Taxonomic Revision of the Genus Lactifluus (Russulales, Basidiomycota) of South Korea

Abstract Lactifluus (Pers.) Roussel is an ectomycorrhizal genus that was recently recognized to be distinct from the genus Lactarius. To date, 226 Lactifluus species have been reported worldwide. Misidentification of Lactifluus species is common because of intraspecific morphological variation, cryptic diversity, and the limited number of taxonomic keys available. Molecular data are indispensable for species delimitation; a multilocus phylogenetic analysis showed that most Asian Lactifluus species are not conspecific with morphologically similar species present on other continents. In particular, Korea has misused European and North American Lactifluus names. In this study, we evaluated the taxonomy of Lactifluus in Korea using both morphological and multilocus molecular (ITS, nrLSU, rpb1, and rpb2) data. We examined 199 Lactifluus specimens collected between 1980 and 2016, and a total of 24 species across the four Lactifluus subgenera were identified. All Korean species are distinct and clearly separated from European and North American species. Five taxa corresponded to previously described species from Asia and the remaining 19 taxa are confirmed as new species. Herein, we provide keys to the Korean Lactifluus species within their subgenera, molecular phylogenies, a summary of diversity, and detailed description of the new species.


Introduction
The ectomycorrhizal genus Lactifluus (Pers.) Roussel was recently recognized as a distinct genus of milkcaps [1][2][3]. Previously, Lactifluus was considered a part of Lactarius, likely because species in both genera exude latex when the fruiting body is bruised. Molecular data showed that the original genus Lactarus was not monophyletic and warranted the creation of the new milkcap genera Lactifluus and Multifurca (the latter somtimes without latex). Upon closer inspection, Lactifluus and Lactarius have some key morphological differences. Although no synapomorphic characteristic has been found to consistently distinguish both genera, the distinction can usually be made on a combination of characters and trends. First, Lactifluus species, relative to Lactarius, are more likely have dry pilei with a velvety to pruinose texture. Also, Lactifluus species never have zonate, hairy, viscid, or scrobiculate pilei. The genus Lactifluus contains many species with veiled velvety pilei, as well as all known pleurotoid milkcap species [1,4]. So far, no sequestrate species are known within the genus Lactifluus. Microscopically, species of Lactifluus are characterized by having spherical cells (sphaerocytes) in the hymenophoral trama and very often cells with thickened cell walls in the pileipellis structure and/or in the hymenium [5]. In many cases, a clear distinction between Lactifluus and Lactarius relies on genetic data.
Lactifluus species are an important resource for human use. Most Lactifluus species are edible and are important food sources in the Northern Hemisphere (e.g., Lf. piperatus and Lf. volemus). Some species are sold commercially in markets of Europe [27,28], Central & North America [29,30], and Asia [27,[31][32][33]. Also, some species, such as Lf. Vellereus, are the source of useful bioactive secondary metabolites, such as sesquiterpenes with lactarane or marasmane skeletons [34]. Lactifluus volemus (Fr.) Kuntze has a large amount of low-molecularweight natural rubber in its latex that was used for revealing the mechanism of natural rubber synthesis [35,36].
Inaccurate description and misidentification of Lactifluus species is common in the literature due to intraspecific morphological variation and the occurrence of cryptic diversity. Verbeken and Nuytinck [4] predict that cryptic species are more common in Lactifluus, a genus with a very large genetic diversity [20], than in Lactarius. Identification of species is complicated by the limited number of taxonomic keys available. To counter these problems, mycologists have utilized DNA sequence data for species identification and phylogenetic studies. The most commonly used genes for identification of milkcaps (genera Lactifluus and Lactarius in the family Rusulaceae) are the internal transcribed spacer (ITS), the large subunit 28S region (nrLSU), and the region between the conserved domain 6 and 7 of the second largest subunit of RNA polymerase II (rpb2) [1,37,38]. Several studies suggested incorporation of the region between domains A and C of the largest subunit of RNA polymerase II (rpb1) and rpb2 sequences for clearer understanding in infrageneric relationships [5,39,40]. However, DNA data have uncovered new problems-some Lactifluus species have been identified to be species complexes and remain difficult to identify [9,10,25,26,41,42].
