The Genus Leccinum (Boletaceae, Boletales) from China Based on Morphological and Molecular Data

Leccinum is one of the most important groups of boletes. Most species in this genus are ectomycorrhizal symbionts of various plants, and some of them are well-known edible mushrooms, making it an exceptionally important group ecologically and economically. The scientific problems related to this genus include that the identification of species in this genus from China need to be verified, especially those referring to European or North American species, and knowledge of the phylogeny and diversity of the species from China is limited. In this study, we conducted multi-locus (nrLSU, tef1-α, rpb2) and single-locus (ITS) phylogenetic investigations and morphological observisions of Leccinum from China, Europe and North America. Nine Leccinum species from China, including three new species, namely L. album, L. parascabrum and L. pseudoborneense, were revealed and described. Leccinum album is morphologically characterized by the white basidioma, the white hymenophore staining indistinct greenish blue when injured, and the white context not changing color in pileus but staining distinct greenish blue in the base of the stipe when injured. Leccinum parascabrum is characterized by the initially reddish brown to chestnut-brown and then pale brownish to brown pileus, the white to pallid and then light brown hymenophore lacking color change when injured, and the white context lacking color change in pileus but staining greenish blue in the base of the stipe when injured. Leccinum pseudoborneense is characterized by the pale brown to dark brown pileus, the initially white and then brown hymenophore lacking color change when injured, and the white context in pileus and stipe lacking color change in pileus but staining blue in stipe when bruised. Color photos of fresh basidiomata, line drawings of microscopic features and detailed descriptions of the new species are presented.


Introduction
The genus Leccinum Gray is a species-rich genus of Boletaceae and is characterized by a whitish or yellow hymenophore, a white to cream context unchanging or staining blue or red when injured, a brown to blackish scabrous to dotted squamules on the surface of the stipe, and comparatively long and smooth basidiospores. Generally, most species of the genus are widely spread in the subarctic, boreal, temperate and Mediterranean regions, with a few secondary expansions to the neotropics [1][2][3][4][5][6][7][8][9][10][11][12]. Species  In this study, we used both morphological data and molecular sequences from the nuclear ribosomal internal transcribed spacer (ITS), the large subunit of the nuclear ribosomal RNA (nrLSU), the translation elongation factor 1-alpha (tef1-α) and the RNA polymerase II second largest subunit (rpb2), together with ecological data to (1) elucidate species diversity of Leccinum in China; (2) evaluate the phylogenetic relationships of species within Leccinum; (3) make morphological and ecological comparisons between closely related species.

Taxon Sampling
Nineteen specimens of the genus Leccinum from China were examined. For each collection, a part of the basidioma was dried with silica gel for DNA extraction. The remaining materials were then air-dried at 45-50 • C using an electric food dehydrator. Specimens studied in this work were deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN). Genera are abbreviated as follows: L. for Leccinum, Le. for Leccinellum, O. for Octaviania, R. for Rossbeevera, Ru. for Rugiboletus, T. for Turmalinea, Ca. for Castanopsis, Li. for Lithocarpus, P. for Pinus and Q. for Quercus.

Morphological Observation
The macroscopic descriptions are based on the detailed field notes and photographs of fresh basidiomata. Color codes of the form "4B2" indicate the plate, row, and color block from Kornerup and Wanscher [58]. For microscopic studies, the microscopic features of each part of the basidioma were observed under microscope (Leica DM2000, Leica Microsystems, Wetzlar, Germany), including basidiospores, basidia, cheilocystidia, pleurocystidia and pileipellis, using 5% KOH as a mounting medium to revive the dried materials. Microscopic studies follow Zhou et al. [59]. In the description of Basidiospores, the abbreviation n/m/p means n basidiospores measured from m basidomata of p collections in 5% KOH solution. The notation of the form (a) b-c (d) stands for the dimensions of the basidiospores; the range b-c contains a minimum of 90% of the measured values, a or d given in parentheses stands for extreme values. Q is used to mean "length/width ratio" of a basidiospore in a side view; Q m means average Q of all basidiospores ± sample standard deviation. Measurements of basidospores, cystidia, basidia and terminal cells in pileipellis are presented as length × width. All microscopic structures were drawn freehand from rehydrated material under the microscope with 10× eyepiece and 100× objective (the total magnification is 1000×).

