﻿Species diversity and taxonomy of Scytinostroma sensu stricto (Russulales, Basidiomycota) with descriptions of four new species from China

﻿Abstract Scytinostroma is species-rich genus in Peniophoraceae, Russulales and has been shown to be polyphyletic. In this study, we performed phylogenetic analyses on the core clade of Scytinostroma based on concatenated ITS1-5.8S-ITS2-nrLSU sequence data. Fifteen lineages including four new species from China, Scytinostromabeijingensis, S.boidinii, S.subduriusculum, and S.subrenisporum, were recognized. The genus Michenera was nested within the Scytinostroma s.s. clade in the phylogenetic tree of Peniophoraceae. Sequences of S.portentosum (type species) and S.hemidichophyticum from Europe formed a strongly supported lineage sister to the S.portentosum sample from Canada. It is supposed that the European “S.portentosum” is S.hemidichophyticum, and the former species is restricted in distribution to North America. Scytinostromaduriusculum is supposed to be a species complex. Samples from Sri Lanka (the type locality) formed a lineage sister to those from China, Thailand and Vietnam (described herein as S.subduriusculum) and two samples from France that might represent an undescribed species. The four new species are described and illustrated, and an identification key to all the 14 Scytinostroma s.s. species worldwide is provided. Until now, seven species of Scytinostroma s.s. have been found in China. Our results increased the knowledge of species diversity and taxonomy of corticioid fungi in China.


Introduction
The genus Scytinostroma Donk sensu lato (Peniophoraceae, Russulales), typified by S. portentosum (Berk. & M.A. Curtis) Donk, is characterized by resupinate, effused basidiomes with a smooth to tuberculate hymenophore, a dimitic hyphal system with dextrinoid and cyanophilous skeletal hyphae, presence of gloeocystidia in most species, and subglobose to ellipsoid, variably amyloid or inamyloid, smooth basidiospores (Bernicchia and Gorjón 2010; Stalpers et al. 2021). It is a widely distributed genus with 42 species level names in Index Fungorum (http://www.indexfungorum.org, accessed on 1 January 2023). Morphologically, Scytinostroma can be easily distinguished from other genera of Peniophoraceae by having a dimitic hyphal system and smooth basidiospores. It is similar to Vararia P. Karst., which usually differs in having typical dichohyphae (Bernicchia and Gorjón 2010).  demonstrated that Michenera Berk. & M.A. Curtis belonged to Peniophoraceae and was closely related to Scytinostroma. The two genera are similar in some aspects, such as the texture of basidiome, a dimitic hyphal system, presence of gloeocystidia, but species of Michenera have larger basidia and larger, inamyloid, thick-walled basidiospores. Based on morphology, Stalpers et al. (2021) transferred the two species of Michenera to Scytinostroma and treated the former genus as a synonym of the latter. Larsson and Larsson (2003) and Miller et al. (2006) showed that five species of Scytinostroma, S. galactinum (Fr.) Donk, S. jacksonii Boidin, S. ochroleucum Donk, S. odoratum (Fr.) Donk and S. portentosum (type species), occurred on five distinct branches that are distantly separated in Peniophoraceae. Leal-Dutra et al. (2018) built the genus Baltazaria Leal-Dutra, Dentinger & G.W. Griff. for S. galactinum and other three species, S. neogalactinum Boidin & Lanq., S. eurasiaticogalactinum Boidin & Lanq. and Parapterulicium octopodites Corner. Our preliminary phylogenetic analyses showed that some specimens recently collected from China clustered with S. portentosum (type species) and several other species, which represented the core clade of Scytinostroma. In order to understand the species diversity within this clade, we carried out phylogenetic analyses of Peniophoraceae based on concatenated ITS1-5.8S-ITS2-nrLSU sequence data, focusing on samples of Scytinostroma s.s. worldwide. Fifteen species-level lineages were recognized in the phylogenetic tree. Among them, four lineages are new and here described and illustrated as S. beijingensis, S. boidinii, S. subduriusculum, and S. subrenisporum spp. nov.

