Taxonomic revision of the genus Lactarius (Russulales, Basidiomycota) in Korea

The genus Lactarius Pers. (Russulales) is a cosmopolitan group of Basidiomycota that forms ectomycorrhizal relationships primarily with both deciduous and coniferous trees. Although the genus has been well-studied in Europe and North America, only fragmentary researches have been carried out on Asian species. In particular, the distribution of Lactarius species in South Korea is poorly understood due to insufficient morphological descriptions and a lack of DNA sequence data. In addition, the misuse of European and North American names has added to confusion regarding the taxonomy of Asian Lactarius species. In this study, the diversity of Lactarius in South Korea was evaluated by employing both morphological and phylogenetic approaches. A multi-locus phylogenetic analysis of 729 Lactarius specimens collected between 1960 and 2017 was performed using the internal transcribed spacer (ITS) region, partial nuclear ribosomal large subunit (nrLSU), partial second largest subunit of RNA polymerase II (rpb2), and minichromosome maintenance complex component 7 (mcm7). 49 Lactarius species were identified in three Lactarius subgenera: L. subg. Russularia (17 spp.), L. subg. Lactarius (22 spp.), and L. subg. Plinthogalus (10 spp.). Among them, 28 Lactarius species were identified as new to science, while just 17 were previously described Lactarius species. Four of the taxa remain un-named due to paucity of materials. A key to Korean Lactarius species, molecular phylogenies, a summary of diversity, and detailed description are provided.


Introduction
Lactarius Pers. is a well known ectomycorrhizal genus that is characterized by the presence of latex (Persoon 1797), hence the common name milkcaps. The genus has long been recognized as one of two major groups (together with the larger genus Russula Pers.) in the family Russulaceae. A recent multi-locus study revealed that species identified as Lactarius spp. actually represent three genera: Multifurca Buyck & V. Hofst (Buyck et al. 2008), Lactarius, and Lactifluus (Pers.) Roussel (Buyck et al. 2010;Verbeken et al. 2011Verbeken et al. , 2012Stubbe et al. 2012).
Until recently, species identification of Lactarius was based on morphological characters such as latex colour and colour change, pileus features, spore ornamentation, the presence/absence of cystidia and pileipellis structures (Hesler and Smith 1979;Heilmann-Clausen et al. 1998). Classification of Lactarius species has historically been problematic, as mycologists often use different morphological characters for infrageneric classification. Pileipellis structure has been the most frequently used character for infrageneric classification in Lactarius. However, Neuhoff (1956) divided the milkcaps into two subgenera based on pileipellis structure, while Hesler and Smith (1979) classified North American species of Lactarius s.l. into six subgenera using pileus and latex features. For European Lactarius, Heilmann-Clausen et al. (1998) described six subgenera using pileipellis structure as well as features of cystidia, while Basso (1999) proposed six subgenera based on the pileipellis structure and ecological complements.
