Three New Species of Lactifluus (Basidiomycota, Russulaceae) from Guizhou Province, Southwest China

Lactifluus is a distinct genus of milkcaps, well known as ectomycorrhizal fungi. The characteristics of the genus Lactifluus include grayish-yellow, orange to orange-brown, or reddish-brown pileus, white latex from the damaged lamellae, discoloring to a brownish color, reticulate spore ornamentation, lampropalisade-type pileipellis, and the presence of lamprocystidia. Guizhou Province is rich in wild mushroom resources due to its special geographical location and natural environment. In this study, three novel Lactifluus species were identified through the screening of extensive fungal resources in Suiyang County, Guizhou Province, China, sampled from host species of mostly Castanopsis spp. and Pinus spp. Based on critical morphology coupled with nuclear sequences of genes encoding large subunit rRNA, internal transcribed spacer, and RNA polymerase II, these new species, Lactifluus taibaiensis, Lactifluus qinggangtangensis, and Lactifluus jianbaensis, were found to belong to Lactifluus section Lactifluus. A comparison with closely related species, Lactifluus taibaiensis was distinguished by its lighter-colored pileus, different colors of lamellae, and more subglobose basidiospores; Lactifluus jianbaensis was identified by the height of the spore ornamentation and its subglobose basidiospores; and Lactifluus qinggangtangensis was characterized by having smaller basidiospores, ridges, and pleurolamprocystid.


Morphological Analysis
Basidiospores were examined in Melzer's [61] reagent and measured in side view. At least 20 mature basidiospores were examined from basidiomata. Other microscopic structures were studied after these structures were soaked in 5% KOH and 1% Congo Red for 10 min. The ornamentation of the spores was observed under a scanning electron microscope (SEM, Coxem EM-30, Daejeon, South Korea). The structures were cut under a stereomicroscope (Leica S9E, Wetzlar, Germany), then observed and measured under a compound microscope (Leica DM 2500, Wetzlar, Germany). The measurements (and Q values) are given as (a) bec (d), in which "a" is the lowest value, "bec" covers a minimum of 90% of the values, and "d" is the biggest value. "Q" stands for the ratio of the length and width of a spore, and "Q ± av" represents the average Q of all spores ± sample standard deviation [61]. Other microscopic structures were treated in 5% KOH for 30 s and then observed in 1% Congo Red. Sections through the stipitipellis were taken from the middle of the stipe [64].

DNA Extraction, Amplification, and Sequencing
Dried specimens were used to extract genomic DNA using an EZgene TM Fungal gDNA Kit (Biomiga, San Diego, CA, USA). Reaction mixtures (20 µL) contained 1 µL template DNA, 7 µL distilled water, and 1 µL (10 µM) of each primer and 10 µL 2 × Taq PCR StarMix with Loading Dye (Genstar, Kangrunchengye Biotech, Beijing, China). Three nuclear gene loci were amplified and sequenced: the universal primers ITS1 and ITS4 were used for amplification of the internal transcribed spacer (ITS) region of the ribosomal DNA, which includes spacer regions ITS1 and ITS2 and the ribosomal gene 5.8S; LROR and LR5 were the primers used for the amplification of LSU, which is a part of the ribosomal large subunit 28S region [65,66]; and RPB2-6F and RPB2-7CR were the primers used for amplification of the region between domains 6 and 7 of the second largest subunit of RNA polymerase II (rpb2) [65,66]. The PCR amplification reactions were performed on a T100 Thermal Cycler (T100™, Bio-Rad, Hercules, CA, USA). The ITS, LSU, and RPB2 regions were amplified by an initial denaturation step at 5 min at 95 • C, 35 cycles of 30 s at 94 • C, 30 s at 55 • C, 40 s at 55 • C, and a final extension stage of 5 min at 72 • C. PCR products were verified by 1% agarose gel electrophoresis and compared with 2 Kb DNA Markers [66]. The verified PCR products were purified and sequenced with the primers mentioned above at Sangon Biotech (Shanghai, China).

