Multiple-marker phylogeny and morphological evidence reveal two new species in Steccherinaceae (Polyporales, Basidiomycota) from Asia

Abstract Two new wood-inhabiting fungi, Mycorrhaphium subadustumsp. nov. and Trullella conifericolasp. nov., are proposed and described from Asia based on ITS, nrLSU and tef1 molecular phylogeny and morphological characteristics. Mycorrhaphium subadustum is characterized by a stipitate basidiocarp, velutinate pileal surface concentrically zoned, hydnoid hymenophore, a dimitic hyphal system in spine trama and monomitic in context, absence of gloeocystidia, presence of cystidioles and the non-amyloid, cylindrical to ellipsoid basidiospores. Trullella conifericola is characterized by a laterally stipitate basidiocarp with flabelliform to semicircular pileus, hirtellous pileal surface with appressed coarse hair and concentrically zoned and sulcate, tiny pores (10–12 per mm), a dimitic hyphal system, absence of any type of cystidia, short clavate basidia and thin-walled, smooth, cylindrical to allantoid basidiospores. Phylogenetic analyses based on a three-marker dataset were performed using maximum likelihood and Bayesian inference methods. The two new species formed isolated lineages with full support in Steccherinaceae. The distinguishing characters of the two new species as well as allied species are discussed, and a key to species of Mycorrhaphium is provided.

Morphological and phylogenetic analyses have provided more accurate identification and contributed to the definition of the taxonomic status of the genera in Steccherinaceae. In recent years, phylogenetic analysis based on multi-marker data has been widely used in the taxonomy of these fungi (He and Dai 2012;Miettinen et al. 2012;Binder et al. 2013;Dai et al. 2014;Miettinen and Ryvarden 2016;Justo et al. 2017;Kotiranta et al. 2017;Westphalen et al. 2018;Yuan et al. 2020).
The species of the Steccherinaceae are widely distributed all over the world. During the investigation of specimens in Steccherinaceae from Asia, several specimens which represent two undescribed species were found. The morphological and molecular features showed that they belong to the genus Mycorrhaphium and Trullella. In this study, we describe them as two new species based on morphological characteristics and threemarker phylogenetic analyses.

Morphological studies
The studied specimens were deposited at the herbarium of the Institute of Applied Ecology, Chinese Academy of Sciences (IFP). Microscopic procedures followed Yuan and Qin (2018). Microscopic observations were made on tissue sections mounted in cotton blue and Melzer's reagent to test for any amyloid and/or dextrinoid reactions (cotton blue: 0.1 mg Methyl blue (SIGMA, PCode: 1001545602) dissolved in 60 g pure lactic acid; Melzer's reagent: 1.5 g KI (potassium iodide), 0.5 g I (crystalline iodine), 22 g chloral hydrate, distilled water 20 mL). The following abbreviations are used in the text: KOH = 2.5% potassium hydroxide; CB = cotton blue; CB+/-= cyanophilous/acyanophilous; IKI = Melzer's reagent; IKI-= neither amyloid nor dextrinoid; L m = mean spore length (arithmetic average of all spores); W m = mean spore width (arithmetic average of all spores); Q = variation in the ratios of L m /W m between specimens studied, and n = total number of spores measured from a given number of specimens. Sections were studied at magnifications up to ×1000 using a Nikon Eclipse E600 microscope (Tokyo, Japan) with phase-contrast illumination, and dimensions were estimated subjectively with an accuracy of 0.1 μm. Microscopic drawings were made with the aid of a drawing tube. The spores' measurements excluded the apiculus, and 5% of the measurements at each end of the range are given in parentheses. The spores' measurements were made with a Nikon SMZ 645 stereomicroscope. Special colour terms are from Kornerup and Wanscher (1981).
PCR reactions were performed in 30 μL reaction mixtures containing 15 μL of 2×Phire Plant PCR buffer, 0.6 μL Phire Hot Start II DNA Polymerase, 1.5 μL of each PCR primer (10 μM), 10.5 μL double deionized H 2 O (ddH 2 O), and 0.9 μL template DNA. The PCR thermal cycling program condition was set as follows: initial denaturation at 95 °C for 5 min, followed by 34 cycles at 95 °C for 30 s, the annealing temperatures were as follows: 58.9 °C for ITS4/ITS5, 47.2 °C for LR0R/LR7, 57.6 °C for 983F/2218R, then 72 °C for 20 s, and a final extension at 72 °C for 7 min. PCR amplification was confirmed on 1% agarose electrophoresis gel stained with ethidium bromide (Stöger et al. 2006) and sequenced at the Beijing Genomics Institute (BGI) with the same primers as used in PCR. The newly generated DNA sequences were assembled and manually modified with the software DNAMAN8 (Lynnon Biosoft, Quebec, Canada). The sequences quality control followed the guidelines by Nilsson et al. (2012). All newly obtained sequences were submitted to GenBank (Sayers et al. 2020). Sequences from allied genera were based on the studies of Miettinen et al. (2012), Yuan (2014) and Westphalen et al. (2019) or found in GenBank (http:// www.ncbi.nlm.gov) using the BLAST option and downloaded (Table 1). DNA alignments were performed using the MAFFT v.7.471 online service (https://mafft.cbrc.jp/ alignment/server/index.html; Katoh et al. 2019). Intron regions of tef1 as well as lowhomology regions of ITS1 and ITS2 were removed before phylogenetic analyses, and the sequence datasets were combined using BioEdit v 7.2.6 (Hall 2005).
Bayesian analysis and Maximum likelihood were applied to the ITS + nrLSU + tef1 dataset. All characters were weighted, and gaps were treated as missing data. Bayesian analysis with MrBayes 3.2.7 (Ronquist et al. 2012) implemented the Markov Chain Monte Carlo (MCMC) technique. The combined dataset was divided into seven partitions: ITS1, 5.8S, ITS2, nrLSU and tef1 1 st , 2 nd as well as 3 rd codon positions. The best-fit models selected were K80+G for ITS1, GTR+I+G for 5.8S, JC+G for ITS2, GTR+I+G for nrLSU, JC for tef1 1 st , TrNef+G for tef1 2 nd and GTR+I+G tef1 3 rd which were determined by the jModelTest 2.1.10 (Darriba et al. 2012) based on the Corrected Akaike Information Criterion (AICc). Four simultaneous Markov chains were run with 10 million generations and starting from random trees and keeping one tree every 100 th generation until the average standard deviation of split frequencies was below 0.01. The value of burn-in was set to discard 25% of trees when calculating the posterior probabilities. Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the trees kept. A Maximum Likelihood (ML) analysis uses the seven-partitions' database which is the same as Bayesian analysis and performed in RAxML v8.2.4 (Stamatakis 2014). The best tree was obtained by performing 1000 rapid bootstrap inferences followed by a thorough search for the most likely tree (Stamatakis et al. 2008). Phylogenetic trees were checked and modified in FigTree 1.4 (Rambaut 2012). The combined dataset and trees were deposited in TreeBASE (No. S27633).