While some Lactarius species are easily identified to the species level using only morphological characters, most Lactarius species are difficult to distinguish due to subtle morphological differences between species (Heilmann-Clausen et al. 1998;Stubbe et al. 2010;Nuytinck and Ammirati 2014). As the sequence data available in public nucleotide databases such as GenBank and UNITE have increased in recent years, the combination of morphological, molecular, and ecological data has greatly improved the accuracy of species identification in the Lactarius (Nuytinck and Verbeken 2003;Lee et al. 2015a). The internal transcribed spacer (ITS) region is commonly used as a fungal barcode (Schoch et al. 2012) and has been instrumental in identifying new Lactarius species Das et al. 2015). The accuracy of phylogenies has been further enhanced through the use of a multi-locus approach, for example the partial nuclear ribosomal large subunit 28S (nrLSU) and the conserved region between domain 6 and 7 of the second largest subunit of RNA polymerase II (rpb2) (Stubbe 2012;Verbeken et al. 2014;Wisitrassameewong et al. 2016).
Based on recent molecular studies, three Lactarius subgenera are currently recognized: Lactarius subgenus Lactarius, L. subg. Russularia (Fr. ex Burl.) Kauffman, and L. subg. Plinthogalus (Burl.) Hesler & A.H. Sm. (Verbeken and Nuytinck 2013;Wisitrassameewong et al. 2014a). Only L. subg. Plinthogalus is well-supported by molecular data in the most recent global phylogeny (Eberhardt and Verbeken 2004). Before the twenty-first century, the taxonomy of Lactarius was well-studied in Europe and North America (Hesler and Smith 1979;Heilmann-Clausen et al. 1998). As a result, European and North American nomenclature was applied to Lactarius species found in other continents or relatively distant geographical regions (e.g. Central and South America) (Pegler and Fiard 1979;Singer et al. 1983) and East Asia (Tanaka 1890;Imai 1935;Hongo 1957aHongo , b, 1971Hongo , 1979. Recent studies based on molecular analysis demonstrated that most Lactarius species are not cosmopolitan and certain species are distributed to specific geographical regions (Le et al. 2007a;Wisitrassameewong et al. 2016). Consequently, many Asian Lactarius species have been misidentified, often using European and American species names. Similar situations have been reported in South Korea (Lee et al. 2014(Lee et al. , 2015aKim et al. 2015). Thus a reevaluation of Korean Lactarius species is warranted. Uyeki (1936) identified the first three Lactarius species in Korea: L. akahatsu Nobuj. Tanaka, L. hatsudake Nobuj. Tanaka and L. piperatus (L.) Pers. (now Lactifluus piperatus (L.) Roussel). Since then 64 additional Lactarius species have been recorded in South Korea, with 49 of these belonging to the Lactarius s. str. (Bok and Shin 1985;Lee et al. 2015bLee et al. , 2018. These species were identified based on phenotypic similarities to European and North American Lactarius and lacked detailed descriptions and molecular data. Recently, several studies found that Asian members of many fungal genera, e.g., Amanita Pers., Laccaria Berk. & Broome, Russula, and Sparassis Fr. as well as Lactarius, are divergent from their European and North American counterparts, and many specimens were reported as new species (Zhao et al. 2013;Lee et al. 2017;Vincenot et al. 2017;Cho et al. 2018;Cui et al. 2018). Thus, it is likely that the diversity of Korean Lactarius taxa is different from what is currently described. Here, we investigated 729 Korean Lactarius specimens collected from 1960 to 2017 using morphological data and phylogenetic analyses of sequences from four markers: the ITS, nrLSU, rpb2 and minichromosome maintenance complex component 7 (mcm7).

