MycoKeys 75: 1-29 (2020) er-reviewed open-access journal doi: 10.3897/mycokeys.75.53673 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Taxonomic revision of Russula subsection Amoeninae from South Korea Komsit Wisitrassameewong'’, Myung Soo Park', Hyun Lee!?, Aniket Ghosh’, Kanad Das*, Bart Buyck°, Brian P. Looney®, Miroslav Caboni’, Slavomir Adam¢ik’, Changmu Kim®, Chang Sun Kim’, Young Woon Lim! I School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 08826, South Ko- rea 2. National Biobank of Thailand (NBT), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Thanon Phahonyothin, Tlambon Khlong Neung, Amphoe Klong Luang, Pathum Thani 12120, Thailand 3 Department of Botany & Microbiology, H.N.B. Garhwal University (A Central Univer- sity), Srinagar, Garhwal, 246174, Uttarakhand, India 4 A.J.C. Bose Indian Botanic Garden, Botanical Survey of India, PO. Botanic Garden, Howrah 711103, India § ISYEB (CNRS, Sorbonne Université, EPHE) Institut de Systématique, Evolution, Biodiversité, Muséum national d'Histoire naturelle, case postale 39, 57 rue Cuvier, F-75231 Paris cedex 05, France 6 Department of Biology, Duke University, Durham, NC 27708, USAT Plant Science and Biodiversity Centre, Slovak Academy of Sciences, Dubravskd cesta 9, SK-845 23, Bratislava, Slo- vakia 8 Microorganism Resources Division, National Institute of Biological Resources, Incheon 22689, South Korea 9 Forest Biodiversity Division, Korea National Arboretum, Pocheon-si, Gyeonggi-do 11186, South Korea Corresponding author: Young Woon Lim (ywlim@snu.ac.kr) Academic editor: Zai-Wei Ge | Received 27 April 2020 | Accepted 9 October 2020 | Published 9 November 2020 Citation: Wisitrassameewong K, Park MS, Lee H, Ghosh A, Das K, Buyck B, Looney BP, Cabon M, Adam¢ik S, Kim C, Kim CS, Lim YW (2020) Taxonomic revision of Russula subsection Amoeninae from South Korea. MycoKeys 75: 1-29. https://doi.org/10.3897/mycokeys.75.53673 Abstract Russula subsection Amoeninae is morphologically defined by a dry velvety pileus surface, a complete ab- sence of cystidia with heteromorphous contents in all tissues, and spores without amyloid suprahilar spot. Thirty-four species within subsection Amoeninae have been published worldwide. Although most Russula species in South Korea have been assigned European or North American names, recent molecu- lar studies have shown that Russula species from different continents are not conspecific. Therefore, the present study aims to: 1) define which species of Russula subsection Amoeninae occur on each continent using molecular phylogenetic analyses; 2) revise the taxonomy of Korean Amoeninae. The phylogenetic analyses using the internal transcribed spacer (ITS) and multilocus sequences showed that subsection Amoeninae is monophyletic within subgenus Heterophyllidiae section Heterophyllae. A total of 21 Russula Copyright Komsit Wisitrassameewong et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) subsection Amoeninae species were confirmed from Asia, Australia, Europe, North America, and Central America, and species from different continents formed separate clades. Three species were recognized from South Korea and were clearly separated from the European and North American species. These species are R. bella, also reported from Japan, a new species described herein, Russula orientipurpurea, and a new species undescribed due to insufficient material. Keywords Amoeninae, Heterophyllae, multilocus phylogeny, Russula orientipurpurea, species delimitation Introduction Russula Pers. is the largest genus in the family Russulaceae, with at least 2,000 de- scribed species worldwide (Adam¢ik et al. 2019). Compared to most other genera of Basidiomycetes, Russula has complex morphological and chemical features (Buyck et al. 2018). The initial period of diversification of the genus has been inferred as occurring in temperate regions of the Northern hemisphere, though there is some debate as to its origin given its high diversity in tropical areas (Looney et al. 2016; Buyck et al. 2018). Recent molecular studies have recognized eight subgenera within the genus: Russula subg. Archaeae Buyck & V. Hofst., R. subg. Brevipedum Buyck & V. Hofst., R. subg. Compactae (Fr.) Bon, R. subg. Crassotunicatae Buyck & V. Hofst., R. subg. Glutinosae Buyck & X.H. Wang, R. subg. Heterophyllidiae Romagnesi, R. subg. Malodorae Buyck & V. Hofst., and R. subg. Russula (Buyck et al. 2018; Buyck et al. 2020). In the field, specimens of Russula subsection Amoeninae Buyck are identified by their velvety pileus surface and stipe, white spore print, mild taste, and stipe flushed with pink, red, or purple. Microscopically, species of Amoeninae display subglobose spores, typically with prominent amyloid and reticulate ornamentation but without amyloid suprahilar spot. Moreover, the basidiomes completely lack gloeocystidia, and both pileipellis and lamellar edges have large subulate hyphal terminations often aris- ing from short unbranched cells (Buyck 1988, 1994). Because of the complete ab- sence of gloeocystidia, Sarnari (1998) altered the rank of this subsection to subgenus, proposing Russula subg. Amoenula Sarnari. Recent multilocus phylogenetic studies, however, have shown that Amoeninae is in fact a small part of R. subg. Heterophyllidiae section Heterophyllae (see position of R. violeipes Quél. and R. mariae Peck in Buyck et al. 2018). To date, 34 species have been published worldwide within Russula subsection Amoeninae (Suppl. material 1: Table S1), some of which were not originally placed in this group (e.g. R. diversicolor Pegler, R. epitheliosa Singer, and R. variegata Romagn.). However, their microscopic features are very similar to those of other Amoeninae spe- cies. Three species have been reported from Europe: R. amoena Quél., R. amoenicolor Romagn., and R. violeipes Quél. (Sarnari 1998). Although no relevant molecular stud- ies have been performed that include species from North America, previous studies us- ing morphological data have listed 14 species for this area (Suppl. material 1: Table S1). Regarding the tropics, R. diversicolor and R. epitheliosa were reported from neotropical Russula subsection Amoeninae from South Korea 3 areas of Latin America (Buyck 1988), as were six species from tropical areas of Africa (Buyck 1994; Buyck and Sharp 2007) and Madagascar (Heim 1938), and two species from arid areas of Australia (Lebel and Tonkin 2007, Hyde et al. 2016). Finally, six spe- cies native to Asia have been described, two from East Asia (Chiu 1945; Hongo 1968), and four from India (Das et al. 2005, 2017; Crous et al. 2016; Hyde et al. 2016). Historically, taxonomic studies of Russula in Asia have been influenced by Euro- pean or North American literature. That is why Russula species from Asia were often as- signed names of morphologically similar counterparts from Europe or North America. However, recent molecular studies have revealed that many Asian Russula species are in fact not conspecific with European or North American species (Park et al. 2014; Lee et al. 2017; Paloi et al. 2018; Song et al. 2018). Although molecular data can provide additional information that may result in more robust phylogenies (Hebert et al. 2003; Savolainen et al. 2005; Hibbett