As for many other groups of macrofungi, Lactifluus research has primarily been conducted in Europe and North America, where there is a long history of mycology. In the twentieth century, the taxonomy of Lactifluus was well studied under the name of Lactarius sensu lato in Europe and North America [43,44]. Subsequently, mycologists studying Asian fungal diversity used the scientific names originating from Europe and North America to refer to morphologically similar individuals (e.g., Pegler & Fiard [45], Singer et al. [46]). Recent molecular studies, however, showed that most Asian Lactifluus species are not conspecific with morphologically similar species present on other continents [8,10,11,25,42]. Recent work on Korean milkcaps revealed new species and raised doubt of previous identifications based on morphological characters [23,24,47]. Such findings warrant a comprehensive reevaluation of Korean Lactifluus. In this study, we examine all available Lactifluus specimens from Korean herbaria to delineate species using morphological and multilocus molecular (ITS, nrLSU, rpb1, and rpb2) data.

Sampling
A total of 199 Lactifluus specimens were obtained from four herbaria in South Korea: Kangwon National University (TPML), Korea National Arboretum (KA), National Institute of Agricultural Sciences (HCCN), and Seoul National University (SFC). These specimens were collected across South Korea from 1980 to 2016, with the initial identification based on morphological characters. When available, the collection information of each specimen was obtained from each herbarium.

Morphological approaches
Morphological characters were recorded from fresh materials, such as size and color of basidiomata, latex features, taste and smell. Specimen colors were recorded during daylight hours with color standard codes from Korneup and Wanscher [48]. For macromorphological characters, we follow the terminology in Verbeken and Walleyn [7]. Most collections were photographed in the field.
All microscopic characters were observed in dried specimens, using a Nikon Eclipse 80i microscope (Nikon, Japan), with the aid of the NIS element BR v3.2 (Nikon, Japan). Measurements and statistics follow Lee et al. [47], and were based on at least 20 spores and 10 hymenial structures (e.g., basidia, cystidia, marginal cells and pseudocystidia). Spore size and ornamentation were measured and observed in Melzer's reagent. The spores were measured in lateral view and the height of ornamentation was examined separately. The Q value represents the length/width ratio of the spore, referring to its shape. The spore ornamentation was photographed using a scanning electron microscope (SEM) at 5000Â and 10,000Â magnification (SUPRA 55VP, Carl Zeiss, Germany). Before SEM photography, dried pieces of lamellae were attached onto aluminum stubs, coated with platinum using a sputter coater (EM ACE200, Leica, Austria). Hymenial and pileal elements were mounted in 5% (w/v) KOH and observed in 5% (w/v) Congo red. The width and contents of pseudocystidia were recorded. For the features of hymenial and pileal cystidia, we recorded size, contents, abundance, and thickness of the cell wall. We documented the following pileipellis characters: overall structure, depth, delimitation, stratification, and features of dermatocystidia, if present.

Molecular approaches
We used a modified CTAB method [49] to extract genomic DNA from dried tissue. Four loci were amplified and sequenced: ITS, nrLSU, rpb1, and rpb2. Molecular approaches were performed in two steps. First, we attempted to amplify and sequence ITS for all samples. Next, we provisionally identified the species based on morphological characters and the ITS-based phylogeny. For each provisional species, one to five representative specimens were selected (more recently collected) for additional amplification and sequencing of nrLSU, rpb1, and rpb2.