Materials and methods
Sampling A total of 729 Lactarius specimens were obtained from five herbaria in South Korea: Seoul National University (SFC), Kangwon National University (TPML), National Institute of Agricultural Sciences (HCCN), National Institute of Biological Resources (NIBR), and Korea National Arboretum (KA). Samples were collected in South Korea from 1960 to 2017. All specimens were initially identified using morphological characters and collection information was obtained from each herbarium where available.

Morphology
Newly collected specimens were preliminary identified using field guides (Romagnesi 1985;Heilmann-Clausen et al. 1998;Kränzlin 2005;Park and Lee 2011) and the Russulales news website (http://www.mtsn.tn.it/russulalesnews/) in order to have a provisional working name. The terminology of macro-morphology is in accordance with Verbeken and Walleyn (2010). We observed latex colour when it was exuded from basidiocarps and colour changes when exposed to the air. The Methuen Handbook of Colour (Kornerup and Wanscher 1978) was used for colour indications.
Microscopic characters were studied and illustrated from dried specimens using an Eclipse 80i light microscope (Nikon, Tokyo, Japan). Specimens were mounted in 3-5% (w/v) KOH and 1% (w/v) phloxine (Largent et al. 1977) to observe hyphae contents. To compare with other published data, we measured at least 20 basidia and cystidia per specimen. All elements on the hymenium and pileal structure were observed using 5% (w/v) Congo red solution (Clémençon 1973) to observe cell walls and to measure the size of elements. The amyloidity of the plage on basidiospores was observed using Melzer's reagent (Largent et al. 1977). The length and width of basidiospores were measured in lateral size using scanning electron microscope (SEM). Pieces of dried lamellae were attached to aluminum stubs using double sided adhesive tape, coated with platinum in a sputter coater (EM ACE200, Leica, Austria), and then examined with a SEM (SUPRA 55VP, Carl Zeiss, Germany). Pileipellis was observed from the middle part of pileus. Twenty basidiospores were measured per collection. Basidiospore size and shape are calculated as (MIN -)[AVa -2 9 SD] -AVa -AVb -[AVb ? 2 9 SD](-MAX), in which MIN = the minimum value, MAX = the maximum value, AVa = lowest mean value for the measured collection, AVb = highest mean value for the measured collection and SD = standard deviation. Q value represents basidiospore ''length/width ratio which corresponds to basidiospore shape and is reported as MIN -QAVa -QAVb -MAX, where QAVa and QAVb are the lowest and the highest mean ratio for a measured specimen, respectively. For species with only one sample, the size and shape are calculated as (MIN -)[AV -2 9 SD] -AV -[AV ? 2 9 SD](-MAX) and MIN -QAV -MAX, in which AV = mean value for the measured collection and QAV = mean ratio for a measured specimen. All average values are in italics. The size of hymenial elements and pileal structure were presented as MIN-MAX and rounded up up to 0.5 lm.

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from fresh or dried material using a modified CTAB extraction protocol of Rogers and Bendich (1994) (Park et al. 2013). Four loci were amplified and sequenced: the ITS, nrLSU, mcm7, and rpb2. PCR amplification and sequencing were conducted in two steps. First, we amplified and sequenced the ITS region from all specimens. Second, we sequenced the nrLSU, mcm7, and rpb2 loci from the representative specimens of each putative species, which was delimitated by the ITS phylogeny and morphological features.
PCR amplifications were conducted using AccuPower Ò PCR premix (Bioneer Co., Daejeon, Korea) in a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). The ITS region was amplified using two primer sets: forward primers ITS1F or ITS5 (White et al. 1990;Gardes and Bruns 1993) and reverse primers ITS4B or Russ3R (Gardes and Bruns 1993;Park et al. 2013). PCR conditions for the ITS are described in Park et al. (2013). The nrLSU region was amplified using ITS3 and LR5 primers (Vilgalys and Hester 1990;White et al. 1990). The PCR conditions for the nrLSU locus were: 5 min initial denaturation at 95°C followed by 35 cycles of 40 s at 95°C, 40 s at 55°C and 60 s at 72°C with a final extension step for 7 min at 72°C. The partial rpb2 locus was amplified using two sets of primers. The first amplification was conducted using the fRPB2-5F and bRPB2-8.2R primers (Liu et al. 1999;Matheny et al. 2007) under the following amplification conditions: 5 min initial denaturation step at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 50°C with 0.3°C ramp per s to 72°C and 1 min at 72°C, and a final extension for 10 min at 72°C. The primers bRPB2-6F and bRPB2-7.1R or bRPB2-7R (Matheny 2005) were used to amplify domain 6 to 7 of the rpb2 in the second amplification. The PCR program consisted of an initial 5 min denaturation step at 95°C, followed by 35 cycles of 40 s at 95°C, 40 s at 58°C and 60 s at 72°C, and a final extension 7 min at 72°C. We developed four Russulaceaespecific mcm7 primers using Primer3 (Rozen and Skaletsky 2000): mcm7-Russ1F (GRA ARG AGT TYA CBC CYA TT), mcm7-Russ2F (GAC TGY CAG AAY GAG AAC GA), mcm7-Russ1R (TGA KAT CSC CAC GRA TYY  TCA T), and mcm7-Russ2R (GCT TTC TTS ACG TCR AYR TG). The first amplification of the mcm7 locus was conducted using either the primers mcm7-Russ1F/mcm7-Russ1R or mcm7-Russ2F/mcm7-Russ2R with the same amplification conditions previously described for nrLSU.
All PCR amplicons were checked on a 1% agarose gel stained with EcoDye DNA staining solution (SolGent Co., Daejeon, Korea). PCR products were purified with the Expin PCR Purification Kit (GeneAll Biotechnology, Seoul, Korea) according to the manufacturer's instructions. DNA sequencing was performed by Macrogen (Seoul, South Korea), using the same set of primers for each locus on an ABI3730 automated DNA Sequencer. Forward and reverse sequences were checked and edited when necessary using FinchTV 1.4.0 (Geospiza, Inc.; http://www.geospiza. com/finchtv) and assembled in Mega 6 (Tamura et al. 2013). Sequences of all DNA regions generated in this study were deposited in GenBank (Supplementary Table 1).