Phylogenetic analyses
Multiple-marker analyses provide an advantage of accurately and promptly discovering a new species or genus (Taylor et al. 2000). Therefore, we used three markers in our dataset which included 75 ITS, 68 nrLSU and 20 tef1. The combined dataset includes two species belonging to the genera Mycorrhaphium and Trullella respectively, and other 69 samples from 23 allied genera. Climacocystis borealis (Fr.) Kotl. & Pouzar was used as the outgroup. The data matrix comprised 163 sequences and had an aligned length of 2121 bases. Bayesian analysis resulted in an average standard deviation of split frequencies = 0.004878. The maximum likelihood and Bayesian analyses produced similar topologies and therefore, only the ML tree is shown in Figure 1. The two new species Mycorrhaphium subadustum and Trullella conifericola were both defined with three markers and they form full-support (100% ML and 1.00 BPP) isolated lineages respectively in this study. The new species M. subadustum clustered together with Mycorrhaphium spp. and form a subclade with American M. adustum. In case of another new species T. conifericola, although the material of T. conifericola Cui 2851 was only provided with ITS sequences, it showed a high similarity of ITS to the other two samples (Yuan 12657 and Yuan 12655) with 99.59% and 98.77% respectively. Furthermore, the morphological and anatomical features, distribution and the coniferous-saprophytic habit suggested it represented an individual which belongs to T. conifericola. Three samples of T. conifericola get together with another six samples from the Trullella clade with support 92% in ML and 1.00 BPP. The phylogenetic tree obtained in this study is similar to that of Miettinen et al. (2012). All the species were divided into 23 main clades which include Antella, Etymology. Subadustum (Lat.), referring to the affinity with M. adustum. Description. Basidiocarps annual, stipitate, solitary or imbricate, corky to soft fibrous, without odor and taste when fresh, light in weight when dry. Pilei semicircular to dimidiate, 2.5-4.5 cm wide and 0.3 cm thick. Pileal surface velutinate, smooth, concentrically zonate, yellowish white to greyish orange (4A2-5B4); margin acute, yellowish white (4A2). Hymenophore hydnoid; spines crowded, evenly distributed, greyish orange (5B4), fibrous, subulate to terete, straight to somewhat flexuous, solitary or confluent, up to 1 mm long, 5-7 per mm; sterile margin smooth, yellowish grey (4B2), up to 2 mm wide. Context yellowish white (3A2), leathery, azonate, homogeneous, up to 0.5 mm thick. Stipe up to 3 cm long, 1 cm wide, straight and base inflated, surface tomentum eventually glabrous, brownish orange (5C4).
Hyphal structure. Hyphal system monomitic in context, dimitic in spine trama; generative hyphae often with clamp connections and simple septate occasionally present; skeletal hyphae thick-walled to subsolid, CB+, IKI-; tissues pale yellow in KOH.