et al. 2016; Adaméik et al. 2019), the use of this type of data may be hampered by misidentifications when there are limited reference databases or when low-resolution markers are used (Hofstetter et al. 2019). The present study aims to: 1) clearly distinguish species of Russula subsection Amoeninae from different continents through a phylogenetic analysis using updated sequence data; and 2) revise the taxonomy of Korean Amoeninae based on materi- als obtained from recent collections from different habitats and areas of the Korean peninsula. Four Amoeninae species were reported in South Korea: R. amoena, R. bella, R. mariae, and R. violeipes (Park et al. 2013; Lee et al. 2015). Russula amoena and R. violeipes were described from Europe, R. bella from Japan, and R. mariae from North America. With the increase in available Russwla sequence data, taxonomists can inves- tigate more precisely the boundaries and distribution of Korean species. ‘Therefore, the present study also aims to verify whether species are conspecific between continents using internal transcribed spacer (ITS) sequences from GenBank and generated for this study in a first analysis and, in a second analysis, using a concatenated dataset of other molecular markers including the second largest subunit of RNA polymerase II (7p62), mitochondrial small subunit ribosomal DNA region (mtSSU), and the transla- tion elongation factor 1-alpha (tef/a). Methods Sampling A total of 15 collections from the Korean peninsula were included in this study. All Korean specimens were deposited in the Seoul National University Fungus Collection (SFC) and The Herbarium Conservation Center of the National Academy of Agri- cultural Science (HCCN). Because of a paucity of available sequence data from other continents, eight additional specimens from USA, four from Europe, and one from India were sequenced; all non-Asian samples are from the Herbarium of Plant Science and Biodiversity Centre of the Slovak Academy of Sciences (SAV) (Table 1 and Suppl. material 2: Table S2). 4 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) Table |. Specimens used for the multi-locus analyses in this study. Sequences produced in this study are presented in boldface. Taxon Herbarium no. Locality rpb2 GenBank accession no. oie Outgroup R. aff. delica 1119/BB 12.086 Italy KU237879 KU237442 KU238020 R. chloroides 572/BB 07.209 Slovakia KU237845 KU237407 KU237990 ae KUBT Other subgenera R. aff. griseobrunnea 741/BB 09.344 New Caledonia KU237877 KU237440 KU238018 R. cfr. Liberiensis 91/BB 06.184 Madagascar KU237760 KU237318 KU237905 R. compacta 228/B 06.295 USA ees KU237324 R. edulis 579/BB 08.167 Madagascar —— KU237412 KU237993 nls KU PHT Russula sp. 570/BB 08.178 Madagascar RES KU237405 KU237988 Closely related groups in subg. Heterophyllidiae R. aff. crustosa 31/BB 06.616 Canada KU237747 KU237305 KU237896 R. aff. madagassensis Madagascar eae KU237906 Tia KU23309 R. amoenolens 577/ BB 08. O75 Italy SOD KU237848 - R. cle. annulata 75/BB 06.048 Madagascar KU237756 KU237314 KU237902 R. cfr. illota 36/ BB 06.380 Mexico KU237750 KU237308 KU237898 R. cf. pseudocarmesina Madagascar KU237739 - aeeranell : R. cfr. vesca 45/BB 06.525 Mexico KU237751 KU237309 KU237899 R. flavobrunnea vat. violaceotincta 71/ BB 06.050 Madagascar KU237754 KU237312 KU237901 R. grisea 449/BB 07.184 Slovakia KU237795 KU237355 KU237939 Bowel KU i lng KUTA R. madagassensis 21/BB 06.146 Madagascar KU237742 KU237300 KU237891 R. maguanensis XHW4765 China MH939989 - MH939983 R. medullata 555/BB 07.252 Slovakia ——— KU237392 KU237976 per KU23802 R. oleifera Tanzania ae KU237919 46/BB 06.530 Mexico KU237752 18/BB 06.161 KU237741 578/ BB 05.160 USA _— KU237310 - KU237299 KU237890 KU237411 7 R. ornaticeps R. prolifica Madagascar R. pulverulenta Rue. ROBOT Tubs [—xewaes [Chim | Mnb900¢ |__| M3988 Subsect. Amoeninae R. aff. mariae SAV F-4484 USA, New York State - MT417192 R. aff. mariae SAV F-4493, USA, New York State - MT417193 MT417213 R. aff. mariae SAV F-4564 USA, New York State MT417194 MT417214 R. alachuana SAV 1252 USA, Florida MT417198 MT417186 MT417204 R. alachuana SAV F-20108 USA, Florida MT417199 MT417187 MT417206 R. amoena SAV F-1352 Slovakia MT417200 MT417185 - R. amoena SAV F-3147 Slovakia MT417202 MT417190 MT417211 R. cf. amoenicolor SAV F-20302 Greece MT417196 MT417188 MT417209 R. cf. amoenicolor SAV F-20324 Greece MT417197 MT417189 MT417210 R. bella SFC20120722-03 South Korea MT199642 MT196930 R. bella SFC20170819-05 South Korea MT199643 MT196931 MT199655 R. bella SFC20170819-10 South Korea MT199644 MT196932 MT199656 R. bella HCCN16818 South Korea KF361734 MT196933 - R. bella HCCN15410 South Korea MT196934 R. bella HCCN21655 South Korea KF361736 MT196935 MT199657 R. bella SFC20170731—02 South Korea MT199645 MT196936 MT199658 R. mariae 546/BB 07.038 USA KU237824 KU237384 KU237968 R. orientipurpurea sp. nov. HCCN19111 South Korea KF361712 MT196923 MT199648 R. orientipurpurea sp. nov. HCCN18725 South Korea KF361710 MT196924 MT199649 Russula subsection Amoeninae from South Korea 5 Taxon Herbarium no. Locality GenBank accession no. 9 of R. orientipurpurea sp. nov. HCCN21685 South Korea KF361714 MT196925 MT199650 R. orientipurpurea sp. nov. SFC20170819-08 South Korea MT199638 MT196926 MT199651 R. orientipurpurea sp. nov. SFC20170725-37 South Korea MT199639 MT196927 MT199652 iio arate MT199653 R. orientipurpurea sp. nov. SFC20170726—47 South Korea MT199641 MT196929 MT199654 R. pseudoamoenicolor India MT199646 MT196937 MT199659 R. violeipes 542/BB 07.273 Slovakia KU237820 KU237380 KU237964 Russula sp. SFC20160726-13 South Korea MT199647 MT196938 MT199660 Russula sp. SAV F-20134 USA, Florida - | = ss MP417205 Russula sp. SAV F-20117 USA, Florida MT417195 - MT417208 Russula sp. SAV F-4063 USA, Tennessee MT417203 MT417191 MT417212 Morphological study Macromorphological characters were described from fresh specimens using the ter- minology of Vellinga (1988). The color standard codes in Kornerup and Wanscher (1978) were followed for describing the colour of the basidiomes. All microscopic characters were measured from dried herbarium samples using an Eclipse 80i light microscope (Nikon, Japan) with immersion lenses at the magnification of 1000x and using the software NIS ELEMENT BR v3.2 (Nikon, Japan). The description tem- plates and terminology of Adam¢cik et al. (2019) were used for the observations of microscopic structures. The exception is that the sterile elements in hymenium have no distinct heteromorphous contents unlike hymenial cystidia of majority of Rus- sula members, i.e. gloeocystidia. Because it is not certain if they correspond to “true gloeocystidia”, we refer to them as hymenial cystidia when observed on lamellae sides and marginal cells in case of lamellar edges. Spore ornamentation was observed using a light microscope, and a scanning electron microscope (SEM, SUPRA 55VP, Carl Zeiss, Germany) at 5,000x and 10,000x magnification. For each collection, statistics of the measurements of microscopic characters were based on 20 measurements per character. Spore measurements excluded ornamentation. We followed the protocols of chemical tests for micro-morphological observation in Adam¢ik et al. (2019). Sta- tistics of microscopic characters