Specimen collection
In situ photos of specimens were taken with a Canon camera EOS 70D (Canon Corporation, Japan). Specimens were dried with a portable dryer, labelled, and then stored in a freezer at minus 40 °C for two weeks to kill the insects and their eggs before proceeding with morphological and molecular studies. Voucher specimens are deposited at the herbarium of Beijing Forestry University, Beijing, China (BJFC).

Morphological studies
Thin, freehand sections were made from dried basidiomes and mounted in 2% (weight/volume) aqueous potassium hydroxide (KOH) and 1% (w/v) aqueous phloxine. Amyloidity and dextrinoidity of hyphae and basidiospores were checked in Melzer's reagent (IKI). Cyanophily of hyphal and basidiospore walls were observed in 1% (w/v) cotton blue in 60% (w/v) lactic acid (CB). Microscopic examinations were carried out with a Nikon Eclipse 80i microscope (Nikon Corporation, Japan) at magnifications up to 1000×. Drawings were made with the aid of a drawing tube. The following abbreviations are used: IKI-= neither amyloid nor dextrinoid, CB+ = cyanophilous, CB-= acyanophilous, SA+ = positive reaction in Sulphobenzaldehyde, SA-= negative reaction in Sulphobenzaldehyde, L = mean spore length, W = mean spore width, Q = L/W ratio, n (a/b) = number of spores (a) measured from the number of specimens (b). Color codes and names follow Kornerup and Wanscher (1978).

DNA extraction and sequencing
A CTAB plant genomic DNA extraction kit, DN14 (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract total genomic DNA from dried specimens, then amplified by the polymerase chain reaction (PCR), according to the manufacturer's instructions. The ITS1-5.8S-ITS2 region was amplified with the primer pair ITS5/ITS4 (White et al. 1990) using the following protocol: initial denaturation at 95 °C for 4 min, followed by 34 cycles at 94 °C for 40 s, 58 °C for 45 s and 72 °C for 1 min, and final extension at 72 °C for 10 min. The D1-D2 region of the nucleic ribosomal LSU was amplified with the primer pair LR0R/ LR7 (https://sites.duke.edu/vilgalyslab/rdna_primers_for_fungi/) employing the following procedure: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 50 °C for 1 min and 72 °C for 1.5 min, and final extension at 72 °C for 10 min. DNA sequencing was performed at Beijing Genomics Institute, and newly generated sequences were deposited in GenBank (https:// www.ncbi.nlm.nih.gov/). BioEdit v.7.0.5.3 (Hall 1999) and Geneious Basic v.11.1.15 (Kearse et al. 2012) were used to review the chromatograms and for contig assembly.
Maximum likelihood (ML) analyses, and Bayesian inference (BI) were carried out by using RAxML v.8.2.10 (Stamatakis 2014) and MrBayes 3.2.6 (Ronquist et al. 2012), respectively. In ML analysis, statistical support values were obtained using rapid bootstrapping with 1000 replicates, with default settings for other parameters. For BI, the best-fit substitution model was estimated with jModeltest v.2.17 (Darriba et al. 2012). Four Markov chains were run for 2,000,000 generations for the dataset; until the split deviation frequency value was lower than 0.01. Trees were sampled every 100 th generation. The first quarter of the trees, which represented the burn-in phase of the analyses, were discarded, and the remaining trees were used to calculate posterior probabilities (BPP) in the majority rule consensus tree.

