Four new corticioid species in Trechisporales (Basidiomycota) from East Asia and notes on phylogeny of the order

Abstract Four new species in Trechisporales from East Asia, Dextrinocystiscalamicola, Subulicystidiumacerosum, S.tropicum and Tubuliciumbambusicola, are described and illustrated, based on morphological and molecular evidence. The phylogeny of Trechisporales was inferred from a combined dataset of ITS-nrLSU sequences. In the phylogenetic tree, Sistotremastrum formed a family-level clade of its own, sister to the Hydnodontaceae clade formed by all other genera. Dextrinocystis, is for the first time, confirmed as a member of Hydnodontaceae. A key to all the accepted genera in Trechisporales is given.

Except for Trechispora, the largest genus in the order, most genera in Trechisporales have mostly few species and some are still monotypic. However, in recent years, many new species have been described, based on both DNA sequence data and morphological characters. Wu et al. (2015) described a cryptic species of Porpomyces mucidus (Pers.) Jülich, based mainly on sequence data. Ordynets et al. (2018) studied the shortspored species of Subulicystidium and recognised eleven new species. Tens specimens of Trechisporales were collected from East Asia by the senior authors in the past three years. The purposes of the present paper are to study these specimens by using morphological and molecular methods and discuss the phylogeny of the Trechisporales, based on expanded sampling.

Morphological studies
Voucher specimens were deposited in the herbaria of Beijing Forestry University, Beijing, China (BJFC) and in the Centre for Forest Mycology Research, U.S. Forest Service, Madison, USA (CFMR). Freehand sections were made from dried basidiomata and mounted in 0.2% cotton blue in lactic acid, 1% phloxine (w/v) or Melzer's reagent. 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. All measurements were carried out with sections mounted in Melzer's reagent. The following abbreviations are used: L = mean spore length, W = mean spore width, Q = L/W ratio, n (a/b) = number of spores (a) measured from given number of specimens (b). Colour names and codes follow Kornerup and Wanscher (1978).
For both Maximum Likelihood (ML) and Bayesian Inference (BI), a partitioned analysis was performed with the following four partitions: ITS1, 5.8S, ITS2 and nrLSU. The ML analysis was performed using RAxML v.8.2.10 (Stamatakis 2014) with the bootstrap values (ML-BS) obtained from 1,000 replicates and the GTR-GAMMA model of nucleotide evolution. The BI was performed using MrBayes 3.2.6 (Ronquist et al. 2012). The best-fit substitution model for each partitioned locus was estimated separately with jModeltest v.2.17 (Darriba et al. 2012) by restricting the search to models that can be implemented in MrBayes. Two runs of four Markov chains were run for 4,000,000 generations until the split deviation frequency value was lower than 0.01. The convergence of the runs was checked using Tracer v.1.7 (Rambaut et al. 2018). Trees and model parameters were sampled every 100 th generation. The first quarter of the trees, which represented the burn-in phase of the analyses, was discarded and the remaining trees were used to build a majority rule consensus tree and to calculate Bayesian posterior probabilities (BPP). All trees were visualised in FigTree 1.4.2 (Rambaut 2014).

