinen and Larsson 2011;Liu et al. 2022;Wu et al. 2022a), is treated as a member of Rickenella clade within Hymenochaetales, with Sidera lenis as the generic type. To date, 16 species are accepted in Sidera (Miettinen and Larsson 2011;Du et al. 2019Du et al. , 2020Liu et al. 2021Liu et al. , 2022.  (Du et al. 2020;Liu et al. 2021Liu et al. , 2022. Morphologically, Sidera is characterized by resupinate, white to cream or buff, mostly waxy fresh basidiomata, mostly poroid (one hydnoid species) hymenophore, a dimitic or monomitic hyphal system with generative hyphae with clamp connections, the presence of rosette-like crystals, and allantoid to lunate, hyaline, thin-walled basidiospores (Miettinen and Larsson 2011;Liu et al. 2021). Species in the genus cause a white rot.
In this study, we focus on Sidera represented by eight resupinate specimens from China, and North America. Phylogenetic analysis based on the ITS and nLSU rDNA sequences is carried out and two new species are described. The current study aims to further explore the species diversity of Sidera in the Asia-Pacific region, and more importantly, to confirm the taxonomic position of Sidera within the Rickenella clade of Hymenochaetales, based on the ITS+nLSU phylogenetic analysis. Morphological characters of all 18 currently accepted species of Sidera are summarized in Table 1. Furthermore, an identification key to accepted species is provided in the paper.  Liu et al. (2022). New species are shown in bold.

Species
Growing habit

Morphological studies
Macro-morphological descriptions were based on field notes and dry herbarium specimens. Microscopic measurements and drawings were made from slide preparations of dried tissues stained with Cotton Blue and Melzer's reagent as described by Dai (2010). Pores were measured by subjectively choosing as straight a line of pores as possible and measuring how many per mm. The following abbreviations are used in the description: 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) = number of spores (a) measured from given number of specimens (b); L = mean spore length (arithmetic average of all the spores); W = mean spore width (arithmetic average of all the spores); 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 Petersen (1996) and then herbarium abbreviations follow Thiers (2018). Voucher specimens from the study were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC).