PCR products were visualized on a 1% agarose gel stained with EcoDye (SolGent Co., Daejeon, Korea) and purified with the Expin PCR purification kit (GeneAll Biotechnology, Seoul, Korea), according to the manufacturer's instructions. All successfully amplified products were sequenced using the respective PCR primers on an ABI3730 automated DNA sequencer by Macrogen (Seoul, Korea). Obtained sequences were checked and manually edited using the software FinchTV v1.4 (Geospiza Inc.) and assembled in MEGA 6 [51]. All concensus sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic analyses
Four datasets (ITS, nrLSU, rpb1, and rpb2) were generated from the representative specimens of each species and used for phylogenetic analyses. Data for relevant species were downloaded from GenBank (shown in Figures 1-4), with the majority being from De Crop et al. [5]. Six Lactarius species were chosen as outgroups: L. fulginosus, L. hatsudake, L. miniatescens, L. olympianus, L. scrobiculatus, and L. tenellus [5]. All DNA datasets were aligned using the online version of MAFFT v7 [52], using the E-INS-I strategy, then trimmed or edited in MEGA 6. Species delimitation was first examined using single locus phylogenies. When significant conflict was not observed among the single locus phylogenies, we concatenated all single locus alignments using MEGA 6. A total of 52 concatenated sequences were generated in this study, representing species in six sections/four subgenera: Lf. subg. Gymnocarpi sect. Luteoli, Lf. subg. Lactariopsis sect. Albati, three sections belonging to Lf. subg. Lactifluus (sect. Gerardii, Lactifluus, and Piperati), and Lf. subg. Pseudogymnocarpi sect. Pseudogymnocarpi.
All phylogenetic analyses were performed in the CIPRES Science Gateway [53]. Maximum likelihood (ML) analyses were done using RAxML 8.2.10 [54]. jModeltest 2.1.7 [55] was used to find best substitution model for each gene in the concatenated dataset. Models found by jModeltest 2.1.7 under Bayesian information criterion (BIC) were: K80 þ I þ G (ITS and nrLSU), HKY þ I þ G (rpb1), and SYM þ I þ G (rpb2). Four parallel runs of Bayesian Inference (BI), each containing one cold and three heated chains, were carried out using MrBayes v.3.2.6 [56]. Each analysis was run for 20 million generations, sampling every 1000 th generation. The convergence and burn-in value were visualized in Tracer 1.6 [57].

Diversity of Lactifluus in South Korea and multigene analysis
Of the 199 Korean Lactifluus specimens, ITS sequences were successfully obtained from 143 specimens. The 56 specimens without sequence data were excluded from the study. A total of 24 putative taxa were identified based on morphological characters and the ITS-based phylogenetic tree. To confirm species identity and to infer the phylogenetic relationships of Korean Lactifluus, three additional loci (nrLSU, rpb1, and rpb2) were amplified and sequenced from the 52 representative specimens of the 24 taxa. The final phylogenetic analyses were performed with datasets of four loci from 134 Lactifluus samples and six Lactarius species as outgroups. The concatenated dataset contained 3,519 bp. The ML and BI analyses generated nearly identical tree topologies with minimal variation in statistical support values. A simplified tree detailing the relationships between the subgenera of Lactifluus is shown in Figure 5. In line with previous studies, the monophyly of the genus Lactifluus is well-supported (ML: 100, BI: 1.0), with four wellsupported subgenera: Lf. subg. Pseudogymnocarpi Table 1. GenBank accession numbers for DNA sequences of four loci, species names, voucher and potential host trees of studied specimens in this study.  Figure 5).

Classification and morphological characterization of Korean Lactifluus species
Latifluus subg. Gymnocarpi, originating from Africa and Central-South America, includes four well-supported sections and two unnamed clades ( Figure 3). The two Korean species found in this study (Lf. albidopectinatus sp. nov. and Lf. pallidotestaceus sp. nov.) belonging to Lf. sect. Luteoli. This section contains species from all continents except Antarctica and South America. Korean species under this section are characterized by capitate elements in the pileipellis and/or marginal cells, brownish discoloring white latex, and absence of true cystidia.
Lactifluus subg. Lactariopsis has four well supported sections, some unnamed clades, and several isolated species (Figure 2). The three Korean species (Lf. multiseparatus sp. nov., Lf. orientivellereus sp. nov., and Lf. pilosus) belong to Lf. sect. Albati. Species in this section are characterized by a whitish and velutinous pileus, a lamprotrichoderm as pileipellis structure, and the presence of pleuromacrocystidia.
