Taxonomic revision of Russula subsection Amoeninae from South Korea

Abstract Russula subsection Amoeninae is morphologically defined by a dry velvety pileus surface, a complete absence 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 molecular 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 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.


Introduction
Russula Pers. is the largest genus in the family Russulaceae, with at least 2,000 described species worldwide . Compared to most other genera of Basidiomycetes, Russula has complex morphological and chemical features . 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 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 arising from short unbranched cells (Buyck 1988(Buyck , 1994. Because of the complete absence 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 species. Three species have been reported from Europe: R. amoena Quél., R. amoenicolor Romagn., and R. violeipes Quél. (Sarnari 1998). Although no relevant molecular studies have been performed that include species from North America, previous studies using 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 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 Tonkin 2007, Hyde et al. 2016). Finally, six species native to Asia have been described, two from East Asia (Chiu 1945;Hongo 1968), and four from India (Das et al. 2005(Das et al. , 2017Crous et al. 2016;Hyde et al. 2016).
Historically, taxonomic studies of Russula in Asia have been influenced by European or North American literature. That is why Russula species from Asia were often assigned 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;Adamčík 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 materials 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 Russula sequence data, taxonomists can investigate 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 (rpb2), mitochondrial small subunit ribosomal DNA region (mtSSU), and the translation elongation factor 1-alpha (tef1α).

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 Agricultural 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).

Morphological study
Macromorphological characters were described from fresh specimens using the terminology 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 1000× and using the software NIS ELEMENT BR v3.2 (Nikon, Japan). The description templates and terminology of Adamčík 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 Russula 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 5,000× and 10,000× 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 Adamčik et al. (2019). Statistics 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 characters 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 specieslevel identification and to infer evolutionary relationships among species. The following primer pairs were used in the amplifications: NSI1 and NLB4 for the ITS 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 tef1α locus (Matheny et al. 2007 (Seoul, South Korea). The obtained sequences were checked and manually edited using the software FINCHTV 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 tef1α regions, sequences of each locus were separately aligned and analyzed after introns were excluded. Seven partitions were assigned; mtSSU, rpb2pos1, rpb2pos2, rpb2pos3, tef1αpos1, tef1αpos2, and tef1αpos3. Substitution models of all partitions were tested using ModelTest-NG (Darriba et al. 2020). The best substitution models for the different loci under BIC were GTR+I+G for mtSSU, K80+I+G for rpb2 partitions, and TrN+I+G for tef1α partitions. Bayesian 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 substitution model for tef1α was replaced by the GTR model for this analysis. The analysis was run for 20 million generations, with sampling every 1,000 th 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 probabilities exceeding 70% and 0.95 as well-supported. The ITS dataset and the combined dataset (rpb2-mtSSU-tef1α) are available in TreeBase (http://treebase.org/treebaseweb/; submission ID 26896 and 22640, respectively). All phylogenetic analyses were executed on the CIPRES Science Gateway (Miller et al. 2010). Three species of R. subg. Malodorae were chosen as outgroup.

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 S2). The phylogenetic analysis of the ITS sequences indicated the existence of more than 21 Russula specieslevel 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 sequences 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 S1). 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 Chinese 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 monophyletic (Fig.1). The European Mediterranean samples have similar morphology with R. amoenicolor. However, phylogenetic analysis showed that they are likely not conspecific. 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 species names (R. mariae and R. alachuana). The four Australian samples formed two wellsupported clades that do not overlap with samples from other continents.
Sequences of three loci (rpb2, mtSSU, and tef1α) 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 tef1α and they were excluded in the phylogenetic 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.

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 comprising mainly attenuated terminal cells of hyphae usually subtended by one or two shorter ellipsoid or subglobose cells. The pileipellis of all three species have dimorphous 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 previously described Russula species. Figs 3A, B, 4A, B, 5

Russula orientipurpurea
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 deeply 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 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 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 × 11-13 mm, centrally attached, cylindrical, surface smooth, longitudinally 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 4 and pale orange to orange white with KOH; taste mild; odor slightly fruity. Spore print cream white to white.

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 species groups in the various ectomycorrhizal genera of Russulaceae, which are often endemic 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 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 previously identified as R. mariae and R. violeipes in South Korea are in fact R. orientipurpurea and R. bella, respectively. We also showed that R. orientipurpurea forms a distinct 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 distinguished 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 unidentified 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 collections. 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. Previous 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 (Sugiyama 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 characterization 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 subsection 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.