Aleurodiscus Bicornis and A. Formosanus Spp. Nov. (Basidiomycota) With Smooth Basidiospores, and Description of A. Parvisporus From NE China

Three species of Aleurodiscus s.l. characterized in having effused basidiomata, clamped generative hyphae and quasi-binding hyphae, sulphuric positive reaction of gloeocystidia, hyphidia, acanthophyses and smooth basidiospores, are described. They are A. bicornis sp. nov., A. formosanus sp. nov. and A. parvisporus. Aleurodiscus bicornis was found from high mountains of NW Yunnan Province of SW China, grew on branch of Picea sp. Aleurodiscus formosanus was found from high mountains of central Taiwan, grew on branch of gymnosperm. Aleurodiscus parvisporus was previously reported only once from Japan and Sichuan Province of China respectively, and is reported in this study from Jilin Province of China. Phylogenetic relationships of these three species were inferred from analyses of a combined dataset consisting of three genetic markers, viz. 28S, nuc rDNA ITS1-5.8S-ITS2 (ITS), and a portion of the translation elongation factor 1-alpha gene, TEF1. The studied three species are phylogenetically closely related with signicant support, corresponds with resemblance of their morphological features.

However, it has long been a complicated and unsettled problem as how to delimit the segregate genera among Aleurodiscus s.l. (Núñez and Ryvarden 1997 Since the phylogenetic relationships of the taxa in Aleurodiscus s.l., together with the family Stereaceae it belongs, are not resolved, we adopt a broad and inclusive generic concept of Aleurodiscus for the studied taxa presented in this study.
During an ongoing survey of corticioid fungi from mainland China and Taiwan, we noted two undescribed and an uncommon Aleurodiscus sp. with effused basidiomata, clamped generative hyphae and quasi-binding hyphae, sulphuric positive reaction of gloeocystidia, hyphidia, acanthophyses and smooth basidiospores. These morphologically resembling species are also phylogenetically closely related.

Materials And Methods
Morphological study Specimens used in this study are deposited in the herbarium of the National Museum of Natural Science of ROC (TNM; Taichung City, Taiwan). Macroscopic and microscopic studies were based on dried specimens. Color names from Rayner (1970) are capitalized. Thin free-hand sections of basidiocarps were prepared for microscopic study. For observations and measurements of microscopic characters, sections were mounted in 5% KOH to ensure rehydration. A blue-black color change with Melzer's reagent (IKI) indicates an amyloid reaction. Cotton blue (CB) was used as mounting medium to determine cyanophily. Sulphoaldehyde (SA) was used to detect a sulphuric reaction of gloeocystidia; a bluish black color change with SA indicates a positive reaction. The following abbreviations are used for basidiospore measurements: L = mean spore length with standard deviation, W = mean spore width with standard deviation, Q = variation in L/W ratio, and n = number of spores measured from each specimen. Apiculi and ornamentation were excluded in spore measurements. Living mycelia were isolated from the woody substratum beneath the basidiocarps, and were cultured on 1.5% malt extract agar (MEA).

DNA extraction and sequencing
Dried specimens or the mycelial colonies cultured on MEA were used for DNA extraction, carried out with a Plant Genomic DNA Extraction Miniprep System (Viogene-Biotek Corp., New Taipei City, Taiwan). Liquid N and TissueLyser II (Qiagen, Hilden, Germany) were used to disrupt and homogenize the fungal tissues before DNA extraction process. The primer pairs ITS1/ITS4 or ITS1F/LR22 were used for the ITS region (White et al. 1990, Gardes and Bruns 1993), and LR0R/LR3 and LR0R/LR5 were used for the 28S region (Vilgalys and Hester 1990). Efdf/1953R and 983F/2218R were used to amplify a portion of the TEF1 gene (Rehner & Buckley 2005;Matheny et al. 2007). PCR products were puri ed and directly sequenced by MB Mission Biotech Company (Taipei City, Taiwan). We examined the technical quality of the newly obtained sequences by comparison to entries in GenBank (Mashima et al. 2016). Sequences were assembled using BioEdit v7.2.5 (Hall 1999). Newly obtained sequences (Supplementary Table 1 In ML analysis, the bootstrap values were calculated with 1000 replicates. Only phylogram inferred from BI analyses was shown since both ML and BI analyses resulted in similar topologies. Statistic supports were shown on nodes of BI tree when BP value ≥ 70 and PP value ≥ 0.7. The nal alignment and phylogenetic trees were submitted to TreeBASE (TBS: S27713; www.treebase.org).

Phylogeny
The 3-gene dataset was composed of 63 taxa and 2923 sites including gaps, 1420 for 28S, 932 for ITS and 571 for TEF1. The absent gene sequences were treated as missing data. GTR+G+I model was t for ML analysis. For BI analysis, partition model was selected, GTR+I+G for 28S, GTR+G for ITS, GTR+I+G+I for TEF1. Average standard deviation of split frequencies fell to 0.008016 after 10 million generations.

Discussion
The studied three Aleurodiscus species share some important morphological characteristics, i.e. effused basidiomata, clamped generative hyphae and quasi-binding hyphae, sulphuric positive reaction of gloeocystidia, hyphidia, acanthophyses and smooth basidiospores. These three species are phylogenetically closely related with signi cant support (Fig. 1), corresponds with resemblance of their morphological features. An independent generic taxon separate from Aleurodiscus s.s., to accommodate these three new species, should be proposed. However, it needs further studies by advanced phylogenetical analysis based on a comprehensive survey of Aleurodiscus s.l.
Aleurodiscus dextrinoideocerussatus Manjón, M.N. Blanco & G. Moreno is distributed in Spain and Italy, resembles A. formosanus in having similar morphological characteristics including acanthophyses with dextrnoid apical protuberances; but differs from the latter in having smaller basidiospores (7 10 × 4 7 μm, Núñez and Ryvarden 1997). In addition, the lateral aculeate branch of basidia in A. formosanus, is not present in A. dextrinoideocerussatus. Aleurodiscus dextrinoideocerussatus is phylogenetically closely related to the presented three new species (Fig. 1).
Aleurodiscus parvisporus was originally reported based on the holotype collected from Niigata Prefecture of Japan (Núñez and Ryvarden 1997). The second specimen of this species was reported from high mountain (3890 m) of Sichuan Province in China (Maekawa et al. 2002). The present study reports two specimens of this species collected from NE China in 2013. All of these four collections were made from branches of angiosperms. Recognition of two specimens (Wu 1307-84 & Wu 1307-88) collected from NE China as A. parvisporus, is based on resemblance of some diagnostic features described from the holotype (Núñez and Ryvarden 1997): effused basidiomata with similar color of hymenial surfaces, thin subiculum and thick hymenial layer, clamped generative hyphae, gloeocystidia, narrow acanthophyses, and smooth basidiospores which are distinctly small in Aleurodiscus s.l. It is hardly to have different species to share these characteristics. Moreover, the collecting site of these two specimens in Jilin Province of China, is geographically close to the holotype collected from Niigata Prefecture of Japan.
However, the characteristics of hyphidia and basidia with lateral aculei present in the specimens (Wu 1307-84 & Wu 1307-88), were not mentioned for the holotype of A. parvisporus. Nevertheless, the hyphidia could be neglected in study, and good basidia may be not found in the holotype as drawing of this structure was lacking in the protologue (Núñez and Ryvarden 1997). This study rst reports DNA sequences of A. parvisporus. Table   Table 1. List of species, specimens and sequences used in this study. Sequences generated in this study are shown in boldface.