Lactifluus subg. Lactifluus is the most diverse subgenus with four well-supported sections and two isolated clades in our analysis. This subgenus lacks African taxa. Three Korean species (Lf. betulicola sp. nov., Lf. luminosus sp. nov., and Lf. stellatus sp. nov.) belong to Lf. sect. Gerardii. These Korean species share characters like spore ornamentation forming a complete reticulum, a palisade as pileipellis structure, the absence of macrocystidia, distant lamellae, and unchanging white latex. Six Korean species (Lf. acicularis, Lf. koreanus sp. nov., Lf. longistipes sp. nov., Lf. orientivolemus sp. nov., Lf. porphyreus sp. nov., and Lf. rugiformis sp. nov.) are members of Lf. sect. Lactifluus. This section contains species occurring throughout Europe, North America, and Asia that were previously referred to as Lf. volemus. Korean species in this section have an orange to orange brown pileus, a lampropalisade as pileipellis structure, pleurolamprocystidia, and brownish discoloring latex. The remaining seven Korean species (Lf. curvativus sp. nov., Lf. quercicola sp. nov., Lf. subquercicola sp. nov., Lf. subviridilacteus sp. nov., Lf. undulatus sp. nov., Lf. viridilacteus sp. nov., and Lactifluus sp. 1) belong to Lf. sect. Piperati. All Korean species in this section are characterized by firm and whitish basidiocarps, thin-walled macrocystidia, a hyphoepithelium as pileipellis structure comprising dermatocystidia in the suprapellis, a low spore ornamentation forming an incomplete reticulum, and white latex.
Lactifluus subg. Pseudogymnocarpi contains four well supported sections and one unnamed clade ( Figure 1). The three Korean species (Lf. holophyllus, Lf. Luteolamellatus, and Lf. pseudohygrophoroides) are members of Lf. sect. Pseudogymnocarpi. This section contains African, European, North American, and Asian species. These Korean species all have a yellowish orange to orange colored pileus, a lampropalisade as pileipellis structure, and lack of true cystidia.    Pileus 25-60 mm diam., convex to depressed with decurved margin; margin even and entire when young, radially folded to pectinate in age; surface dry, slightly tomentose, white (1 A-) often tinged with faint ochraceous to light brown. Lamellae adnate, sometimes slightly decurrent, rather crowded, never forked, up to 3 mm broad, with 2-4 series of lamellulae between lamellae, whitish. Stipe 30-70 Â 10-20 mm, cylindrical, sometimes tapering downwards, slightly velvety; surface whitish to concolorous with pileus; base with white tomentosum. Latex abundant, white changing slowly to brownish.
Habitat: Scattered to gregarious on soil in mixed forests dominated by Quercus.
Comments: Lactifluus albidopectinatus has a whitish colored pileus, which is a rare character of species in sect. Luteoli. This species is phylogenetically closely related to Lf. brunneoviolascens (Figure 3), a species found in Europe, but differs in the size and shape of basidiospores. The ellipsoid basidiospores of Lf. brunneoviolascens are smaller (8-9 Â 5.5-6.5 mm) than the subglobose spores of Lf. albidopectinatus [58] and the terminal elements of the pileipellis are more distinctly capitate.  Pileus 30-50 mm diam., convex to depressed with decurved margin; margin even and entire; surface dry, velutinous, grayish red (7B3) to reddish brown (8D6) with whitish to faint ochraceous tinges. Lamellae adnate to broadly adnate, rather crowded, up to 5 mm broad, with 2-4 series of lamellulae between lamellae, whitish to pale cream. Stipe 45-80 Â 8-15 mm, cylindrical, sometimes tapering upwards, sometimes irregularly compressed; surface slightly velvety, concolorous with pileus; base with dense white tomentosum. Latex abundant, white changing brownish slowly, staining lamellae brownish.
