A contribution to the genus Steccherinum (Steccherinaceae, Polyporales): Introducing two new species and two new combinations of the genus

Two new wood-inhabiting fungi from China, Steccherinum juniperi and S. incrustans, in the family Steccherinaceae are described and illustrated based on morphological and molecular analyses. The species S. juniperi was found growing on the rotten wood of Juniperus in Qinghai Province, China, while S. incrustans was collected on rotten angiosperm wood in Yunnan Province, China. The characteristics of S. juniperi include annual, resupinate basidiomata with a buff yellow fresh pore surface that becomes apricot orange when bruised, angular pores of 3–6 per mm, subicular generative hyphae sometimes covered with crystals, the presence of encrusted skeletocystidia in tube trama only, fusiform to slim clavate cystidioles, and ellipsoid basidiospores measuring as 3–4 × 2–3 μm. The characteristics of S. incrustans include annual, resupinate basidiomata with a buff yellow or pinkish buff to clay buff dried pore surface, angular pores (8–10 per mm), generative hyphae in trama frequently covered with crystals, the presence of encrusted skeletocystidia in tube trama and hymenium, and ellipsoid basidiospores (3.5–4.5 × 2.5–3.5 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 Steccherinum, and they are compared with morphologically similar and related species of this genus, respectively. In addition, two new combinations from Junghuhnia, transferred to Steccherinum as S. austrosinense and S. nandinae, are proposed based on examination of their type materials and phylogenetic analysis.

Steccherinum is characterized by the resupinate to effuse-reflexed basidiomata with poroid or odontioid to hydnoid hymenophore, a monomitic or dimitic hyphal structure with . /fmicb. . thick-walled skeletal hyphae; most species have clamped generative hyphae, encrusted or smooth skeletocystidia, and smooth, thinwalled, ellipsoid basidiospores (Maas Geesteranus, 1974;Eriksson et al., 1984;Miettinen et al., 2012). Miettinen et al. (2012) carried out a multigene phylogenetic analysis (ITS + nLSU + mtSSU + atp6 + tef1) for Steccherinaceae and proposed the monophyletic Steccherinum clade (Figure 4 in Miettinen et al., 2012). Liu and Dai (2021)  During investigations on the diversity of wood-rotting fungi from China, three resupinate polypore specimens were collected from Yunnan Province and Qinghai Province. Their morphology corresponded to the concept of Steccherinum. To confirm their affinity, phylogenetic analysis based on the ITS and nLSU rDNA sequences was carried out. Both morphological characteristics and molecular evidence demonstrated that these three specimens represent two new species of Steccherinum, which we describe in the present study. In addition, we studied the type specimens of Junghuhnia austrosinensis F. Wu et al. and J. nandinae F. Wu et al. They were transferred to Steccherinum based on morphological and phylogenetic analyses.
. Materials and methods

. . Morphological studies
Macro-morphological descriptions were based on dry herbarium specimens and field notes. Microscopic measurements and drawings were prepared 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+ = cyanophilous in 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 = spore length (arithmetic average of all the spores); W = 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 amplification, 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 (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 the ITS was as follows: initial denaturation at 95 • C for 3 min, followed by 35 cycles at 94 • C for 40 s, 54 • C for 45 s, and 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 (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 Liu and Dai (2021). New sequences generated in this study, along with reference sequences retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/; Table 1), 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 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 3.70 (https://www.mesquiteproject.org/; Maddison and Maddison, 2021). The sequence alignment was deposited at TreeBase (Submission ID: 30018). According to Miettinen et al. (2012), Junghuhnia crustacea (Jungh.) Ryvarden also belongs to the family Steccherinaceae and is not close to the Steccherinum clade, thus sequences of Junghuhnia crustacea obtained from GenBank were used as out-groups to root the trees in the ITS + nLSU analysis.
/fmicb. .  for 1 million generations until the split deviation frequency value <0.01, and the trees were sampled at every 1,000 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 the Bayesian posterior probabilities (BPP) of the clades. Maximum parsimony analysis was applied to the ITS + nLSU dataset sequences. The approaches to phylogenetic analysis were conducted as described by Liu et al. (2022), and the tree was constructed using PAUP * 4.0β10 (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 branch swapping, and 1,000 random sequence addition maxtrees were set to 5,000. Branches of zero length were collapsed, and all the parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1,000 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 generated.
A total of 24 models of evolution were scored using PAUP * 4.0β10 (Swofford, 2002). Optimal substitution models for the combined dataset were then determined using the Akaike information criterion implemented in MrModeltest 2.3 (Posada and Crandall, 1998;Nylander, 2004). The model GTR + I + G was selected in the ML and BI analyses.

. . Phylogenetic results
The combined ITS + nLSU dataset included sequences from 47 specimens representing 27 species (Table 1). The dataset had an aligned length of 2,044 characters, of which 1,544 were constant, 96 were variable but parsimony-uninformative, and 404 were parsimony-informative. MP analysis yielded an equally parsimonious tree (CI = 0.524, HI = 0.476, RC = 0.403, RI = 0.768, TL = 1,288). ML analysis resulted in the best tree, and Bayesian and MP analyses resulted in a similar topology to the ML analysis, with an average standard deviation of split frequencies of 0.007184 (BI). Hence, the ML tree is shown combined with the support values from the MP and BI analyses.
The phylogeny (Figure 1) inferred from the ITS and nLSU sequences demonstrated that the new species (Steccherinum juniperi and S. incrustans) and new combinations (S. austrosinense and S. nandinae) clustered in Steccherinum clade, and thus, they are described and proposed herein. Etymology-Incrustans (Lat.): referring to the species having encrusted generative hyphae in trama.

. . Taxonomy
Fruiting body-Basidiomata annual, resupinate, difficult to separate from the substrate, soft corky when fresh, hard corky when dry, up to 11 cm long, 2 cm wide, and ∼1.5 mm thick at the center; pore surface buff yellow or pinkish buff to clay buff upon drying; sterile margin indistinct; pores angular, 8-10 per mm; dissepiments thin, entire; subiculum very thin to almost absent, paler than tubes, nearly 0.2 mm thick; tubes concolorous with poroid surface, up to 1.3 mm long.
Fruiting body-Basidiomata annual, resupinate, difficult to separate from the substrate, soft corky when fresh, hard corky when dry, up to 10 cm long, 2 cm wide, and ∼2.5 mm thick at the center; pore surface buff yellow when fresh, apricot orange when bruised, buff to honey yellow upon drying; sterile margin distinct, cream and nearly 1 mm width; pores angular, 3-6 per mm; dissepiments thin, entire; subiculum very thin to almost absent, paler than tubes, nearly 0.5 mm thick; tubes concolorous with poroid surface, up to 2 mm long.

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: https://www.ncbi.nlm. nih.gov/genbank/, ON182084, ON182087, OP956031, OP956076, and OP956077.
Author contributions Z-BL: investigation, software, data curation, visualization, and writing-original draft. Q-YZ and MZ: data curation and visualization. JS: visualization, supervision, writing-reviewing and editing, project administration, and funding acquisition. All authors contributed to the manuscript and approved the submitted version.

Funding
The research was supported by the National Natural Science Foundation of China (Nos. 32270016 and 32070016).