are expressed as the mean + standard deviation with extreme values in parenthesis. The mean values are indicated by underline. When multiple samples were available, individual measurements of all microscopic charac- ters of a species were obtained from at least three samples and diagnostic characters of species were further used to compare with the remaining samples. Molecular studies DNA was extracted from fresh or dried basidiomes using a modified CTAB extraction method (Roger and Bendich 1994). Four molecular markers were used for species- level identification and to infer evolutionary relationships among species. The fol- lowing primer pairs were used in the amplifications: NSI1 and NLB4 for the ITS 6 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) region (Martin and Rygiewicz 2005), bRBP2-6F1 and RPB2-7R for the partial rpb2 locus (Matheny et al. 2007), MS1 and MS2 for part of the mtSSU region (White et al. 1990), and EF1-983F and EF1-2218R for the partial tefZa locus (Matheny et al. 2007). The PCR conditions were: initial denaturation of 5 min at 95 °C, 35 cycles that varied for each marker (60 s at 95 °C, 40 s at 50 °C, and 60 s at 72 °C for ITS; 40 s at 95 °C, 40 s at 58 °C, and 60 s at 72 °C for rpb2; 30 s at 94 °C, 30 s at 55 °C, and 60:s:at 72°C for mtSSU;.30 § at 94°C, 30's at. 56°C, and’ 60 $.at*72°C for tefla), and final incubation of 7 min at 72 °C. All PCR products were checked on 1% aga- rose gel stained with EcoDye DNA staining solution (SolGent Co., Daejeon, South Korea) and purified with the Expin PCR purification kit (GeneAll Biotechnology, Seoul, South Korea) following the manufacturer’s instructions. DNA sequencing was conducted using an ABI3730 automated DNA Sequencer by Macrogen (Seoul, South Korea). The obtained sequences were checked and manually edited using the soft- ware FINCHTYV v1.4 (Geospiza, Inc.), and then assembled manually using MEGA 7 (Kumar et al. 2016). Phylogenetic analysis For species delimitation of the Korean samples, ITS sequences of R. subsect. Amoeninae were downloaded from GenBank and aligned with the newly generated ITS sequences using MAFFT v7, with the E-INS-I strategy (Katoh and Standley 2013). Russula grisea and R. virescens were used as outgroup based on the results of previous studies (Buyck et al. 2008; Park et al. 2013). Maximum Likelihood (ML) analyses were conducted using RAxML 8.2.10 (Stamatakis 2014) and the GTR + G model with 1000 rapid bootstrap replicates. For rpb2, mtSSU, and tef/a regions, sequences of each locus were separately aligned and analyzed after introns were excluded. Seven partitions were as- signed; mtSSU, rpb2pos1, rpb2pos2, rpb2pos3, teflapos|, teflapos2, and teflapos3. Substitution models of all partitions were tested using Model'Test-NG (Darriba et al. 2020). The best substitution models for the different loci under BIC were GTR+1+G for mtSSU, K80+1I+G for rpb2 partitions, and TrN+I+G for tef/a partitions. Bayes- ian inference (BI) analysis was performed with MRBAYES v. 3.2.6 (Ronquist and Huelsenbeck 2003), with four independent runs of four chains each. The TrN substi- tution model for tefZa was replaced by the GIR model for this analysis. The analysis was run for 20 million generations, with sampling every 1,000" generation. At the end of the run, the average standard deviation of split frequency of runs was 0.001412. The convergence and burn-in values of runs were then checked in Tracer 1.6 (Rambaut et al. 2014). We considered clades with the bootstrap values and posterior probabili- ties exceeding 70% and 0.95 as well-supported. The ITS dataset and the combined dataset (7p62-mtSSU-tef1a) are available in TreeBase (http://treebase.org/treebaseweb/; submission ID 26896 and 22640, respectively). All phylogenetic analyses were ex- ecuted on the CIPRES Science Gateway (Miller et al. 2010). Three species of R. subg. Malodorae were chosen as outgroup. Russula subsection Amoeninae from South Korea 7 Results Phylogenetic analysis The ITS region was amplified and sequenced from 22 specimens for this study. A total of 152 ITS sequences belonging to Amoeninae were downloaded from Gen- Bank and used in the analysis (Suppl. material 2: Table $2). The phylogenetic analy- sis of the ITS sequences indicated the existence of more than 21 Russula species- level clades: eight Asian species (two names undetermined), five European (one undetermined), five North American (three undetermined), two Australian, and one Central American (undetermined) (Fig. 1). However, none of the African or Malagasy species were included in this analysis as no ITS sequences were available. The Korean samples represented three phylogenetic species, and they were grouped with Asian samples and clearly separated from specimens of Australia, Europe, and North America. A total of 72 ITS sequences were confirmed as R. bella: 5 from this study and 67 from GenBank. All of these sequences are from specimens in East Asia, i.e. from South Korea, China, and Japan. Of the ITS sequences in the R. bella clade, 31 were initially misidentified as the European R. violeipes, 35 ambiguously labelled as “Russula sp.”, and one labelled as Russula cf. violeipes. A total of 33 specimens for which ITS sequenc- es were newly generated or retrieved from GenBank belonged to a new species clade, R. orientipurpurea. The twenty nine ITS sequences from GenBank originated from South Korea, China, and Japan. Of these, 22 were mislabelled as the North American R. mariae and seven were labelled as “Russula sp.” (Suppl. material 1: Table $1). The R. orientipurpurea clade was closely related to R. mariae, but they were clearly separated (Figs 1, 2). One specimen (SFC20160726-13) formed a unique clade with three Chi- nese specimens and we define it as an undetermined species. Two Indian species were positioned as a sister clade of the Russula sp. clade. Russula amoena, R. amoenicolor, R. andaluciana, and R. violeipes were monophy- letic (Fig.1). The European Mediterranean samples have similar morphology with R. amoenicolor. However, phylogenetic analysis showed that they are likely not con- specific. Therefore, we named them as “R. cf. amoenicolor” in this study. The North American samples formed five clades. Of these, only two clades are labelled with spe- cies names (R. mariae and R. alachuana). The four Australian samples formed two well- supported clades that do not overlap with samples from other continents. Sequences of three loci (7p62, mtSSU, and ¢ef1a) were obtained for 28 samples (Table 1) and combined with 102 sequences of 34 samples obtained from GenBank. Two short introns were detected only in zef2a and they were excluded in the phyloge- netic analysis. The results of this multilocus phylogenetic analysis were similar to those of the ITS analysis. Subsection Amoeninae formed a well-supported monophyletic group (Fig. 2). Three species were found for South Korea, and the East Asian Russula species were clearly separated from European and North American species. @ Asia & Europe @ America @ sustralia ga Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) R. bella HCCN15410 KOR R. bella SFC20120722-03 KOR R. bella SFC20170819-05 KOR R. bella SFC20170731-02 KOR R. bella SFC20170819-10 KOR R. bella HCCN16818 