Sequence Alignments and Phylogenetic Analyses
The newly generated sequences of each locus were blasted in GenBank, and the most closely related sequences (nucleotide identities >95%) were downloaded for further alignment. Sequences were aligned separately for each of the loci using MAFFT v7.130b with the E-INS-I strategy and manually optimized on BioEdit v7.0.9 [66,67]. Two datasets, the ITS dataset and the multi-locus (nrLSU + tef1-α + rpb2) dataset, were analyzed using RAxML and Bayesian methods, respectively. For the multi-locus dataset, single-gene analyses were conducted to assess incongruence among individual genes using the ML method (results not shown). Because no well-supported bootstrap value (BS > 70%) [55] conflict was detected among the topologies of the three genes, their sequences were then concatenated together for further multi-locus analyses.
For ML analyses, the multi-locus and ITS datasets were analyzed using RAxML (https://www.phylo.org/, accessed on 26 August 2021) under the model GTRGAMMA [68]. Statistical supports for the phylogenetic analyses were determined using nonparametric bootstrapping with 1000 replicates. For BI analyses, the parameter model was selected by the Akaike information criterion (AIC) as the best-fit likelihood model with Modeltest 3.7 (Free Software Foundation, Boston, MA, USA) [69]. The models employed for each of the four loci were GTR + I + G for ITS, nrLSU and tef1-α, and SYM + I + G for rpb2. Posterior probabilities (PP) were determined twice by running one cold and three heated chains in parallel mode, saving trees every 1000th generation. Other parameters were kept at their default settings. Runs were terminated once the average standard deviation of split frequencies went below 0.01 [70]. Chain convergence was determined using Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/, accessed on 26 August 2021) to confirm sufficiently large ESS values (>200). Subsequently, the sampled trees were summarized after omitting the first 25% of trees as burn-in using the 'sump' and 'sumt' commands implemented in MrBayes.

Molecular Phylogenetic Analysis
A total of 57 sequences, including fifteen for nrLSU, fifteen for tef1-α, fourteen for rpb2 and thirteen for ITS, were newly generated in this study and aligned with sequences downloaded from GenBank. Sequences retrieved from GenBank and obtained in this study for the multi-locus phylogenetic analyses are listed in  [71,72]. ML and Bayesian analyses produced very similar estimates of tree topologies, and thus only the tree inferred from ML analysis is displayed ( Figure 1). The monophyly of Leccinum was highly supported (BS = 100% and PP = 1) in our analyses. Four main clades were recovered, and three of them correspond to the three known subsections, viz. L. subsect. Leccinum, L. subsect. Fumosa and L. subsect. Scabra of L. sect. Leccinum [14]. Three new species, namely L. album, L. parascabrum and L. pseudoborneense, were revealed in our multi-locus phylogenetic analyses. Leccinum parascabrum formed the remaining clade with BS = 100% and PP = 1, while L. pseudoborneense and L. album nested in L. subsect. Scabra and clustered together with L. flavostipitatum E.A. Dick & Snell, L. subradicatum and L. variicolor with low supported lineage (BS = 54%).    For the ITS dataset, as revealed by den Bakker et al. [57] and our primary analysis, the ITS1 region contains a minisatellite, which is characterized by the repeated presence of CTATTGAAAAG and CTAATAGAAAG core sequences and mutational derivatives. Moreover, some species contain a minisatellite in the ITS2 region, e.g., the newly described species L. album (GenBank Acc. No.: MZ392872 for clone 1 and MZ392873 for clone 2), with a region of 212 bp that consists of tandem repeats (see Supplementary Material for details). Though there is length variation in either the ITS1 or ITS2 spacers, it can also provide some phylogenetic signals. We performed phylogenetic analyses of the ITS dataset. In this dataset (Supplementary File S2), 51 samples were included. The length of the dataset was 1416 bp, of which 377 were parsimony informative. Leccinellum albellum (Peck) Bresinsky & Manfr. Binder was chosen as outgroup. ML and Bayesian analyses also produced very similar estimates of tree topologies, and only the tree inferred from ML analysis is displayed (Figure 2). The monophyly of Leccinum was also well supported (BS = 100% and PP = 1) in our analyses. Three new species viz. L. album, L. parascabrum and L. pseudoborneense) were revealed. Leccinum album is closely related to L. pseudoborneense yet without statistical support, while L. parascabrum forms an independent lineage. Species to which L. parascabrum is phylogenetically related remain as yet unknown.