DNA extraction, PCR and sequencing
Total genomic DNA was extracted from dried specimens by a CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Company, Limited, Beijing, China) according to the manufacturer's instructions with some modifications (Li et al. 2014). The ITS regions were amplified with primers ITS4 and ITS5 (White et al. 1990). The nLSU regions were amplified with primers LR0R and LR7 (Vilgalys and Hester 1990). The polymerase chain reaction (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, 58 °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 30 sec, 48 °C for 1 min, and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min (Zhao et al. 2015). 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
Phylogenetic trees were constructed using ITS + nLSU rDNA sequences, and phylogenetic analyses were performed with the Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Inference (BI) methods. Sequences of the species and strains were primarily adopted from ITS-based and 28S-based tree topology as described by Miettinen and Larsson (2011) and Liu et al. (2022). New sequences generated in this study, along with reference sequences retrieved from GenBank (Table 2), were aligned by MAFFT 7 (Katoh et al. 2019; http://mafft.cbrc.jp/alignment/server/) using the "G-INS-i" strategy and manually adjusted in BioEdit v.7.2.5 (Hall 1999). 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 (Maddison and Maddison 2021; https://www.mesquiteproject.org/). The sequence alignment was deposited at TreeBase. Sequences of Exidia candida Lloyd and Exidiopsis calcea (Pers.) K. Wells outside Hymenochaetales obtained from GenBank were used as outgroups to root the tree in the ITS + nLSU analysis.
Maximum Parsimony analysis was applied to the ITS + nLSU dataset sequences. The approaches to phylogenetic analysis utilized those conducted by Liu et al. (2022), and the tree was constructed using PAUP* version 4.0 beta 10 (Swofford 2002). 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 (Felsenstein 1985). 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 (Stamatakis 2014) and RAxML-HPC through the CIPRES Science Gateway V. 3.3 (Miller et al. 2010; http://www.phylo.org). Statistical support values (BS) were obtained using nonparametric bootstrapping with 1000 replicates. The BI analysis was performed with MrBayes 3.2.7a (Ronquist et al. 2012). Four Markov chains were run for two runs from random starting trees for 5 million generations until the split deviation frequency value < 0.01, and the trees were sampled at 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 the Bayesian Posterior Probabilities (BPP) of the clades.
A total of 24 models of evolution was scored using PAUP* version 4.0 beta 10 (Swofford 2002). Optimal substitution models for the combined dataset were then determined using the Akaike Information Criterion (AIC) implemented in MrModeltest 2.3 (Posada and Crandall 1998;Nylander 2004). 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), 50% (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 (Rambaut 2018) was used to visualize the resulting tree.
Besides, we collected two Sidera lenis on rotten wood of Picea in Yunnan Province, China: Dai 22834 (BJFC 037407) and Dai 22854 (BJFC 037427). This is the first time the species has been reported in China. We have uploaded ITS and nLSU sequences of the two specimens to GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and added them to our phylogenetic analysis (Fig. 1).   Diagnosis. Sidera americana is characterized by annual, resupinate basidiomata with silk sheen when dry, round pores (9-11 per mm), a dimitic hyphal system, and allantoid basidiospores measuring 3.5-4.2 × 1 μm.
Etymology. Americana (Lat.): referring to the species occurring in North America. Basidiomata. Annual, resupinate, soft and without odor or taste when fresh, soft corky when dry, up to 14 cm long, 6 cm wide, and approximately 2 mm thick at center; pore surface white when fresh, becoming cream to buff with silk sheen when dry; sterile margin indistinct; pores round, 9-11 per mm; dissepiments thin, lacerate; subiculum very thin to almost absent; tubes concolorous with poroid surface, up to 2 mm long.
Etymology. Borealis (Lat.): referring to the species occurring in boreal areas of China.
Basidiomata. Annual, resupinate, soft corky and without odor or taste when fresh, corky when dry, up to 5 cm long, 2 cm wide, and less than 1 mm thick at center; pore surface white to cream or pale buff when fresh, becoming cream to pinkish buff when dry; sterile margin indistinct, white, cottony, thinning out; pores angular, 6-7 per mm; dissepiments thin, entire; subiculum very thin to almost absent; tubes concolorous with poroid surface, less than 1 mm long.

Spores.
Basidiospores allantoid, hyaline, thin-walled, smooth, occasionally with one or two guttules, IKI-, CB-, (3.5-)3.9-4.1(-4.2) × (0.8-)1-1.    Description. See Liu et al. (2022). Liu et al. (2022) described Sidera tibetica as a new species based on Tibetan specimens and a photo of holotype. Subsequently, more specimens of the species from Belarus and China (Guangxi, Yunnan and Zhejiang) were collected and we took many photos of the fungus at different stages of growth on different hosts to make it easier for taxonomists to recognize the fungus in the field.
Sidera americana and S. borealis are described from North China and North America; like most other species of Sidera, the two new species grow mostly on gymnosperm wood in temperate or boreal forests, but they are distinguished from existing species in the genus by morphology, geographic distribution and DNA sequences.
Boreal areas of China have the most important virgin forests in the country, and such forests provide favorable environments for some special wood-decaying fungi, e.g. Heterobasidion Bref., Skeletocutis Kotl. & Pouzar and Sidera, because fewer morphological characteristics existed among different species of each genus, and many species in the traditional definition are, in fact, the species complex. In recent years, the introduction of molecular systematics has greatly improved our understanding of the diversity of wood-rotting fungi in the boreal forests. Numerous new species have been found there (Dai et al. 2007Yuan and Dai 2008;Tian et al. 2013;Li et al. 2014;Chen et al. 2015Chen et al. , 2016Cui et al. 2019;Wang et al. 2021;Wu et al. 2021Wu et al. , 2022b, and we believe that more boreal new species will be found in the future.