Discussion
The phylogenetic profiling showed that the new species Mycorrhaphium subadustum as well as Trullella conifericola are nested in the Steccherinaceae which belongs to the residual polyporoid clade (Miettinen et al. 2012;Binder et al. 2013;Zmitrovich 2018;Westphalen et al. 2019) where they emerge robustly supported isolated lineages. Furthermore, these lineages are supported by morphological characteristics.
Mycorrhaphium was recommended by Maas Geesteranus (1962) and typified by M. adustum. The two samples of the new species M. subadustum (Yuan 12976 and Dai 10173) clustered in Mycorrhaphium clade, were both collected on fallen branches of angiosperm from northeast of China. The similarity of ITS and nrLSU sequences between the two samples of M. subadustum are 99.00% as well as 99.64% respectively, and they form a full-support isolated lineage which is closely related to M. adustum, the type species of the genus. The type material of M. subadustum Yuan 12976 have a 95.56% similarity of ITS sequences with the American M. adustum KHL12255. Morphologically, M. subadustum is similar to M. adustum in having the velutinate and concentrically zonate pileal surface, presence of clamps and simple septa, a dimitic hyphae system in spine trama and monomitic in context, absence of cystidia as well as gloeocystidia and the non-amyloid basidiospores. However, M. adustum often have a dark-colored pileal margin, which is distinctly different from the yellowish white ones of M. subadustum. Anatomically, the new species can be differentiated from M. adustum by the slender generative hyphae in context (3-5 μm vs. 4-6.3 μm), cyanophilous hyphae and presence of cystidiols (Maas Geesteranus 1962;Ryvarden 1989;Westphalen et al. 2019).
Mycorrhaphium embraced nine species (http://www.indexfungorum.org, 2020) and among which there are others two species described from Asia: Mycorrhaphium sessile H.S. Yuan & Y.C and M. stereoides Maas Geest. M. sessile is a species described from China, but the characteristics such as the sessile basidiocarps and presence of gloeocystidia can differentiate it from M. subadustum (Yuan and Dai 2009). Mycorrhaphium stereoides is related to M. subadustum in having stipitate basidiocarps, hyd-noid hymenophore, a monomitic hyphal system in context and dimitic in spines, but differs from it by the presence of gloeocystidia and the larger basidiospores (4-6.3 × 2.5-3.8 μm) (Maas Geesteranus 1971). The North Europe Mycorrhaphium pusillum (Brot.) Maas Geest. is closely related to M. subadustum in having the stipitate basidiocarps as well as pale colored and zonate pileal surface, but differs it by the presence of gloeocystidia, absence of clamps and the broader basidiospores (Q = 1.52 in M. pusillum vs. 2.13-2.17 in M. subadustum) (Tervonen et al. 2015). Mycorrhaphium ursinum Decock & Ryvarden is a new species from African; its habit of growing on the soil can be distinguished from M. subadustum. Ryvarden (1989) as well as Mossebo and Ryvarden (2003) have provided keys to a part of species in Mycorrhaphium and after which several new taxa have been described. We provide a new key to the whole described species (except M. ursinum) of the genus in this study.
In the phylogenetic tree, nine samples of Trullella species which include the new species T. conifericola form the clade with strong support (92% ML and 1.00 BPP). Trullella is agenus which was originally proposed as 'Trulla' by Miettinen and Ryvarden (2016) and renamed by Zmitrovich (2018). Trullella conifericola is quite an extraordinary species in the genus because of its coniferous-saprophytic habit. The type species of Trullella, T. dentipora (Ryvarden & Iturr.) Zmitr., was described from South America. Trullella dentipora, together with the other species of the genus, inhabits dead angiosperm trees (e.g. Quercus and Cecropia peltata) (Patouillard 1902;Murrill 1907;Miettinen and Ryvarden 2016). Morphologically and anatomically, T. conifericola resembles others Trullella spp. in having sessile or laterally stipitate basidiocarps, mostly small and regular pores, a dimitic hyphal structure, nearly monomitic in the context, and curved cylindrical spores. However, the new species can be distinctly differentiated from others species by having a hirtellous pileal surface with appressed coarse hair, larger spores than those of previous Trullella species (L m = 4.94 μm and W m = 2.09 μm in T. conifericola vs L m = 4.00-4.77 μm and W m = 1.39-1.91 μm in others Trullella spp.), and inhabiting fallen gymnosperm branches. Trullella composed of six species as of now, and the key to these species was provided by Miettinen and Ryvarden (2016).
Besides, the genera Mycorrhaphium and Trullella together with Austeria, Flabellophora and Nigroporus form a large clade in the phylogenetic tree with strong support (85% ML and 1.00 BPP), and share similar morphological features, including zonate or sulcate pileal surfaces, tiny pores or dense spines and a context that is entirely or almost monomitic. They form a distinct subgroup in the Steccherinaceae.