KF361784 KOR R. bella HCCN21655 KF361786 KOR 96" R. violeipes KOR R. sp. roots Pyrola japonica JPN R. ve CHN R. ct. violeipes GU371290 CHN . Sp. roots Monotropa hypopithys JQ396501 CHN . Sp. roots Abies homolepis AB848571 JPN . Sp. roots Pinus amamiana LC364244 JPN . Sp. roots Pinus densiflora AB571503 KOR . Sp. roots Pinus thunbergii AB587767 KOR R. sp. roots Pinus thunbergii LC033912 JPN + 55 sequences (see supplementary table2) R. bella AwAAAA 94s" Macowanites sp. KY697596 AUS io 76/0.99 R. variispora EU019934 AUS | ®- variispora 81/0.99 *P )R. sp. AB854679 THA a R. cf. rosacea AB459514 THA R. intervenosa MK414579 IND ‘R. violeipes’ roots Pinus pinaster KY693648 ESP 91/-|— R. sp. forest soil FN610951 FRA R. sp. forest soil FN610993 FRA R. amoenicolor AY061655 os/* R. rostraticystidia EU019938 AUS R. sp. JF960823 AUS Tasmania R. sp. SAV F-4063 USA TN R. sp. roots Oreomunnea mexicana KM595029 PAN R. sp. KX441127 CHN R. orientipurpurea SFC20170821-22b KOR R. orientipurpurea SFC20170725-37 KOR R. orientipurpurea SFC20170819-08 KOR R. orientipurpurea SFC20170726-47 KOR R. orientipurpurea HCCN18725 KF361760 KOR 85/0.93 R. orientipurpurea HCCN21685 KF361764 KOR| - ‘R. mariae’ HCCN23016 KF361766 KOR ‘R. mariae’ KY681436 CHN R. sp. ECM JQ991809 CHN R. sp. KX441071 CHN R. sp. GQ359821 JPN R. sp. roots Pinus massoniana AB982038 CHN 7410.83 +21 sequences (see supplementary table2) R. aff. mariae SAV F-4484 USA NY R. aff. mariae SAV F-4493 USA NY R. aff. mariae SAV F-4564 USA NY R. mariae MF161263 USA MA 88/0.99 R forest soil FM999598 USA OH R. sp. FM999501 USA OH ‘R. nitida’ EU598164 USA TN R. mariae EU598199 USA TN R. aff. mariae Castanea dentata RT JX030253 USA NY R. sp. roots Quercus rubra GU907783 USA NJ + 6 sequences (see supplementary table2) R. amoenicolor R. rostraticystidia R. orientipurpurea R. mariae complex R. violeipes AY061726 R. sp. roots Fagus sylvatica KX886145 CHE R. violeipes JF908655 ITA 7710.94 R. amoena SAV F-3147 SVK 89/0.99 —— R. amoena SAV F-1352 SVK R. sp. ECM MF946183 USA FL 97/0.99 R. sp. roots Pinus taeda AJ633575 USA NC 80/0.98 R. sp. roots Pinus clausa KX899245 USA FL + 9 sequences (see supplementary table2) R. andaluciana MK105628 ESP j ogo lh. andalucana Miiosee7 Esp |. andaluciana Russula sp. SFC20160726-13 KOR 93/" 1R. sp. MK748180 CHN R. sp. MK748183 CHN 98/0.99 oe fas R. violeipes |R. amoena Russula sp. Ee R. sp. MK748181 CHN R. pseudoamoenicolor KX234819 IND 92/0.96 R_ pauriensis MF535185 IND 96/0.98] 74/-_)R. sp. SAV F-20117 USA FL 91/0.98 va 85/0.99 R. cf. amoenicolor SAV F-20302 GRC ay ( R. sp. SAV F-20108 USA FL R. sp. MH211804 USA FL 96/*' R. sp. SAV F-20134 USA FL R. cf. amoenicolor SAV F-20324 GRC R. sp. roots Pinus pinaster JQ975980 ESP |R. cf. amoenicolor R. sp. SAV F-20113 USA FL R. sp. MK532805 USA IN R. sp. MH211876 USA FL R. sp. roots Hexalectris brevicaulis HQ667813 MEX R. alachuana R. grisea KT934006 GER R. virescens DQ422014 BEL 0.03 Figure |. Maximum Likelihood (ML) tree based on internal transcribed spacer (ITS) sequences of most Russula subsect. Amoeninae and closely related species. Species in boldface are described in this study. ML bootstrap values >70 and Bayesian Inference posterior probability >0.90 are shown. Stars indicate clades with 100 ML bootstrap values and 1.0 Bayesian posterior probabilities. Russula subsection Amoeninae from South Korea x R. bella HCCN15410 KOR R. bella SFC20120722-03 KOR @ asia | ‘oor’ + R bella HCCN16818 KOR ! R. bella SFC20170819-05 KOR 4 e709 R. bella SFC20170819-10 KOR I R. bella HCCN21655 KOR I l l I l qs Europe &3 America R. bella SFC20170731-02 KOR +» - R. alachuana SAV F-20108 USA (FL) - R. alachuana SAV F-20113 USA (FL) Subsection Amoertinae 97/* Russula sp. SFC20160726-13 KOR fi R. pseudoamoenicolor AG-15-739 IND +» -R. cf. amoenicolor SAV F-20302 GRC 87/0.99 R. cf. amoenicolor SAV F-20324 GRC 88/* R. sp. SAV F-20134 USA (FL) *7| R. sp. SAV F-20117 USA (FL) R. sp. SAV F-4063 USA (TN) sg/- -R. orientipurpurea SFC20170726-47 KOR R. orientipurpurea HCCN18725 KOR aa ||| R. orientipurpurea HCCN21685 KOR R. orientipurpurea SFC20170819-08 KOR R. orientipurpurea SFC20170725-37 KOR R. orientipurpurea SFC20170821-22b KOR R. orientipurpurea HCCN19111 KOR 96/0.9| R. aff. mariae SAV F-4564 USA (NY) R. aff. mariae SAV F-4493 USA (NY) 3d0.93 | FR. aff. mariae SAV F-4484 USA (NY) R. mariae BB 07.038 USA R. violeipes BB 07.273 SVK R. amoena SAV F-3147 SVK Section Heterophyllae 98/* a=. = Se = ee ee eB eB eB eB eB BE BE BE EB BE EK SK = - ” o x s._ 92! R, amoena SAV F-1352 SVK 97/0.99 R. madagassensis BB 06.146 MDG +p R. aff. madagassensis BB 06.255 MDG R. cf. annulata BB 06.048 MDG 7 R. sp BB 08 061 MDG sf 10.99 R. cf. pseudocarmesina BB 06.030 MDG 70/0.93 R. cf. roseoaiba BB 06.105 MDG R. prolifica BB 06.161 MDG R. aff. crustosa BB 06.616 CAN fer ie ra R. mustelina SA 09.88 SVK ~* R. aff. virescens BB 09.021 NCL Subgenus - R. medullata BB 07 252 Heterophy. lidiae R. grisea BB 07.184 SVK a R. ornaticeps BB 06 530 CAN 100/* R. ionochiora BB 07 338 FRA R. vesca 06 525 MEX +p R. langei BB 07 792 FRA R. flavobrunnea var. violaceotincta BB 06 050 MDG R. oleifera BB 98 024 BDI 98/" o4y* R. pulverulenta BB 05 160 USA i R. amoenolens BB 08 675 ITA R. cf. illota BB 06 380 MEX “p R. maguanensis XHW 4765 CHN R. substriata XHW 4785 CHN oad R. aff. griseobrunnea BB 09 344 NCL R. compacta BB 06 295 USA 86/" il R. edulis BB 08 167 MDG gor R. cf. liberiensis BB 06.184 MDG oor R. sp. BB 08.178 V R. sp. BB 08.066 MDG _»—R. aff. delica BB 12.086 ITA n R. herrerae BB 06.532 MEX R. chloroides BB 07.209 SVK -— 0.02 meee ee ee ee ee Figure 2. Maximum Likelihood (ML) tree based on the second largest subunit of RNA polymerase II (7p62), mitochondrial small subunit ribosomal DNA region (mtSSU), and the translation elongation factor 1-alpha (tef1a) sequences of Russula subsect. Amoeninae species and representatives from closely related subgenera. Spe- cies in boldface are described in this study. ML bootstrap values >70 and Bayesian Inference posterior probabili- ty >0.90 are shown. Stars indicate clades with 100 ML bootstrap values and 1.0 Bayesian posterior probabilities. 10 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) Morphological analysis The three species found in South Korea have the typical morphological characters of subsection Amoeninae: pruinose dry pileus surface, mild taste, and stipe flushed with pink, red, or purple. In fact, they have almost completely white stipes, usually with only a faint pink color (Fig. 3). Microscopically, these species display moderately large spores with crested and subreticulate to reticulate ornamentation (Fig. 4), an absence of cystidia with well-defined contents reactant with sulfovanillin, and a pileipellis com- prising mainly attenuated terminal cells of hyphae usually subtended by one or two shorter ellipsoid or subglobose cells. The pileipellis of all three species have dimor- phous hyphal terminations, some with long subulate or lageniform terminal cells and others with shorter, cylindrical, or ellipsoid terminal cells (Figs 5—7). The three Korean species were easily distinguished in the field. The morphological features of the Korean R. bella are consistent with the original description of the species (Table 2). The other