Bull. Natl. Mus. Nat. Sci., Ser. B, 47(1), pp. 1–12, February 22, 2021 Russula ryukokuensis sp. nov., an Outstanding Species of the Genus Russula (Russulaceae) Having Minute Basidiomata from Japan Yoshito Shimono1*,2, Taiga Kasuya3*,** and Kentaro Hosaka4 1 Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-machi, Tsu, Mie 514–8507, Japan 2 Osaka Museum of Nature History, 1–23 Nagai-koen, Sumiyoshi-ku, Osaka, Osaka 546–0034, Japan 3 Department of Biology, Keio University, 4–1–1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223–8521, Japan 4 Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan *Both authors contributed equally to this work. **E-mail: [email protected] (Received 23 September 2020; accepted 23 December 2020) Abstract. Russula ryukokuensis, a new species of Russula is described from mixed broad-leaved trees and coniferous forests in Japan based on macro- and microscopic features with molecular data. Russula ryukokuensis is characterized by small-sized, orange to reddish basidiomata, convex to plano-convex pileus with shallowly tuberculate to sulcate margin, and whitish to slightly light orange lamellae with strongly acrid taste. Detailed macro- and micro-morphological descriptions of R. ryukokuensis are given, and its taxonomic and phylogenetic positions were confirmed using DNA sequences. Morphological observations and molecular phylogenetic analysis revealed that this species belongs to the subsection Emeticinae of the section Russula in the subgenus Russula. Keywords : Agaricomycetes, ITS and LSU sequences, mycobiota, phylogeny, Russulales, taxon- omy. Introduction (Atri et al., 1993, 1997; Buyck and Horak, 1999; Miller and Buyck, 2002; Chou and Wang, 2005; Species of the genus Russula Pers. are widely Das and Sharma, 2005; Buyck et al., 2008, 2018; distributed in diverse forests from the tropical to Das et al., 2010, 2013a, 2013b, 2014, 2017; the frigid zones of all continents except Antarc- Shin, 2010; Dutta et al., 2015; Li et al., 2015; tica and are well-known as ectomycorrhizal fungi Ghosh et al., 2016, 2017; Melera et al., 2016; (Kirk et al., 2008). Currently, ca. 100 species of Jabeen et al., 2017). However, species diversity the genus Russula are recorded in Japan (Imazeki of Japanese Russula inferred from molecular and Hongo, 1989; Ikeda, 2013). Recently, num- phylogeny is still not comprehensively studied. bers of molecular studies have been conducted During recent taxonomic and phylogenetic stud- using internal transcribed spacer (ITS) region ies on Japanese Russula (Shimono et al., 2004, and nuclear large subunit (LSU) gene of ribo- 2014, 2018), we collected remarkably small somal DNA sequences to determine taxonomic (4–10 mm across), orange to reddish basidiomata and phylogenetic positions of Russula species with strongly acrid taste of the genus in central to western parts of Honshu. © 2021 National Museum of Nature and Science The aim of this study was to clarify the taxo- 2 Yoshito Shimono et al. nomic and phylogenetic positions of this out- cation of Russula followed Romagnesi (1985, standing Russula species having minute basidi- 1987), with some references to Bon (1987, omata. In this paper, we describe this Russula 1988), Singer (1986), Sarnari (1998, 2005) and species as a new to science based on morphologi- Buyck et al. (2018). Color annotations of fresh cal observations and molecular phylogenetic materials were determined based on Kornerup analysis using rDNA ITS and LSU sequences. and Wanscher (1978). Fresh basidiomata of each specimen were dried using a food dehydrator (Snackmaster Express FD-61, Nesco/American Materials and Methods Harvest, WI, USA) under 46°C. Preparation of specimens We collected Russula specimens having Micro-morphological observations orange to reddish, minute basidiomata with acrid Microscopic features of specimens were taste in Shiga, Mie, Hyogo and Hiroshima Pre- observed from dried material mounted in 10% fectures, central to western Honshu of Japan dur- KOH and Melzer’s reagent, using an Olympus ing our fieldwork from 2009 to 2020. Addition- BX-53 light microscope (Olympus, Tokyo, ally, for molecular phylogenetic analysis, Japan) under Nomarski interference contrast. specimens of R. decolorans (Fr.) Fr., R. kan- More than 20 randomly selected basidiospores saiensis Hongo and R. omiensis Hongo collected were measured under a light microscope at from our fieldwork in Japan were also investi- 1000× magnification. For basidiospores, the gated (Table 1). Specimens examined in this factor Q (mean of quotient of length and width in study were deposited at the herbaria of the any one spore) was also calculated to indicate National Museum of Nature and Science (TNS), spore shape. In addition, the surface features of Tsukuba, Japan and the Osaka Museum of Natu- basidiospores were observed by scanning elec- ral History (OSA), Osaka, Japan. tron microscopy (SEM). For SEM, a small por- tion from pileus was put onto