Habitat: Scattered to gregarious on soil in mixed forests dominated by Quercus. Comments: In the field, Lf. pallidotestaceus is easily recognized by its medium-sized, apricot colored velutinose pileus, brown staining latex, and warty basidiospores. This species differs from the only other Korean species in the same section (Lf. albidopectinatus) by the apricot colored pileus and the distinctly capitate marginal cells. This species is phylogenetically closely related to Lf. luteolus from North America (Figure 3). Lactifluus pallidotestaceus has subglobose to broadly ellipsoid basidiospores and lacks cheilocystidia, while Lf. luteolus has ellipsoid basidiospores and cheilocystidia in the lamellar edge [43]. Lactifluus luteolus also lacks the reddish colors in the pileus, which is off-white to buff.    slightly tapering downwards, often irregularly compressed, central, sometimes eccentric; surface dry, velvety, and concolorous with pileus. Latex transparent changing to white in a few second, changing pale yellow slowly, staining lamellae brownish.
Habitat: Scattered to gregarious on soil in mixed forests dominated by Betula.
Additional studied material: SOUTH KOREA: Gangwon-do, Inje-gun, Wondae-ri birch forest, alt. Comments: Lactifluus multiseparatus is characterized by whitish basidiomata with transparent latex that changes yellowish when isolated and has brownish discoloration on lamellae. Another white Korean species (Lf. orientivellereus) is phylogenetically closely related to Lf. multiseparatus (Figure 2). However, the former species is distinguished from the latter species by unchanging white latex and less interconnection of spore ornamentation. Pileus 80-170 mm, convex when young, later infundibuliform and deeply depressed in the center; margin inrolled when young, expanding in age, sometimes wavy; surface dry, velvety, white (A1-) tinged with ocherous to yellowish brown. Lamellae slightly decurrent, very crowded, up to 3 mm broad, sometimes forked, with abundant lamellulae of different lengths, white turning grayish yellow when bruised. Stipe 10-45 Â 10-25 mm, cylindrical to slightly tapering downwards, central, sometimes eccentric; surface dry, velvety, and concolorous with pileus. Latex white, unchanging.
Habitat: Solitary to gregarious on soil in deciduous forests dominated by Betula.
Comments: Lactifluus luminosus can be easily recognized by its strikingly bright yellow pileus. This species has the lightest basidiomata among the three Korean species (Lf. luminosus, Lf. betulicola, and Lf. stellatus) in the Lf. sect. Gerardii. In addition, the   Habitat: Solitary to gregarious on soil in mixed forests dominated by Quercus and Pinus.
Comments: Lactifluus stellatus is a unique species in the sect. Gerardii, and this species can be characterized by the uneven dark brown color of pileus with striking whitish spots, the strong rugulose surface, and the low ornamentation of reticulate basidiospores. This species is phylogenetically closely related to Lf. gerardii, Lf. leae, and Lf. betulicola, but can be distinguished from the aforementioned three species by the height of spore ornamentation. The basidiospore ornamentation Lf. gerardii (0.5-0.8 mm), Lf. leae (0.5-1.0 mm) and Lf. betulicola (up to 0.7 mm) is distinctly higher than that of Lf. betulicola (maximum 0.3 mm) [41].

Key to the species of Lactifluus subgenus
Lactifluus section Lactifluus -Grayish-yellow, orange to orange-brown or reddish-brown pileus; white latex discoloring brownish on lamellae; reticulate spore ornamentation; lampropalisade type pileipellis; presence of lamprocystidia  Pileus 35-85 mm diam., convex when young, becoming applanate with slightly depressed center to infundibuliform when mature, rugulose when young; margin entire, sometimes wavy; surface dry, slightly velutinous, pruinose when young, light orange (6A4) to grayish red (7B6), getting paler toward margin, sometimes with orange brown tinges. Lamellae subdecurrent to decurrent, crowded, up to 6 mm broad, sometimes forked near the stipe, with numerous lamellulae of different length, cream colored, discoloring grayish brown to dark brown when bruised. Stipe 45-75 Â 5-15 mm, cylindrical to slightly tapering upwards, centrally attached, surface dry, sometimes velutinous, pruinose when young, concolorous to pileus or darker tinged often with brownish. Latex abundant, sticky, white, slowly changing to brown.