Phylogenetic analyses
ITS sequences were aligned with Lactarius reference sequences from GenBank and UNITE using MAFFT v. 7.380 (Katoh and Standley 2013). Ambiguously aligned positions were determined using Gblock (Castresana 2000) and excluded from further analyses. Maximum likelihood (ML) analyses were performed using RAxML v. 7.03 (Stamatakis 2006) and the GTR ? G model with 1000 bootstrap replicates. A jModelTest 2.1.7 (Darriba et al. 2012) was used to determine the model of character evolution. Bayesian inference (BI) analyses were executed with MrBayes v. 3.2.6 (Ronquist and Huelsenbeck 2003) using the model K80 ? I ? G. The BI analyses were performed with four independent runs in which each run had four chains. Each run had 20 million generations at a sampling frequency of 1000. All analyses were done on the CIPRES Science Gateway (Miller et al. 2010).
Phylogenetic relationships among Korean Lactarius species were determined using sequences of the four loci. Phylogenetic tree was constructed using only sequences from Korean Lactarius species because no reference sequences of the mcm7 locus were available. Sequences of each locus were aligned and analyzed separately. Sequence alignments were performed using the same method as that of the ITS described above. Alignment properties of each gene were analyzed using MEGA v. 6 (Tamura et al. 2013). ML and BI analyses were performed on the CIPRES Science Gateway with the same criteria used for the ITS. The BI analysis models for the nrLSU, mcm7, and rpb2 loci were K80 ? I ? G, HKY ? I ? G, and SYM ? I ? G, respectively. Finally, a combined analysis of the ITS, nrLSU, mcm7, and rpb2 sequences was conducted using the same methods and models previously assigned for each gene. Clades were considered to be independent evolutionary lineages when strongly supported (bootstrap support exceeding 70 and the posterior probability exceeding 0.95).

Diversity of Lactarius in South Korea
Among 729 Korean Lactarius specimens examined, ITS fragments of 650-700 bp were successfully sequenced from 525 specimens. The other 204 specimens were excluded from the study due to missing information or poor condition. ITS sequences from 525 sepcimens in Korea was analysized with 238 reference ITS sequences and found to represent 49 species (Fig. 1). To confirm species identity and infer the phylogenetic relationships of Korean Lactarius, three additional loci were amplified and sequenced for 128 specimens representing the 49 species: nrLSU (540-550 bp), rpb2 (630-670 bp), and mcm7 (420-440 bp). The ITS, nrLSU, rpb2, and mcm7 sequence  Hongo, L. tabidus Fr., and L. vietus (Fr.) Fr. The other 32 taxa formed distinct branches not corresponding to any of the described species. Four of these taxa lack morphological data or photographs of basidiomata, and thus remain un-named until additional data are collected. The other 28 species were confirmed as new to science after morphological comparison with closely related species.