double-sided adhe- Macro-morphological Observations sive tape on a specimen holder and coated with Each specimen of Russula was photographed, platinum-palladium using a JFC-1600 Ion Sput- and macroscopic observation was conducted in ter Coater (JEOL, Tokyo, Japan). Specimens fresh state. Morphologies of the fruiting bodies were examined with a JSM-6480LV SEM were recorded according to Ikeda (2013), Ima- (JEOL) operating at 20 kV. zeki and Hongo (1989) and Shimono et al. (2014, 2018). In this paper, infrageneric classifi- Table 1. List of sequences newly generated from Japanese specimens of Russula Sampling Voucher GenBank accession number* Species Collector Locality date number ITS LSU Russula decolorans 2002 Aug. 24 Y. Goto Japan: Yamanashi OSA-MY-9216 Not obtained LC269001 R. kansaiensis 2011 Nov. 9 T. Kasuya Japan: Ibaraki TNS-F-43739 MN989318 MN989319 R. omiensis 1996 Apr. 20 Y. Shimono Japan: Kyoto OSA-MY-1763 LC269002 LC269002 R. omiensis 2013 Mar. 24 Y. Shimono Japan: Kyoto OSA-MY-9217 LC269003 LC269003 R. omiensis 2013 Mar. 24 Y. Shimono Japan: Kyoto OSA-MY-9218 LC269004 LC269004 R. omiensis 2013 Mar. 24 Y. Shimono Japan: Kyoto OSA-MY-9219 LC269005 LC269005 R. ryukokuensis 2009 Oct. 11 Y. Kotera Japan: Shiga OSA-MY-9225 LC269011 LC269011 R. ryukokuensis 2013 Jul. 20 T. Ueda Japan: Shiga TNS-F-70424 MH037291 MH037293 R. ryukokuensis 2016 Sep. 19 T. Kasuya Japan: Shiga TNS-F-70425** MH037292 MH037294 *Identical accession numbers for ITS and LSU indicate a single rDNA sequence containing both regions. ** Holotype Russula ryukokuensis sp. nov. from Japan 3 DNA extraction, PCR, and sequencing ITS and LSU datasets. Phylogenetic analysis was DNA extraction, PCR and DNA sequencing performed in MEGA 7 software (Kumar et al., were carried out according to the methods intro- 2016) with the maximum likelihood (ML) duced by Shimono et al. (2004, 2007, 2014, method. For the ML analysis, clade robustness 2018). Briefly, fungal DNA was extracted from was assessed using a bootstrap analysis with the lamellae of fresh basidiomata using Indicat- 1000 replicates (Felsenstein, 1985). Sequences of ing FTA Cards (Whatman International Ltd, R. decolorans and R. lilacea Quél. which were Maidstone, UK) based on the manufacturer’s pro- strongly supported as the sister to the subgenus tocol. PCR amplifications of the ITS (ITS1-5.8S- Russula Pers. emend. Romagn. in previous stud- ITS2) region and the LSU gene of rDNA were ies (Shimono et al., 2004, 2018), were selected carried out using one prepared FTA disc 2 mm for outgroups. The final alignment is available diam., according to the manufacturer’s instruc- from TreeBASE (http://www.treebase.org/) as a tion. The ITS1F/ITS4B (Gardes and Bruns, NEXUS file under the accession number 25896. 1993) and the ITS1/ITS4 (White et al., 1990) primer pairs for the rDNA ITS region, and the Results and Discussion BN1/TW14 (Shimono et al., 2004) and the NL1/ NL4 (Mori et al., 2000) primer pairs for the Phylogenetic analyses rDNA LSU gene were used for amplification and Among Japanese Russula specimens having sequencing. PCR reactions were performed using orange to reddish, minute basidiomata with acrid KOD FX Neo DNA polymerase (Toyobo, Tokyo, taste, ITS and LSU sequences of three samples Japan) in 25 mL reaction volumes containing 5 μl collected from Ryukoku University Forest in of 0.2 mM dNTP, 12.5 μl of PCR buffer, and 0.5 Shiga Prefecture were successfully generated U KOD FX Neo. PCR conditions were 94°C for (Table 1). The ITS dataset had an aligned length 2 min, followed by 40 cycles at 98°C for 10 s, of 769 characters including gaps consisting of 49 55°C for 30 s, 68°C for 1 min, and a final 6 min at ingroups and three outgroups. The maximum 68°C. The DNA sequencing was performed at likelihood analysis of the ITS dataset resulted in SolGent Co. Ltd. (Daejeon, South Korea) using one ML tree with the highest log likelihood an ABI 3700 automated DNA Sequencer (－4258.54). The resulting ML topology is (Applied Biosystems Inc., Foster City, CA, shown in Fig. 1. From the ML analysis of the USA). ITS dataset, 49 sequences of Russula were divided into two major clades A and B (Fig. 1). Phylogenetic analyses Species included in the major clade A are mem- A total of eight ITS and nine LSU sequences bers of the section Russula Pers. of the subgenus were newly generated from our specimens (Table Russula, while the major clade B contains spe- 1). Additional 44 ITS and 25 LSU sequences cies belonging to the subgenus Tenellula were retrieved from the GenBank and UNITE Romagn. (Romagnesi, 1985). Major clade A cor- databases for phylogenetic analysis (Table 2). responded with Clade 5 of Miller and Buyck DNA sequences were initially aligned using (2002), Clade A of Li et al. (2015) and Clade VII Muscle v.3.6 (Edgar, 2004a, 2004b), followed by of Buyck et al. (2018). The LSU dataset had an manual alignment in the data editor of BioEdit aligned length of 610 characters including gaps ver.