two Korean species, however, do not entirely agree with the description of any previ- ously described Russula species. Taxonomy Russula bella Hongo, Memoirs of Shiga University 18:50 (1968) Figs 3A, B, 4A, B, 5 Diagnosis. Pileus medium-sized, 20-50 mm diam., plano-convex to convex when young, applanate with depressed center to infundibuliform when mature, often lo- bate, margin with short striation, sometimes cracking in age; cuticle smooth, pruinose, viscid and shiny when wet, cuticle peeling approximately to half of the pileus radius or sometimes almost to the center, color variable, typically darker at the disk, greyish red (11D5), brownish violet (11D6) to bluish red (12A6-A8, 12B7), few with yel- lowish brown spots, margin pink (11A5) to greyish rose (11B5). Lamellae 2-3 mm deep, brittle, adnate, approximately 11-19 per cm near the pileus margin, moderately distant to crowded, yellowish white to pale cream; lamellulae absent; edge entire and concolorous. Stipe 17-34 x 5—9 mm, centrally attached or eccentric, cylindrical to slightly tapered towards the base, surface dry, longitudinally striate, whitish with a pinkish flush; solid when young, becoming hollow in age. Context 1-3 mm thick near the stipe, white, unchanging after cutting, turning greenish with FeSO » turn- ing quickly yellow with KOH, pale violet with PDAB; taste mild; odor slightly fruity. Spore print pale cream to white. Basidiospores (n = 60, 3, 3) (6—)6.5—7.1—7.7(—8.8) x (5—)5.3-5.7—6(—6.6) um, broadly ellipsoid, Q = (1.11-)1.17—1.25-1.33(-1.49), ornamentation thin, 0.4— 1 pm high ridges forming an incomplete reticulum (2—7 in a 3 wm circle) with some dispersed isolated warts (0O—2 warts in a 3 pm circle), suprahilar spot not amy- loid, smooth. Basidia (25.1—)30—35.3-40.5(-48) x (7—)8.5—-9.4-10.5(-11.5) um, Russula subsection Amoeninae from South Korea ee . | j ; ¥ A Aff j y / X ‘ A .: | . y | . ~~ ost ; Se Oe a | Pe. as J] ] \\ ‘ a sp. ~~ _ : : Le oS. 7? . 8 2 Re ta a y . ies eae oe : Tse : Pies ia po ’ A eg i ‘i y * Dae 2 2 Figure 3. Basidiomes of Korean Amoeninae from this study. A, B Russula bella (A-SFC20170802-03, B-SFC20170819-05) C=E R. orientipurpurea (SFC20170819-08) F, G Russula sp. (SFC20160726-13). Scale bars: 20 mm. 4-spored, narrowly clavate, with guttate or granular contents. Basidiola (27.3—)28.4— 32-35.5(—41.7) x (8.1-)8.7—-9.4-10.3(-10.6) um, narrowly clavate, with guttate or granular contents. Hymenial cystidia on lamellae sides inconspicuous, widely dis- persed, up to 100 per mm’, (37.5—)52—63.5—75(—90) x (5.5—)7.5—9-10.5(-11.5) um, clavate to subcylindrical, originating from subhymenium, apically obtuse, thick-walled with walls up to 0.8 um; contents optically empty, negative in sulfovanillin. Lamellar edge with dispersed basidia, true gloeocystidia (with differentiated contents) absent; marginal cells very abundant, resembling terminal cells in the pileus, typically narrowly lageniform or subulate (24—)38.5-50.8—63(—83) x (4—)5.5—6.8—7.5(—9.5) tum; shorter clavate to subcylindrical with obtuse apex present, (9—)12.5—15.7—18.5(—23.5) x (3.5— )4.5—-5.7—7(—7.5) um. Pileipellis orthochromatic in Cresyl blue, trichoderm, sharply V2 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) Table 2. Morphological characteristics of Asian R. subsect. Amoeninae species. c & ee re s Pileus size (mm) 50-100 | 25-60 60 Pileus colour bright red pink V grey V brown purple violet V green bright yellow cream or pale yellow Stipe colour almost white V V partly pink partly purple partly violet V Soi S77] 7 7, os 5.3-6.0 | 65-7 | 7.3-8.2 | 66.9 5-6 | 5.6-6.2 Tnean (eng wid] [71x57 [7567] [73x64] 69-63 [73-69 _[eax59 PQualue | ]1.17-1.33]1.07-1.19| 100-15 [1.09-1.19[1.00-1.17[1.03-1.33] —[}.10-1.19 we TT 4 ornamentaion | eiculne [| |_| ¥ |_|} 4 [eight | fom foeoa | os [asa | 2 | 2 | oma Cystidia on 44-55 | 52-75 | 29-34 | 80-110 [745-101] 55-135 | 90-117 | 45-60 | 66.5-91.5 lamellae sides 557 [2-22 | 10-21 | 9-12 [125-165 aia ee wa | = = a ee ole [subulare orlageniform| | | PP v Sm rm a a ON a Cg Gc SU | | ND gy SF | | | ee ae eS ee | ee Se aie es Cystidia on 38.5-63 | 32-39 | 70-100 | 48-88 42.5-56.5 mgliagledess 55-7 55-7 Dee eM OL Oe cn TC tog a0 oO sf 37 | a0 | o10 | oo | 45-6 Short cells number 2-4 1-2 subterminal width -12 -14 (um) TC centre width (um) 3-4 References: a-Hongo 1968, b-Crous et al. 2016, c-Das et al. 2005, d-Das et al. 2017, e-Hyde et al. 2016, Chiu 1945, Size unit of pileus is mm and the other microscopic characters are pm. Species in boldface are described in this study. delimited from the underlying context, 280-400 um thick, with a well-defined, gelati- nized, 150-200 um thick suprapellis of ascending or erect hyphal terminations form- ing a trichoderm, subpellis 130-200 um thick, dense, horizontally oriented, sldender and gelatinized hyphae; acid-resistant incrustrations absent. Hyphal terminations near the pileus margin unbranched, often slightly flexuous, either long and attenu- ated or subcylindrical and obtuse, the attenuated ones more abundant, with terminal cells (20.5—)47—61.4—-76(-85) x (3.5—)5—5.9-7(-8) pm, subulate or narrowly lageni- form, rarely fusiform, apically acute to subacute, thin-walled, usually followed by 1-2 shorter and often more inflated cells before the branching; subcylindrical ones shorter, with terminal cells (7.5—)33.5—51.2-69(—107.5) x (2—)3.5—4.7—6(—7) um, frequently Russula subsection Amoeninae from South Korea 13 Figure 4. Basidiospores with scanning electron microscopy images (A, C, E) and drawing images (B, D, F) A, B Russula bella (SFC20170819-05) C, D R. orientipurpurea; (SFC20170821-22b) E, F Russula sp. (SFC20160726-13). Scale bars: 2 um. originate from branched cells, sometimes with one shorter unbranched subterminal cell. Hyphal termination near the pileus center also dimorphous, the attenuated ones with terminal cells (10.5—)18.5—50.5—82.5(—104.5) x (3.5—-)4.5-6.0-7.5(-10) um, mainly subulate, occasionally narrowly fusiform, apically acute and sometimes acute- pointed, often with thickened walls (up to 0.8 um), shorter cylindrical hyphae with 14 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) Pe Figure 5. Microscopic features of Russula bella (SFC20170819-05) A basidia B basidiola C clavate mar- ginal cells D hymenial cystidia on lamellae sides E subulate marginal cells F hyphal termination at pileus margin G hyphal termination at pileus centre. Scale bars: 10 um. terminal cells (9—)12.5—18.6—24.5(—32) x (2.5—)3-4—5(—6) um; followed by 1-3 un- branched shorter cells, subterminal cells not usually not distinctly wider. Pileocystidia absent. Cystidioid or oleipherous hyphae in subpellis or context absent. Russula subsection Amoeninae from South Korea LS Ecology. gregarious to scattered on soil in mixed forest with Quercus aliena, Pinus densiflora, and Populus sp. Studied materials. SourH Korea. Jeollanam-do, Haenam-gun, Mt. Duryun, 614 m elev., 34°27'24"N, 126°37'07"E, Yang Seop Kim, 5 September 1985, HCCN1457A (HCCN!); Chungcheongbuk-do, Danyang-gun, Mt. Sobaek, 790 m elev., 36°57'29"N, 128°26'35"E, Soon Ja Seok, 13 July 2007, HCCN15410 (HCCN!); Gyeonggi-do, Suwon- si, Seonggyungwan University, 58 m elev., 37°17'42"N, 126°58'22"E, Soon Ja Seok, 1 Au- gust 2008, HCCN16735 (HCCN!); Gangwon-do, Wonju-si, 275 m elev., 37°19'59"N, 127°54'35"E, Soon Ja Seok, 6 August 2008, HCCN16818 (HCCN!); Gyeonggi-do, Suwon-si, Seonggyungwan University, 48 m elev., 37°17'28"N, 126°58'24"E, Hye Yeong Choi, 5 August 2011, HCCN21655 (HCCN!); Daejeon-si, Yuseong-gu, 105 m elev., 36°23'48"N, 127°20'13"E, Myung Soo Park, 31 July 2012, SFC20120731-02 (SFC!); Gyeongsangbuk-do, Ulleung-gun, Mt. Seonginbong, 420 m elev., 37°30'50"N, 130°52'10"E, Seung-Yoon Oh, Won Ju Kim, Young Woon Lim, 14 August 2012, SFC20120814-23 (SFC!); Chungcheongnam-do, Seosan-si, Yonghyeon Natural Recrea- tion Forest, 151 m elev., 36°45'53"N, 126°36'10"E, Young Ju Min, Won Ju Kim, Hyun Lee, 10 October 2012, SFC20121010-06 (SFC!); Seoul, Gwanak-gu, Seoul National University, 103 m elev., 37°27'26"N, 126°56'59"E, Komsit Wisitrassameewong, 31 July 2017, SFC20170731-02 (SFC); ibid., 19 August 2017, SFC20170819-05 (SFC)). Comments. Russula bella is morphological similar to Rk pseudoamoenicolor, R. punicea, and R. violeipes. Russula pseudoamoenicolor has a more vividly colored pileus and a larger basidiome and hymenial cystidia than those of R. bella (Hyde et al. 2016). Russula punicea differs from R. bella in the shape of hymenial cystidia; the former has acute cystidia, whereas the latter has obtuse cystidia (Chiu 1945; Hongo 1968). Russula violeipes differs from R. bella in the yellowish to greenish smeared violet color of the pileus (Kranzlin 2005). Moreover, R violeipes has larger basidia (45-65 x 11-14 um) and pleurocystidia (80-115 x 12-15 um) than those of R. bella (Kranzlin 2005). Russula orientipurpurea Wisittr., H. Lee & Y.W. Lim, sp. nov. MycoBank No: 835272 Figs 3C-E, 4C, D, 6 Material examined. Holotype. Soutu Korea. Jullanam-do, Yeosu-si, Dolsando is- lands, 202 m elev., 34°35'24"N, 127°47'57"E, Komsit Wisitrassameewong, Jae Young Park, 25 July 2017, SFC20170725-37 (Holotype, SFC!). Etymology. ‘orientipurpurea’ refers to the origin of the species, East Asia, and its typical purple color of pileus. Diagnosis. Pileus surface with pale cream with flushed pale purple to purple stains; spores with almost complete to complete reticulum; subfusiform to fusiform hymenial cystidia. Pileus medium-sized, 52-60 mm diam., plano-convex to applanate with the deep- ly depressed center, margin inconspicuously striate up to 2 mm, acute, even; surface smooth, pruinose, slightly waxy, matt, slightly viscid when wet, cuticle peeling 1/2 16 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) Ei F G Figure 6. Microscopic features of Russula orientipurpurea (SFC20170725-37) A basidia B basidiola C clavate marginal cells D hymenial cystidia on lamellae sides E subulate marginal cells F hyphal termi- nation at pileus margin G hyphal termination at pileus centre. Scale bars: 10 um. to 3/4 of the pileus radius, color pale cream to cream, with darker shade of cream towards the center, typically flushed with pale or darker purple stains, sometimes with radial stripes of greyish ruby (12E5). Lamellae 4-5 mm deep, adnate, moderately Russula subsection Amoeninae from South Korea ales distant, approximately 11-18 per cm near the pileus margin, white to pale yellow (3A3), furcations sometimes present near the stipe, lamellulae occasional, edge even. Stipe 40-50 x 11-13 mm, centrally attached, cylindrical, surface smooth, longitudi- nally striate, color white and sometimes with a greyish red (11D4-D5) flush; hollow. Context 2—3 mm thick at half of the pileus radius, white, rather firm but fragile in stipe when mature, turning slowly greenish with FeSO, and pale orange to orange white with KOH; taste mild; odor slightly fruity. Spore print cream white to white. Basidiospores (n = 80, 4, 4) (6.3-)6.9-7.3-7.8(-8.6) x (5.4-)6-6.4-6.9(— 7.6) ym, subglobose to broadly ellipsoid, Q = (1.06—)1.09-1.14—1.19(—1.28), orna- mentation of thin to moderately thick, 0.6—1.4 um high ridges forming an incomplete or complete reticulum (3-7 in a 3 pm circle), in a 3 um circle isolated warts rare (0-1 in a 3 um circle), suprahilar spot not amyloid, small, surrounded by fine and less promi- nent reticulation. Basidia (28—)32—36.2—40(-45) x (7.5-)9.5-10.9-12.5(-14.5) um, 4-spored, clavate. Basidiola (27.8—)33.4—37.7-42.1(—43.4) x (9-)9.7-10.5-11.4(— 12.2) um, narrowly clavate, with guttate or granular contents. Hymenial cystidia on lamellae sides widely dispersed to dispersed, 200-700 per mm’, (64—)74.5-87.9— 101(-131) x (8.5-)10.5-12.8-15(—18.5) um, mostly fusiform or narrowly fusiform, occasionally clavate, originating from subhymenium, emergent or not beyond basidia, apically constricted but obtuse, usually with thickened walls (up to 0.8 um), contents optically empty, negative in sulfovanillin. Lamellar edge with dispersed basidia, true gloeocystidia (with differentiated contents) absent; marginal cells very abundant, re- sembling terminal cells in the pileus, usually narrowly lagenifom or subfusiform, api- cally narrowed but obtuse (28.5—)48—67.8—88(—121) x (3—)5.5-8.2—11(—14.5) um; shorter narrowly clavate to clavate, (10.5—)13.5—18—22.5(—27.5) x (3.5-)5-6.5—-8(- 10.5) um. Pileipellis orthochromatic in Cresyl blue, sharply delimited from the un- derlying context, 170-240 um thick, with a well-defined, gelatinized, 30-60 um thick suprapellis of ascending or erect hyphal terminations forming a trichoderm, subpellis 180-250 ym thick, dense, horizontally oriented, gelatinized and branched cylindri- cal to inflated hyphae; acid-resistant incrustrations absent. Hyphal terminations near the pileus margin unbranched, apically often flexuous, either long and attenuated or subcylindrical, short and obtuse, the attenuated ones (39.0—)55.5—72.3-89.0(— 112.0) x (2.5—-)5.0-6.1—7.2(—8.5) pm, subulate or narrowly fusiform, sometimes slightly moniliform, apically subacute, thin-walled, subterminal cells shorter, cylin- drical ones with terminal cells (10—)16.5—25.3-34(-57) x (3-)4—5.2-6(-7.5) um, apically sometimes slightly constricted, apically obtuse; followed by 0-2 unbranched short, equally wide cells, sometimes originate from branched cells. Hyphal termination near the pileus center also dimorphous, the attenuated ones prevailing with terminal cells (10.5—)18.5-50.5—82.5(—104.5) x (3.5-)4.5-6.0-7.5(-10) um, subulate, thin- walled, apically subacute, cylindrical ones with terminal cells (13.5—)19-42.9-66.5(— 85.5) x (3.5—-)4.5—-5.6-6.5(—9.0) um. Pileocystidia absent. Cystidioid or oleipherous hyphae in subpellis or context absent. Ecology. solitary to scattered on soil in mixed forest with Quercus and Pinus trees. Studied materials. SourH Korea. Chungcheongnam-do, Gongju-si, Mt. Mu- seong, 341 m elev., 36°35'52"N, 127°01'59"E, Hyun Lee, Seung-Yoon Oh, 26 18 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) July 2012, SFC20120726-37 (Paratype SFC!); Incheon-si, Gangwha-gun, Mt. Goryeo, 228 m elev., 37°44'54"N, 126°26'01"E, Young Woon Lim, 4 August 2012, SFC20120804-09 (Paratype, SFC!); Seoul, Gwanak-gu, Mt. Gwanak, 154 m elev., 37°27'06"N, 126°56'34"E, Hyun Lee, Won Ju Kim, 25 August 2012, SFC20120825- 02 (Paratype SFC!); ibid, 202 m elev., 37°27'34'"N, 126°56'19"E, Hyun Lee, Myung Soo Park, 31 August 2012, SFC20120831-04 (Paratype SFC!); ibid, 238 m elev., 37°26'53"N, 126°54'11"E, Hyun Lee, Komsit Wisitrassameewong, 