Taxonomy
Leccinum album X. Meng, Yan C. Li & Zhu L. Yang, sp. nov., (Figures 3g-h and 4). MycoBank: MB 838917. Diagnosis: This species differs from other species in Leccinum in the combination of the entirely white pileus, the white pileal context not changing color when injured, the white hymenophore staining indistinct greenish blue when hurt, the white stipe coarsely covered with initially white and then darkened verrucose squamules, and the white stipe context always staining greenish blue at the base when injured.
Commentary: Leccinum album is characterized by the white pileus, the white hymenophore staining indistinct greenish blue when hurt, the white stipe densely covered with initially white and then darkened scabrous squamules, the white context in pileus not changing color when injured, and the white context in stipe unchanging or only staining distinct greenish blue at base when injured. Morphologically, L. album is close to L. holopus, L. cyaneobasileucum Lannoy & Estadès and Le. albellum (Peck) Bresinsky & Manfr. Binder in similar pileus colors. However, L. holopus, originally described from Europe (Germany), differs from L. album in its medium to large basidiomata (pileus 4-10 cm wide), becoming more viscid pileus with age, pure white or dirty white to pale buff or pale pallid pileus always with a glaucous green tinge, long hymenophoral tubes measuring 9-15 mm long, narrow and subcylindrical hymenial cystidia measuring 30-50 × 7.5-12.5 µm, narrow pileipellis hyphae measuring 3.5-5 µm wide, and association with trees of the genus Betula (Betulaceae) [80][81][82]. Leccinum cyaneobasileucum, originally described from France, is different from L. album in its white or greyish brown to light brown pielus, woolly stipe surface, slender basidiospores with Q m ≥ 3, relatively narrow hymenial cystidia measuring 32-44 × 5.5-7.5 µm, narrow pileipellis hyphae measuring 2-6.5 µm wide, and association with trees of the genus Betula [83]. Leccinellum albellum, originally described from New York, is characterized by its basidiomata not changing color when bruised and narrow basidiospores measuring 13-20 × 4-6 µm [16,17,30].
Phylogenetically, L. album is related to L. variicolor and L. pseudoborneense in the analyses of the multi-locus and ITS datasets, respectively (Figures 1 and 2). However, L. variicolor differs from L. album in its white to grey or cream pileal context staining vinaceous to brown when bruised, white stipe context staining pink to coral red in the upper part and greenblue in the lower part when bruised and association with plants of Betula [81]. Leccinum pseudoborneense is different from L. album in its pale brown to dark brown pileus, white context in pileus and stipe staining blue when bruised, narrow basidiospores measuring (11) 12-19 (20)  Diagnosis: This species differs from other species in Leccinum by its initially reddish brown to chestnut-brown and then brown to pale brownish or even dirty white pileus, white pileal context lacking color change when injured, white to pallid and then light brown hymenophore lacking color change when injured, and the white stipe context staining greenish blue at the base when injured.