Phylogenetic analyses
The concatenated ITS1-5.8S-ITS2-nrLSU dataset contained 58 ITS and 52 nrL-SU sequences from 61 samples, representing 33 ingroup taxa and the outgroup (Table 1), and had an aligned length of 2628 characters. jModelTest suggested GTR+I+G, K80+I, HKY+I+G, GTR+I+G to be the best-fit models of nucleotide evolution for ITS1, 5.8S, ITS2, and nrLSU markers, respectively, for the Bayesian analysis. The average standard deviation of split frequencies of BI was 0.005711 at the end of the run. BI analyses resulted in almost identical tree topologies with the ML analysis. Only the ML tree is provided in Fig. 1 with the likelihood bootstrap values (≥ 50%, first) and Bayesian posterior probabilities (≥ 0.95, second) labelled along the branches.
In the tree, Scytinostroma s.s. clade received a moderately strong support value in ML analysis (bootstrap value = 56) but a strong value in BI (Bayesian posterior probabilities = 1). Four new distinct lineages corresponding to Scytinostroma beijingensis, S. boidinii, S. subduriusculum and S. subrenisporum spp. nov. were recognized. Sequences of S. portentosum and S. hemidichophyticum from Europe formed a strongly supported lineage sister to the S. portentosum sample from Canada. Samples of S. duriusculum from France and those from Sri Lanka (the type locality) formed a lineage sister to S. subduriusculum. Scytinostroma incrustatum (S.H. He, S.L. Liu & Nakasone) K.H. Larss. and S. artocreas (Berk. & M.A. Curtis) K.H. Larss.,which were formerly placed in Michenera, were nested within Scytinostroma s.s. clade. Table 1. Species and sequences used in the phylogenetic analyses. New species are set in bold with type specimens indicated with an asterisk (*).  cracked or deeply cracked with age; margin thinning out, adnate, fimbriate, white or concolorous with hymenophore surface. Context yellow.
Additional specimens examined. Notes. Scytinostroma beijingensis is characterized by having two kinds of gloeocystidia and short branched skeletal hyphae in hymenium, and growing on Pyrus. In the phylogenetic tree (Fig. 1) (Boidin and Lanquetin 1987). Scytinostroma subrenisporum and S. acystidiatum can be easily distinguished from S. beijingensis by the absence of gloeocystidia (Zhang et al. 2023). Scytinostroma caudisporum is unique in the group for its distinctly large basidiospores (15-30 × 3-3.5 µm, Boidin and Lanquetin 1987). Etymology. Named to honor Dr. Jacques Boidin (Lyon, France) who contributed much to the taxonomy of Scytinostroma.

Discussion
Previous studies showed that Scytinostroma is polyphyletic (Larsson and Larsson 2003). In this study, we performed phylogenetic analyses of Peniophoraceae based on ITS1-5.8S-ITS2-nrLSU sequences of samples of Scytinostroma s.s. and representative taxa of other genera. Species of Scytinostroma s.s. including the type, S. portentosum, formed a moderately supported clade in the likelihood analysis but a strongly supported clade in the Bayesian analysis. For the moment, we prefer to treat this clade as a monophyletic genus and believe that the support values could be higher if more samples of Peniophoraceae are included. Since a sequence of S. portentosum (type species, described from Pennsylvania) from Canada was distinct from the strongly supported sister lineage comprised of sequences of S. portentosum and S. hemidichophyticum from Europe, we suppose that all the European sequences represent S. hemidichophyticum and S. portentosum is restricted in distribution to North America. The two species, S. artocreas and S. incrustatum transferred from Michenera by Stalpers et al. (2021) based on morphological evidence, were nested within the Scytinostroma s.s. Scytinostroma duriusculum is a cosmopolitan species and has been reported from many countries in subtropical and tropical areas (Boidin and Lanquetin 1987;Bernicchia and Gorjón 2010). However, our phylogenetic analyses demonstrated that it could be a species complex, because three lineages were recognized from the samples of France, Sri Lanka, and China, Thailand and Vietnam.
On the one hand, species in Scytinostroma s.s. clade have some common morphological characters, for example, simple-septate generative hyphae, and ovoid, reniform to subglobose basidiospores with amyloid reactions in Melzer's reagent. However, this doesn't mean species with inamyloid basidiospores could not belong to Scytinostroma s.s. On the other hand, the shape of skeletal hyphae, presence of gloeocystidia and encrusted cystidia, and size of basidiospores varies in different species. Based on our phylogenetic and morphological study results, we recognized 14 species of Scytinostroma s.s.worldwide. Until now, seven species have been reported from China, all of which were newly described in the present study and other recently published papers Wang et al. 2020;Zhang et al. 2023). It seems that China, especially its temperate areas, is rich in species diversity of Scytinostroma s.s. Although Scytinostroma s.s. is well studied in the present paper, the species diversity, taxonomy and phylogeny of Scytinostroma s.l. and related genera are still unresolved. A comprehensive study on this issue is urgently needed.
A key to species of Scytinostroma s.s. worldwide