19 August 2017, SFC20170819-08 (Paratype SFC!); Gyeongsangnam-do, Hapcheon-gun, Mt. Gaya, 631 m elev., 35°47'59"N, 128°05'46"E, Jae Young Park, Komsit Wisitrassameewong, Ki Hyeong Park, 26 July 2017, SFC20170726-47 (Paratype SFC!); Gyeongsangbuk- do, Ulleung-gun, Nari basin, 395 m elev., 37°31'03"N, 130°52'11"E, Jae Young Park, Nam Kyu Kim, 21 August 2017, SFC20170821-22b (Paratype SFC’). Comments. Russula orientipurpurea is common in mixed forests in South Korea. This species was misidentified as the North American R. mariae (Park et al. 2013). The spores in R. orientipurpurea have more complete ridge connections (3-7 lines in the 3 wm circle) and a smaller number of warts (0-1 warts in the 3 um circle) than those in R. mariae (lines 1-4, warts 4-6). Russula orientipurpurea is morphologically similar to R. intervenosa. However, the dark red pileus centere, broader hymenial cystidia (up to 18 um), and the thicker pileipellis (250-600 pm) of R. intervenosa (Crous et al. 2016) distinguishes this species from R. orientipurpurea (see Table 2). Russula sp. Figs 3E, G, 4E, FE 7 Diagnosis. Pileus medium-sized, 60 mm diam., applanate with deeply depressed center, margin distinctly striate, crenulate, radially cracking; cuticle dry, viscid when moist, pruinose, peeling to 1/2 of the pileus radius, color greyish violet (18E5-E6), with dark violet (18F4-F6) patches, towards margin violet grey (18D2) to dull violet (18D3). Lamellae 3 mm deep, adnate, dense, pale cream; lamellulae rare, forked near the stipe; edge smooth and concolorous. Stipe 55 x 8-10 mm, centrally attached, tapering downwards base, surface dry, longitudinally-striated, white and flushed with purple. Context 2 mm thick at half of the pileus radius, white, unchanging; taste and odor not recorded. Spore print cream white. Basidiospores (n = 20, 1, 1) (6.4-)6.5-6.8-7(-7.2) x (5.5-)5.6-5.9-6.2(— 6.5) um, Q = (1.08—)1.1—1.14-1.19(-1.24), subglobose to broadly ellipsoid, orna- mentation of thin to moderately thick, 0.8—1.4 um high ridges forming an incomplete reticulum (2-6 in a 3 um circle), isolated warts rare (0-2 in a 3 um circle), suprahi- lar spot not amyloid, small. Basidia (32—)34—36.2—38.5(-40) x (9-)9.5—10.5-11(— 11.5) um, 4-spored, clavate. Basidiola (24—)25.8—31-35(-42.5) x (8.3-)8.9-9.9- 10.8(—11.4) um, narrowly clavate, with guttate or granular contents. Hymenial cys- tidia on lamellae sides widely dispersed to dispersed, 100-500 per mm’, (63)66.5— 79-91.5(—109) x (10.5—)12.5—14.5—16.5(—18) pm, narrowly fusiform or lageniform, originating from subhymenium and emergent beyond basidia, apically acute or obtuse Russula subsection Amoeninae from South Korea 19 Oia — Figure 7. Microscopic features of Russula sp. (SFC20160726-13) A basidia B basidiola C clavate mar- ginal cells D hymenial cystidia on lamellae sides E subulate marginal cells F hyphal termination at pileus margin G hyphal termination at pileus centre. Scale bars: 10 pm. 20 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) but narrowed, thick-walled (walls up to 0.9 um), contents optically empty, negative in sulfovanillin. Lamellar edge with dispersed basidia, true gloeocystidia (with differ- entiated contents) absent; marginal cells very abundant, resembling terminal cells in the pileus, narrowly lageniform or subulate (36.5—)42.5—49.6—-56.5(—70.1) x (5—)5.5— 6.3—7(—7.5) pm, narrowly lageniform or subulate, apically acute; narrowly clavate to clavate with optuse apex, (11.5—)14.5-17.8-21.2(—23.2) x (4—)5.2-6.2-7(-7.5) um. Pileipellis orthochromatic in Cresyl blue, sharply delimited from the underlying con- text, 250-400 um thick, with gelatinous matter, cystidoid hyphae prevailing, shorter cylindrical terminal hyphae present, parallel or repent scattered, pileocystidia absent, with a well-defined, gelatinized, 70-120 um thick suprapellis of ascending or erect hy- phal terminations forming a trichoderm, subpellis 230-350 um thick, dense, horizon- tally oriented, gelatinized and branched cylindrical hyphae; acid-resistant incrustrations absent. Hyphal terminations near the pileus margin unbranched, usually only slightly flexuous, either long and attenuated or subcylindrical, short and obtuse; the attenuated ones more frequent, with terminal cells (45.0—)60.0—-72.6-85.0(—96.5) x (3.5-)4.5— 5.1-6.0(—6.5) um, subulate, apically acute, thin-walled, shorter ones with terminal cells (18—)20.5—28.7—37.0(—48.0) x (3.0—)3.5—4.4—-5.0(—6.0) pm, cylindrical or sub- cylindrical, apically obtuse but often constricted, thin walled; followed by (0—)1—2(— 3) unbranched shorter and equally wide cells. Hyphal termination near the pileus center similar but shorter and narrower, attenuated longer ones with terminal cells (38.5—)43.5-53.4-63.0(—73.0) x (2.5—)3-3.5—4.0(—4.5) um; shorter cylindrical ones with terminal cells (11.5—)13.5-16.4-19.0(—22.0) x (3.0-)3.5—4.1-4.5(-5.5) um. Pileocystidia absent. Cystidioid or oleipherous hyphae in subpellis or context absent. Ecology. solitary on soil in deciduous forest, near Quercus mongolica. Material examined. SoutH Korea. Incheon-si, Ongjin-gun, Jangbongdo islands, 72 m elev., 37°31'55"N, 126°21'10"E, Jae Young Park, Nam Kyu Kim, Suldbold Jar- galmaa, 26 July 2016, SFC20160726-13 (Holotype, SFC). Comments. This species is morphologically similar to Rk. mukteshwarica, but phy- logenetically close to R. pseudoamoenicolor and R. pauriensis. Russula mukteshwarica and R. pauriensis differ from Russula sp. in the greenish yellow region at the pileus center, which is present in the first two species but absent in the latter (Das et al. 2005, 2017). Russula pseudoamoenicolor has a discolorous lamella edge (Hyde et al. 2016), whereas Russula sp. has a concolorous lamellae edge. Russula sp. has a purple pileus similar to that of R. violeipes; however, spore ornamentation height (up to 0.7 pm) and pleurocystidia size (80-115 x 12-15 um) distinguishes Russula sp. from R. violeipes (Kranzlin 2005). Key characters to Asian species in R. subsect. Amoeninae 1 Basidio meavith-piikishepteus 2. tess 9a Maecenas Sava ears eee en aatd 2 Basidiome with violet or purple pilems'..2 054 2.4 oct es ae sceeeeeeesetetere eee dee 4. 2. Obtuse hymenial cystidia, subreticulate spore ornamentation, in mixed Pinus Bi OH CTPUS TOTS SES 01st FOE case he Geka Ns ee a, oA Oates ee Ee R. bella = Acute hymenial cystidia, subreticulate to reticulate spore ornamentation....3 Russula subsection Amoeninae from South Korea 2A 3 Reticulate spore ornamentation, large hymenial cystidia (46-60 x 9-12 um), broad hyphal terminations (6-9 um wide) in the pileipellis ......... R. punicea — Subreticulate spore ornamentation, small hymenial cystidia (29-34 x 10-12.5 um), narrow hyphal termination (2.5-4.5 um wide) in the pileipe liste cacao Ratlam Ue rmatlat uth Malle iano Miavateaeaans R. intervenosa + Reticulate spore ornamentation, pileus with pale cream to yellowish grey COlOnAnapUTplish tin ees. Ames Lap tateenetseei nad uroune R. orientipurpurea e Subreticulate spore ornamentation, pileus with vivid color «0.0.0... eee 5 5 Pileus with violet/greenish/yellowish shades, stipe with purplish flush, large andibroadhymenialicystiditay ss. ts,.0asscecceiacecasshswceitgnes dotaveedstemsetbeaeseaiaee cass 6 — Pileuswathout-oreenish-or yellowish ‘shades... .5:ceutoedsbsdeteibeohuze descents 7. 