Habitat: Solitary or scattered on soil in Quercus forests.   Comments: Lactifluus longistipes can be easily distinguished from other species in sect. Lactifluus by its long stipe relative to the pileus size. The ratio of stipe length/pileus diameter is between 1.5 and 2. This species also has the lowest spore ornamentation among the Korean species of Lf. sect. Lactifluus. This species is phylogenetically closely related to Lf. volemus and Lf. subvolemus from Europe ( Figure 4). However, these two European species have larger basidiospores and higher spore ornamentation (up to about 1.5 mm in Lf. volemus and up to about 1.6 mm in Lf. subvolemus) than those of Lf. longistipes (up to 0.6 mm) [26].
Habitat: Scattered on soil in the mixed forests dominated by Quercus and Pinus.
Habitat: Scattered on soil in Pinus forest or mixed forests dominated by Quercus and Pinus.
Habitat: Scattered on soil in mixed forests dominated by Quercus.
Comments: Lactifluus rugiformis can be recognized by the overall reddish-brown colors, the strongly rugulose pileus margin, the cream to pale orange lamellae, and the short stipe relative to the pileus size. The ratio of stipe length/pileus diameter is between 0.4 and 0.7. This species is phylogenetically closely reated to Lf. longipilus from Thailand ( Figure 4). However, the terminal elements in the pileipellis (10-130 mm) of Lf. longipilus are longer than Lf. rugiformis [10].  Etymology: "curvativus" means slightly curved margin. This name refers to the in-rolled to decurved margin of this species.
Habitat: Solitary or gregarious on soil in Quercus forests or mixed forests dominated by Quercus.

Discussion
Through a comprehensive morphological and multilocus phylogenetic analysis of 199 Lactifluus specimens collected over 37 years, a total of 24 Lactifluus species were identified. Our study shows that Lactifluus is not only well distributed in tropical ecosystems and the Southern Hemisphere, but can also be diverse in temperate forest in the Northern Hemisphere. Surprisingly, only five species were previously described species, while at least 18 others were confirmed as new species (one putatively new species requires additional data). The five previously recognized species were all described from Asia: two species (Lf. acicularis and Lf. pilosus) were originally reported from Thailand [8,10] and three (Lf. holophyllus, Lf. luteolamellatus and Lf. pseudohygrophoroides) were described from South Korea [23]. As expected, many specimens were previously misidentified, being assigned to morphologically similar species from Europe and North America. Our results strongly support that most Asian Lactifluus species are not conspecific with morphologically similar taxa from other continents [5,8]. Such was also the case for Lactarius species in Korea [47]. Therefore, European and North American species are not likely to be distributed in Asia, and most of the recorded species based on morphological surveys in Korea (and other parts of Asia) are likely incorrect. For example, Lf. piperatus, a European species was reported in Korea without detailed morphological descriptions [61], and in our study, sequence data of a European specimen (voucher 2001 08 19 68, France) did not match data from any Korean specimens ( Figure 4). Specimens labeled as Lf. piperatus in Korea have been identified as one of three new species: Lf. quercicola, Lf. subquercicola, and Lf. undulatus.
Previously, 21 Lactifluus species were reported in Korea [22][23][24]62], yet only five were found in this study. Of the 16 remaining recorded species, only three of them were originally reported from Asia, while seven were described from Europe, and six from North America. The three Asian species recorded in Korea are Lf. ochrogalactus (Hashiya) X.H. Wang, Lf. pinguis (Van de Putte & Verbeken) Van de Putte, and Lf. subpiperatus Hongo. Lactifluus ochrogalactus and Lf. pinguis were recently reported in Korea [24,62], while the specimens labeled as Lf. subpiperatus turn out to be a new species in this study, Lf. subviridilacteus. Although we did not identify Lf. subpiperatus in our study, the existence of this species in Korea cannot be ruled out as it is present in Japan.