Morphological characters of Korean Lactarius species
Lactarius subg. Russularia contains four well supported clades and two unclustered species (Fig. 2). Most species in this subgenus have transparent to watery white or white latex and show no discoloration. A few species have white latex turning pale yellow or strong yellow. Some species have an orange brown pileus or a hispid stipe. Microscopically, the presence of cheilo-and pleuro-macrocystidia and pileipellis structure are variable among species. In this subgenus, clade 1 consists of L. citrinus and L. lutescens, which is part of the ''chrysorrheus group''. They have common characters such as a zonate and dry pileus, white latex which turns lemon yellow, and a cutis as pileipellis structure (Fig. 2). Five species (L. mitratus, for description see below, L. neglectus, L. qinlingensis, L. subatlanticus, and L. subhirtipes) in clade 2 have an orange-brown pileus and slender or small basidiomata. Lactarius sp. 3 and L. fuscozonarius, see below, form an unsupported clade with long branches which is sister to clade 2 ( Fig. 2). Clade 3 contains six species (L. cinnamomeus, L. conglutinatus, L. tabidus and the new taxa described below, namely L. microbuccinatus, L. pectinatus and L. subomphaliformis). Species in this clade are characterized by an azonate pileus, a striate pileus margin and incompletely reticulate spore ornamentation with plage inamyloid to slightly amyloid in the distal part. L. orientaliquietus and L. subzonarius in clade 4 are placed at the base of this subgenus and belong to the ''quietus group'' (Figs. 1 and 2). Both species have a zonate pileus and white to watery pale cream latex (Fig. 2).
Lactarius subg. Lactarius contains three clades and seven unclustered species (Fig. 2). These species have a zonate pileus, scrobiculate pileus and stipe, hispid pileus margins, visicid to slimy pileus, and ixocutis to ixotrichoderm pileipellis. Relationships within this subgenus are unclear because of paraphyly and low branch support. Phylogenetic placement of L. betulinus (see taxonomic part) is unclear, but morphologically this species fits well in L. subg. Lactarius because of the sticky pileus surface with inconspicuous zonation and an ixotrichoderm type pileipellis. Eight species in clade 5 have a variety of morphological features. Lactarius species with lilac, violet, and olive discolouration of latex were included in this clade (Fig. 2). Five species in clade 6 are classified as L. section Deliciosi (Fr.) Redeuilh et al. All species have orange or reddish latex, except for L. spadiceus and L. aurantiozonatus (see taxonomic part below) with white and unchanging latex (Fig. 2). These species are associated with conifers such as Pinus and Abies. Lactarius austrotorminosus and L. cremicolor (see below) are weakly supported by share characters such as a fibrillose to hispid pileus and white latex. Clade 7 contains two species (L. orientitorminosus, see below, and Lactarius sp. 1). They have a common character such as hispid pileus. L.
albidoarmeniacus (see below) has a slightly tomentose pileus and is a sister to clade 7. The other unclustered new species L. inquinatus, L. pohangensis, and L. reticulisporus (see below) are placed at the base of the major clade of subg. Lactarius/Russularia. Lactarius subg. Plinthogalus has two supported clades and one unclustered species (Fig. 2). The dry, velvety and azonate pileus (typical for the subgenus) are shown in all species except L. fulvescens (see taxonomic part). Microscopically, Korean species of L. subg. Plinthogalus share a winged spore ornamentation, a trichopalisade, presence of cheiloleptocystidia, and absence of macrocystidia. Clade 8 contains five taxa that are characterized by unchanging white latex (Fig. 2). Clade 9 consists of four species (L. albidocinereus, L. incarnatus, and the other two new taxa, namely L. alboroseus and L. aurantiacopallens) which have pinkish discolouring latex. Finally, L. atromarginatus, which exhibits white latex with lilac discolouration, is placed at the basal position of clade 9 (Figs. 2,3,4).
Habitat: Scattered to gregarious on moss covered soil in mixed forests dominated by Pinus.
Comments: Lactarius fuscozonarius is characterized by the orange white to pastel red coloured, zonate and fibrillose pileus surface, and watery white to milky latex. This species is distinguished from other species of L. subg. Russularia in Korea by the absence of macrocystidia and the almost completely reticulate basidiospore. Lactarius politus Wisitr. & K.D. Hyde from Thailand is the phylogenetically closest species to L. fuscozonarius ( Fig. 1). However, the Thai species has much darker colour and heavier basidiospore ornamentation (up to 2.5 lm) than L. fuscozonarius .