6 Small spores with high ornamentation (up to 2 um), lamellae yellowish, Famiellhilaerabsetut:9ys-2. frie aetescecetinegeatteecnsieet eet mn terc ete ees R. pauriensis — Large spores with low ornamentation (up to 0.75 um), lamellae yellowish to erecinishs lamellae PLeSEMibe. fi. accycomo enya sSaneuesesoeneestaiees R. mukteswarica 2p Lamellar edge discolorous (pastel violet), spore ornamentation up to 2 um Ich teas RO pr Peery Pea Pe Pe oP ee Pea We R. pseudoamoenicolor i Lamellar edge concolorous, spore ornamentation up to 1.2 um high............ A eae A Map A da Re ye ha ee Russula sp. (SFC20160726-13) Discussion The phylogenetic analyses showed that subsection Amoeninae forms a well-supported monophyletic group. Moreover, the species of Asian, Australian, European, and North American origin form separate clades. Similar results have been reported for other spe- cies groups in the various ectomycorrhizal genera of Russulaceae, which are often en- demic to specific geographical regions or a single continent (Cho et al. 2018; De Crop et al. 2018; Wang et al. 2018; Lee et al. 2019; Looney et al 2020). Their host plants may have acted as bridges for species dispersal and diversification (Looney et al. 2018), and geographic distance and climate disjunctions may have caused species divergence (Cabon et al. 2019; Lee et al. 2019). The present study included recently obtained sequences of well-known species from different continents except Africa, which will provide useful information for understanding the diversity in this section. Four Amoeninae species had been previously reported from South Korea based on morphological characteristics: two European, one North American, and one Japanese species (Lee et al. 2015). Based on ITS, large ribosomal subunit (LSU), and rpb2 data, Park et al. (2013) found that the Korean Amoeninae species were R. mariae (North American) and R. violeipes (European). The differences between those results and the present study may be traced back to the limited amount of sequences available at the time. The dramatic increase in the number of sequences available in public databases and the data obtained in the present study allow us to conclude that the species previ- ously identified as R. mariae and R. violeipes in South Korea are in fact R. orientipurpu- rea and R. bella, respectively. We also showed that R. orientipurpurea forms a distinct 22 Komsit Wisitrassameewong et al. / MycoKeys 75: 1-29 (2020) clade that is quite distantly related to North American species, R. mariae. Moreover, the color of the pileus and geographical disjunction distinguishes R. orientipurpurea from R. mariae. Phylogenetic analysis of LSU sequence data showed a close relationship among R. bella, R. mariae, and R. violeipes (Shimono et al. 2004). Although there are no ITS sequences available of R. bella from Japan, one LSU sequence of R. bella was identical to that of Korean samples (Park et al. 2013). The European R. violeipes is clearly distin- guished from R. bella (Figs 1, 2). Therefore, considering the available morphological and molecular information, we conclude that all Korean samples previously designated as R. violeipes are in fact R. bella. Russula sp. (SFC20160726-13) was confirmed to be identical to the three uniden- tified Chinese samples. The Chinese samples are from Taishan of Shandong Province, which is geographically close to South Korea. They formed a distinct clade and might be a new species. However, there are limited specimens to describe it as a new species. It would be better to introduce this new species after observing more specimens. The occurrence of a previously reported species from South Korea (Lee et al. 2015), R. amoena, was not confirmed in this study. This species was originally described from Europe (Sarnari 1998). Two European specimens of R. amoena were included in our phylogenetic analyses, but none of the Korean samples match with these European col- lections. Thus, so far, R. amoena has not been confirmed in South Korea. Most Korean specimens of R. bella and R. orientipurpurea were collected from mixed forests with pine and oak trees, which are very common in South Korea. Previ- ous studies have reported that R. bella is commonly found as ectomycorrhizal root tips of the conifer species Abies homolepsis (Miyamoto et al. 2014), Pinus amamiana (Sugiy- ama et al. 2019), P densiflora (direct GenBank submission), P thenderbergii (Obase et al. 2011; Nakashima et al. 2016), and P yunnanensis (Xie et al. 2010). Some sequences of R. orientipurpurea were also obtained from the roots of other Pinus spp. (Wen et al. 2015). This indicates that these two species are associated with conifers, but symbiotic relationships with deciduous trees cannot be excluded as the information available in the literature is scarce. Russula sp. (SFC20160726-13) seems rare and was collected in a forest dominated by Quercus mongolica. Since we have limited sample and data, additional specimens are needed to complete a morphological and ecological charac- terization of the species. Ecological information can be useful for the identification of morphologically similar ectomycorrhizal fungi species (Nuytinck and Verbeken 2003; Lee et al. 2019). Further investigations focused on ecological information are necessary to obtain a better understanding of the three Korean Russula species. In conclusion, the East Asian Russula species in subsection Amoeninae are distinct from the European and North American species. Three species were identified from South Korea based on molecular and morphological data. However, the molecular data available in GenBank are still limited and comprise only some Russula species in sub- section Amoeninae. The amount of ITS data for this group has continuously increased, but protein-coding gene sequences are still insufficient. An overall increase in sequence information would allow for a better understanding of the phylogenetic relationships and global diversity of this group. Russula subsection Amoeninae from South Korea 23 Acknowledgements This study was supported by the National Institute of Biological Resources (NIBR 201801105), Korea National Arboretum (KNA1-1-25, 19-2), and the Korean govern- ment (NRF-2015M3A9B8029237). The research of Miroslav Cabon and Slavomir Adam¢ik was funded by Slovak Grant APVV 15-0210. References Adamc¢cik S, Jan¢ovicova BB (2018) The Russulas described by Charles Horton Peck. 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The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.75.53673.suppl1 Supplementary material 2 Table S2. Sequences used for the ITS analyses in this study Authors: Komsit Wisitrassameewong, Myung Soo Park, Hyun Lee, Aniket Ghosh, Kanad Das, Bart Buyck, Brian P. Looney, Miroslav Cabon, Slavomir Adamcik, Chang- mu Kim, Chang Sun Kim, Young Woon Lim Data type: ITS sequences Explanation note: Sequences used for the ITS analyses in this study. ITS sequences gener- ated in this study are presented in boldface. (T) indicates the type specimen. Species names in bracket are the original species epithet in GenBank or Park et al. (2013). Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.75.53673.suppl2