For infrageneric classification of Korean Lactifluus, color and color change of latex, pileipellis type, and cystidium type are useful characters. Lactifluus species show various latex discoloration patterns when exposed to air, and this informative to distinguish some groups. The morphological characters of the new species discovered in Korea correspond well with the species in the known clades of Lactifluus [5]. Korean species belonging to Lf. sect. Lactifluus and Lf. sect. Gymnocarpi have brownish discoloration of latex without exception. On the other hand, some groups have diverse patterns of latex discoloration. Most species in the Lf. sect. Piperati have greenish discoloration of latex, which is true for two Korean species, but the other four show no discoloration. Korean species of Lf. sect. Pseudogymnocarpi exhibit mixed patterns-two species with brownish discoloration of white latex and one species with unchanging white latex.
In previous studies, the type of pileipellis was noted as an important character to delimit sections or subgenera within Lactifluus [7,44]. Lactifluus species in sect. Lactifluus and sect. Pseudogymnocarpi share the same type of pileipellis. However, various pileipellis types are observed in species of sect. Albati and sect. Gerardii. For species native to Korea, we found only one type of pileipellis for each section; lamprotrichoderm type of sect. Albati and palisade type of sect. Gerardii. The type of cystidia and presence/absence of cystidia is also useful to delimit some sections such as sect. Lactifluus (pleurolamprocyatidia) and sect. Luteoli (no true cystidia). The presence of only one type of cystidia for each section of Korean species highlights its informativeness for identification. Further work is needed to determine if cystidia characters are useful for species identification in other parts of the world. The characters of the context (color, dicolouration, smell, and taste) are largely unknown in the Korean species, although in some groups they might reveal additional relevant differences. They certainly need more attention in future.
In addition to the three characters mentioned above (latex color and color change, pileipellis type, cystidia type), the shape of basidiomata also is an important character of Lactifluus species. All species of Lactifluus investigated in this study are agaricoid. So far, pleurotoid Lactifluus species are known in three Lactifluus subgenera: Lactariopsis, Gymnocarpi, and Lactifluus. While pleurotoid species are reported from all continents except Europe [11,41,[63][64][65][66][67][68], they have thus far not been observed in Korea. Previous studies show that the pleurotoid basidiomata has multiple evolutionary origins [65,66]. As pluerotoid species are reported from Japan and China [11,69], whether they exist in Korea requires further investigation.
Ecological information of ecotomycorrhizal fungi can be useful for their identification and forest management. Sequence data in GenBank may include information on species distribution and host. From the same sequence data with the new species found in this study, we could confirm that many new species are also distributed in other countries of Asia: Lf. orientivellereus from Japan [70], Lf. betulicola from Japan [9], Lf. longistipes from China and Far East Russia (GenBank), Lf. quercicola from China (GenBank) and Japan [71], Lf. subquercicola from China and Japan (GenBank), and Lf. undulata from Thailand and Vietnam [42]. Hosts that interacted with these species were similar to those studied in Korea (Table 1). An ITS sequence (AB568438) consistent with Lf. quercicola was obtained from the root of orchid Cephalanthera longibracteata, Japan [72]. Thus, Lf. quercicola may be one of the mixotrophic hosts of Cephalanthera longibracteata.
In conclusion, we confirm the presence of 24 Lactifluus species in Korea, including at least 18 species new to science, based on morphological and multilocus phylogenetic approaches. Our results show that identification of Lactifluus species using morphological data alone can be unreliable due to considerable overlap of characters among species. In addition to morphological observation of defining characters, a molecular approach is necessary for infrageneric classification and species identification in Korean Lactifluus. Since many specimens used in this study were missing information regarding host plants, the ecological relevance of all Korean Lactifluus species could not be examined. Further investigation is necessary to better understand the general ecology of Lactifluus species.