Phylogenetic inference
The ITS-nrLSU sequence dataset contained 50 ITS and 51 nrLSU sequences from 58 samples representing 45 ingroup taxa and the outgroup (Table 1). Fourteen ITS and 15 nrLSU sequences were generated for this study. jModelTest suggested GTR+G, SYM+I+G, GTR+I+G and 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. BI analysis resulted in an almost identical tree topology compared to the ML analysis  Larsson et al. (2004) and no significant conflicts were found between the two analyses. Only the ML tree is shown in Fig. 2 with ML bootstrap values ≥ 50% and Bayesian posterior probabilities ≥ 0.95 labelled along the branches.
In the tree (Fig. 2), two large clades, corresponding to Hydnodontaceae and Sistotremastrum family, were strongly supported. Except for Sistotremastrum, the other eight genera sampled were nested within the Hydnodontaceae clade. The genera Brevicellicium, Fibrodontia, Porpomyces and Subulicystidium were strongly supported as monophyletic lineages. Dextrinocystis calamicola, the first species sequenced in the genus, formed a sister lineage to Tubulicium with relatively strong support (ML-BS = 78%, BPP = 1). The three species of Scytinopogon were nested within the Trechispora lineage. Subulicystidium acerosum and S. tropicum formed distinct lineages in the genus, while Tubulicium bambusicola is closely related to T. raphidisporum. Etymology. "calamicola" refers to growing on Calamus. Basidiomata. Annual, resupinate, effused, thin, soft, easily separated from the substrate, at first as irregular small patches, later confluent up to 15 cm long, 2 cm wide. Hymenophore surface smooth, orange white (5A2) to greyish-orange [5B(3-5)], finely cracked with age; margin thinning out, fimbriate, slightly paler than hymenophore surface, becoming indistinct with age.
Remarks. The thin whitish basidiomata on a palm tree, distinctly thick-walled cystidia with a dextrinoid reaction in Melzer's reagent, presence of small cystidia-like branches and short cylindrical basidiospores indicate that the new species is a member of Dextrinocystis. Two species, D. capitata (D.P. Rogers & Boquiren) Gilb. & M. Blackw. and D. macrospora (Liberta) Nakasone have been reported in the genus, both of which differ from D. calamicola by having much larger basidiospores (11-14 × 3-4 µm for D. capitata in Gilbertson and Blackwell 1988;12-19 × 4.5-7 µm for D. macrospora in Liberta 1960) and a distribution in America. In the phylogenetic tree, D. calamicola formed a sister lineage to Tubulicium with relatively strong support (Fig. 2). Etymology. "acerosum" refers to the presence of numerous needle-like crystals. Basidiomata. Annual, resupinate, effused, very thin, easily separated from the substrate, up to 6 cm long, 2 cm wide. Hymenophore surface smooth, more or less arachnoid, white (5A1) to orange grey (5B2); margin undifferentiated.

Discussion
Nine genera in the Trechisporales were included in the present analyses and the results mostly agree with previous studies (Larsson 2007;Birkebak et al. 2013;Telleria et al. 2013a). Most of the sampled genera were retrieved as monophyletic except Scytinopogon, which was nested within the Trechispora lineage (Fig. 2). A Dextrinocystis species was sequenced for the first time and its position in Hydnodontaceae was confirmed. As indicated by the morphology (Burdsall and Nakasone 1983;Gilbertson and Blackwell 1988;Moreno and Esteve-Raventós 2007;Nakasone 2013), the genus is closely related to Tubulicium. However, Tubulicium is morphologically heterogenous, with different basidiospores (Moreno and Esteve-Raventós 2007;Hjortstam and Ryvarden 2008) and only species with fusiform to vermicular basidiospores were sequenced. Moreover, Dextrinocystis is well distinguished from Tubulicium by its distinctly dextrinoid cystida and cylindrical basidiospores (Gilbertson and Blackwell 1988;Nakasone 2013). Thus, at present, the authors prefer to retain them as separate genera until more species are sequenced.
Subulicystidium is a well-circumscribed genus characterised by the unique cystidia encrusted with rectangular crystals and fusiform to vermicular basidiospores (Bernicchia and Gorjón 2010; Ordynets et al. 2018). Although all the sampled species formed a strongly supported lineage in the tree (Fig. 2), the species S. oberwinkleri Ordynets, Riebesehl & K.H.Larss. was not congeneric with other species and excluded from our analyses. Ordynets et al. (2018) showed that S. oberwinkleri formed a distinct basal lineage in the ITS-nrLSU tree. The phylogenetic position of the species in Trechisporales needs to be further studied.
Key to accepted genera in Trechisporales