MycoBank: MB 827901
Etymology: 'lutescens' means becoming yellow which refers to the yellowing milk of the species.
Habitat: Scattered to gregarious on soil in forests dominated by Pinus. Comments: Lactarius citrinus (see above) and L. lutescens are members of the chryssorheus group. They are distinguished by their host trees; the former is associated with Quercus and the latter with Pinus. For a comparison with other species of the chrysorrheus group, refer the comments of L. citrinus mentioned above.

MycoBank: MB 827902
Etymology: 'microbuccinatus' means small and trumpetshaped which refers to small-sized and funnel-shaped like trumpet of the basidiomata.
Habitat: Solitary or scattered on soil in mixed forests dominated by Quercus.
Habitat: Scattered on soil in mixed forests dominated by Abies.
Comments: Lactarius mitratus is characterized by the knob-like head on the apex of pleuromacrocystidia. This species is phylogenetically closely related to L. hirtipes Ying (Fig. 1), However, L. hirtipes has lanceolate macrocystidia while L. miratus has capitate macrocystidia (Wang and Liu 2002).

MycoBank: MB 827904
Etymology: 'pectinatus' means the form of a comb and refers to the radially striated and rugulose pileus margin.
Habitat: Scattered on humid soil in mixed forests dominated by Pinus and evergreen Quercus.

MycoBank: MB 827933
Etymology: 'betulinus' means birch tree. The name refers to the presence of the species in Betula forests and the putative ectomycorrhizal association with Betula.
Habitat: Scattered to gregarious on soil in forests dominated by Quercus.
Comments: Lactarius ciliatus is characterized by the smallsized basidiomata, reddish white to dull red colours, tomentose pileus, and white latex changing to yellowish buff. This species is similar to small-sized species in L.
subg. Russularia. The former can be distinguished from all small-sized species in L. subg. Russularia by its yellowing latex and hairy pileus.

MycoBank: MB 827935
Etymology: 'cinereo' means grey and 'roseus' means light red. The name refers to the colour of the basidiomata.
Habitat: Scattered to gregarious on soil in mixed forests dominated by Abies and Quercus. Comments: Lactarius cremicolor is morphologically similar to L. yazooensis Hesler & A.H. Sm. originated from North America. However, this species differs from L. yazooensis by its hygrophanous pileus and abundant cheilomacrocystidia (Hesler and Smith 1979). Moreover, L. yazooensis has nearly zebroid spore ornamentation while L. cremicolor has incomplete reticulum (Hesler and Smith 1979).

MycoBank: MB 827937
Etymology: 'cyaneo' means blue and 'cinereus' means grey. The name refers to colour change of the latex.
Habitat: Solitary or scattered on soil in Quercus forests.
Habitat: Solitary or scattered on soil or litter layer in forests dominated by Quercus.
Comments: Lactarius inquinatus is characterized by a white to yellowish white pileus, lamellae staining pale reddish brown when bruised, and watery white latex. This species is similar to L. evosmus Kühner & Romagn., but L. evosmus differs by having lower basidiospore ornamentation with less connected ridges and a distinct sweetish apple-like smell (Heilmann-Clausen et al. 1998). Lactarius zonarius (Bull.) Fr. also is similar morphologicaly to L. inquqinatus, but the former differs from the latter by its paler colour and subreticulate basidiospores (Heilmann-Clausen et al. 1998).

MycoBank: MB 827940
Etymology: 'orienti' means eastern and 'torminosus' refers to the European Lactarius torminosus. This name refers to the similar morphology with L. torminosus and its origin in East Asia (South Korea).
Habitat: Solitary or scattered on soil in mixed forests dominated by Quercus.

MycoBank: MB 827941
Etymology: 'parallelus' means parallel. The name defines the zebroid like ornamentation which consists of parallel ridges.
Habitat: Scattered to gregarious on soil in Pinus densifolia forests with numerous Tricholoma matsudake.
Comments: Lactarius pohangensis is characterized by the pale yellow to orange white pileus with darker center, watery white latex, and the association with Pinus trees. This species is similar to L. alpinus Peck. However, the latter differs by having thin macrocystidia (4-7 lm in width), the presence of a papilla on the pileus, and its association with Alnus (Hesler and Smith 1979).