Holotype: CHINA. Etymology: Latin "parascabrum" refers to its similarity to L. scabrum. Basidiomata small to medium-sized. Pileus 2.5-12.5 cm in diam., hemispherical when young, subhemispherical to convex or applanate when mature, reddish brown (12E8) to chestnut-brown (8C7-8) when young, brown (6C6) to pale brownish (7D7-8) or even dirty white (6A2) when mature; surface tomentose; context 6-13 mm thick in the center, white (1A1), not changing color when bruised; Hymenophore adnate when young, adnate to slightly depressed around apex of stipe; surface white to pallid (1A1) when young, and becoming light brown (6B4) when mature, not changing color when injured; tubes 6-14 mm long, 0.5-1.5 mm wide, creamy white (1A1), not changing color when bruised. Stipe 12-14 × 1.1-2.2 cm, clavate, swollen downwards, always staining greenish blue at base when injured; surface white (1A1), covered with initially white (1A1) to light beige (5A4) and then brownish (7D8) squamules; context white (1A1), staining greenish blue (25B6-7) at base when injured; basal mycelium white (1A1). Commentary: Leccinum parascabrum is characterized by the initially reddish brown to chestnut-brown and later brown to pale brownish or even dirty white pileus, the white pileal context not changing color when injured, the white to pallid and then light brown hymenophore not changing color when injured, the white stipe context with greenish blue color change at the base when injured, and the relatively large basidiospores measuring 16-20 (-21)  However, L. duriusculum, originally described from Europe, can be distinguished from L. parascabrum by its pale grey-brown to dark greyish or reddish brown pileus, white context staining violaceous pink when bruised but yellow-green to blue-green in the base of stipe, relatively small basidiospores measuring 11.5-15.5 × 4.5-6 µm [84]. Leccinum griseonigrum, originally described from North America, differs from L. parascabrum in its avellaneous to dingy cinnamon-buff pileus, white pileal context staining blue when bruised, relatively small basidiospores measuring 13-16 × 4-5.5 µm, and association with trees of the genus Populus [16]. Leccinum scabrum differs from L. parascabrum in its wrinkled pileus, pale white hymenophore, pinkish discoloration when injured, and never bluish color change at the base of stipe [13,14,81]. Leccinum uliginosum, originally described from North America, is different from L. parascabrum in its dark fuscous to drab-grey pileus, white context in pileus becoming reddish and then fuscous when bruised, relatively small basidiospores measuring 14-18 × 3.5-5 µm, and small and inconspicuous hymenial cystidia [17]. Leccinellum pseudoscabrum differs from L. parascabrum in its initially red to purplish brown and then blackish brown context color change when injured, and the palisadoderm pileipellis composed of subglobose cells [14]. Leccinum parascabrum also shares the similar colors of pileus and hymenophore and the bluish color change at the base of stipe with L. variicolor. However, L. variicolor is different from L. parascabrum in its white to grey or cream pileal context staining vinaceous to brown when bruised, white stipe context staining pink to coral red in the upper part and green-blue in the lower part when bruised, relatively small basidiospores measuring (10) 13.5-17.5 (-20.0) × 5.0-6.5 (7.0) µm with Q = 2.4-3.1, and association with plants of Betula sp. [81]. In our phylogenetic analysis of the multi-locus and ITS datasets (Figures 1 and 2), L. parascabrum formed independent clades within Leccinum. It might represent a distinct section or subsection. However, formal change of the infrageneric division of this clade should await more molecular and morphological data from additional taxa. Species to which it is phylogenetically related remain as yet unknown.
Leccinum pseudoborneense X. Meng & Yan C. Li & Zhu L. Yang, sp. nov., (Figures 3d-f and 6). MycoBank: MB 838915. Diagnosis: This species differs from other species in Leccinum in its nearly glabrous and pale brown to dark brown pileus, white context in pileus lacking color change when injured, white context in stipe staining blue when bruised, initially white and then brown hymenophore not changing color when injured, white stipe covered with ochraceous to dark brown squamules, and trichodermal pileipellis composed of 3-6 µm wide interwoven hyphae.