MycoBank: MB 827943
Etymology: 'puniceus' means reddish violet which refers to the reddish violet discolouration of latex.
Habitat: Solitary or scattered on soil or litter layer in forests dominated by Quercus.
Habitat: Scattered to gregarious on soil in mixed forests dominated by Pinus and Quercus.
Comments: Lactarius spadiceus forms a clade with members of section Deliciosi. Before this study, only two species (L. porninsis Rolland and L. splendens Hesler & A.H. Sm.) in this section have been known to have white unchanging latex (Nuytinck et al. 2017). From this study, two species (L. aurantiozonatus, see above, and L. spadiceus) with unchanging white latex are added. Among four species with white latex, L. porninsis can be distinguished from L. spadiceus by its smaller basidiospores (6.3 -9.6 9 5.2 -7.3 lm) and the absence of scrobicules on the stipe (Heilmann-Clausen et al. 1998). The pileus of L. spendens is more vivid and azonate (Hesler and Smith 1979). Microscopically, this species lacks cheilocystidia on lamellar edges. Lactarius aurantiozonatus can be easily distinguished from L. spadiceus by its orange yellow and vivid pileus colour. The other closely related species in the phylogeny, L. rubrilacteus Hesler & A.H. Sm., has an orange brown pileus with concentric zonation and reddish latex (Hesler and Smith 1979).

MycoBank: MB 827946
Etymology: 'alboroseus' means pink or pale rose, referring to the pink discolouration of latex.
Habitat: Solitary or scattered on loam or sandy loam soil in forests dominated by Quercus.
Habitat: Solitary or scattered on soil in lowland of mixed forests dominated by Acer, Fraxinus, and Quercus. Comments: Lactarius aurantiacopallens is morphologically similar to two new species, L. alboroseus (see above) and L. incarnatus (see below). This species has a thinner pileipellis (90-150 lm thick) than those of L. alboroseus (150-180 lm thick) and L. incarnatus (150-190 lm thick).
Habitat: Solitary or scattered on humid soil near brooks in forests dominated by Quercus. Comments: Lactarius incarnatus is one of the Korean species with pinkish discoloring latex and can be confused with three Asian species (L. albidocinereus, L. alboroseus, see above, and L. aurantiacopallens, see above). The basidiospores of L. albidocinereus have modest ornamentation and more interconnections between ridges compared to L. incarnatus (Shi et al. 2018). Immediate latex discoloration of L. alboroseus and a thin pileipellis of L. aurantiacopallens are characters that distinguish these species from L. incarnatus (See the descriptions of the species in the present paper).
Habitat: Solitary or scattered on soil with moss or short grass under very old Acer and Quercus trees in mixed forests.
Comments: Lactarius ustulatus is easily recognized by its dry surface, dark reddish brown with darker center, wrinkled pileus, contrasting lamellae colour, and unchanging white latex. This species can be confused with L. cucurbitoides. However, the latter often has a darker lamellae and smaller basidiospores (7.0 -8.4 9 6.8 -8.0 lm) than those of L. ustulatus (Lee et al. 2015b). morphological characters were previously used for inframirabilis D. Stubbe et al. and L. sect. pseudofuliginosi) and one subalpine species, L. ferruginascens K. Das et al., in the subg. Plinthogalus have ixotrichoderm type pileipellis (Verbeken 2000;Stubbe et al. 2007;Das et al. 2017).
In conclusion, we confirmed 49 species of Lactarius from Korea, including at least 28 species new to science, based on morphological and multilocus phylogenetic approaches. Our results show that identification of Lactarius species using morphological data alone is often unreliable due to considerable overlap of characters among species. In addition to the combination of defining characters such as pileipellis, macrocystidia, spore ornamentation, and latex colour, the application of molecular methods is necessary for infrageneric classification and species identification in Korean Lactarius. Since many previously collected specimens had no associated information regarding host plants, the ecological relevance of Lactarius species remains unclear. Therefore, further investigation of the host plants of Lactarius is required in order to better understand the general ecology of Lactarius species.