Habitat Commentary: Leccinum pseudoborneense is characterized by the nearly glabrous and pale brown to dark brown pileus, the white context in pileus not changing color when injured, the white context in stipe staining blue when bruised, the initially white and then brown hymenophore not changing color when injured, the white stipe covered with ochraceous to dark brown squamules, and the trichodermal pileipellis composed of 3-6 µm wide interwoven hyphae. Leccinum pseudoborneense is similar to L. borneense (Corner) E. Horak, originally described from Malaysia, in that they share a brown pileus, bluish color change of the context in stipe when bruised, and similar size of basidiospores. However, L. borneense differs from L. pseudoborneense in its yellow to olive yellow hymenophore staining blue when hurt, pale yellow to yellow pileal context staining blue when hurt, and deep yellow context in stipe staining blue but sometimes with reddish tint at base when injured [6,84]. Leccinum pseudoborneense is phylogenetically close to L. album in our phylogenetica analyses (Figures 1 and 2). However, L. album has a white basidioma, white hymenophore staining indistinctly greenish blue when hurt, white context in pileus not changing color when injured, white context in stipe unchanging or only staining distinctly greenish blue at base when injured, and relatively broad basidiospores measuring 15-19 × 5-7 µm.

Discussion
The genus Leccinum was defined and recognized variously by different mycologists. In an early molecular study, Leccinum was shown to be polyphyletic and proposed to be restricted to the sections Leccinum and Scabra by Binder and Besl [4]. Subsequently, Bresinsky and Besl [32] erected a genus Leccinellum Bresinsky & Manfr. Binder, to accommodate L. section Luteoscabra, including species with yellow hymenophores and/or context. In this study, the phylogenetic inferences based on the multi-locus dataset of nrLSU, tef1-α and rpb2 largely coincide with those of Binder and Besl [4], Bresinsky and Besl [32] and den Bakker et al. [33]. Thus, we adopt the treatment of Bakker et al. [33] and treat Leccinum in a strict circumscription, which only includes species of L. sect. Leccinum (Singer's infrageneric classification with L. sect. Scabra merged to this section). Species in Leccinum are characterized by the white context lacking color changes or staining blue, gray or reddish tints when injured and the cutis-like pileipellis composed of interwoven filamentous hyphae Eleven Leccinum species with specimen citations have been reported from China before this study, of which five species (L. ambiguum, L. atrostipitatum, L. olivaceopallidum, L. potteri and L. subgranulosum) were originally described from North America, five species (L. aurantiacum, L. holopus, L. roseofractum, L. scabrum and L. versipelle) were originally described from Europe, and only one taxon (L. subleucophaeum var. minimum) was originally described from China. Our molecular phylogenetic analyses along with morphological studies identified the existence of L. quercinum, L. scabrum, L. subleucophaeum var. minimum and L. versipelle in China. The distribution of other reported species have not yet been found, based on morphological and/or molecular data. In addition, three species new to science (L. album, L. parascabrum and L. pseudoborneense) and two species new to China (L. melaneum and L. schistophilum) were revealed in our study, based on molecular and morphology evidence. In conclusion, there are nine species of Leccinum in China.
Most species of Leccinum exhibit strong mycorrhizal host specificity. The host specificity along with climate type and edaphic factors appear to be important factors determining the distribution of different species. In China, L. melaneum, L. scabrum, L. schistophilum and L. versipelle are found in temperate forests and associated with plants of Betula platyphylla on acidic soils. Leccinum album, L. parascabrum, L. pseudoborneense and L. subleucophaeum var. minimum are found in tropical forests and associated with plants of Fagaceae (Castanopsis calathiformis, Ca. hystrix, Ca. indica, Ca. orthacantha, Cyclobalanopsis glauca, Lithocarpus mairei, Li. truncatus and Quercus griffithii) and/or Pinaceae (Pinus kwangtudgensis and P. yunnanensis) on acidic soils. It is noteworthy that L. parascabrum can be found in acidic or slightly alkaline habitats. Leccinum versipelle is found in subtropical forests and is associated with plants of Populus yunnanensis on acidic soils. The combination of the color of basidioma, the morphology of pileal surface, the size of basidiospores, the morphology of stipe, the color changes when injured, the climate type, the edaphic factors and the host preferences is very important in distinguishing species in this genus.