Sequence Alignment and Phylogenetic Analysis
The quality of the newly obtained sequences of three new specimens was checked manually by observing the chromatogram with BioEdit [67]. Three datasets (ITS, nrLSU, and RPB2) were generated from the representative (voucher) specimens of each species and used for phylogenetic analyses. Following preliminary analyses that placed the new species within Lactifluus subgenus Lactifluus, phylogenetic analyses were performed with the newly generated sequences and the sequences retrieved from GenBank [68], derived from the BLAST search (best match) of related Lactifluus species, complemented with other GenBank sequences of species of the sections within Lactifluus subgenus Lactifluus identified by De Crop [8] (Table 1). In this way, we selected 110 sequences of Lactifluus sect. Lactifluus, nine sequences of Lactifluus sect. Tenuicystidiati, three sequences of Lactifluus sect. Allardii, three sequences of Lactifluus sect. Ambicystidiati, nine sequences of Lactifluus sect. Gerardii, and six sequences of Lactifluus sect. Piperati, with Auriscalpium vulgare, Bondarzewia montana, and Stereum hirsutum being selected as outgroups [26]. Lactifluus generated from maximum likelihood analysis ITS, LSU, and RPB2 sequence data. Bootstrap support values for maximum likelihood and max mum parsimony greater than 50% and posterior probabilities from Bayesian inference ≥0.95 ar given above the branches. The new species are presented in bold type.

Results
We generated 23 new sequences from the Lactifluus species studied, eight from each of the ITS and nLSU regions of rDNA and seven from the RPB2 region (Table 1). In the phylogenetic trees, ML and BI analyses produced highly similar topologies with comparable support values. The results inferred in the multilocus phylogeny ( Figure 1) strongly supported the recognition of three new species, namely, Lactifluus taibaiensis, Lactifluus jianbaensis, and Lactifluus qinggangtangensis, based on phylogenetic studies with three regions (ITS, LSU, and RPB2). fit partition model (edge-linked) using the BIC criterion. The best-fit models were identified according to BI criteria (BIC): SYM + I + G4: ITS, K2P + I + G4: LSU, K2P + I + G4: RPB2. BI phylogenies were inferred using MrBayes 3.2.6 under a partition model (two parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. The phylogenies from ML and BI analyses were displayed using FigTree v1.4.3 [77].

Results
We generated 23 new sequences from the Lactifluus species studied, eight from each of the ITS and nLSU regions of rDNA and seven from the RPB2 region (Table 1). In the phylogenetic trees, ML and BI analyses produced highly similar topologies with comparable support values. The results inferred in the multilocus phylogeny ( Figure 1) strongly supported the recognition of three new species, namely, Lactifluus taibaiensis, Lactifluus jianbaensis, and Lactifluus qinggangtangensis, based on phylogenetic studies with three regions (ITS, LSU, and RPB2).

Taxonomy
Lactifluus taibaiensis W.P. Zhang, A.M. Chen, and X.H. Xu, sp. nov., is shown in Figure 2. The MycoBank ID is 842968. The etymology refers to the collection site "Taibai", and the holotype is HKAS 122860. Pilei are 35.60-44.50 mm diameter and convex to planoconvex with a broadly depressed center. Velvet is mainly distributed on the edge, and orange (W3C) (#FFA500) is in the center when young, gradually becoming applanate to infundibuliform or concave; the surface is drying, smooth, dry, rugulose, velvety, and darker toward the center. The edge bends inward and is integral and brittle in consistency. Lamellae are decurrent, white (W3C) to cream (#FFFFCC), thick and brittle, and 2.00-3.40 mm broad; the edge is concolorous to marginate, furcate, and with different lengths. The attachment to the stipe varies from adnate to adnate with a decurrent tooth to decurrent and is milk white (#FEF-CFF) after being bruised, with no discoloration reaction. The stipe is 46.50-81.10 × 9-14 Pilei are 35.60-44.50 mm diameter and convex to planoconvex with a broadly depressed center. Velvet is mainly distributed on the edge, and orange (W3C) (#FFA500) is in the center when young, gradually becoming applanate to infundibuliform or concave; the surface is drying, smooth, dry, rugulose, velvety, and darker toward the center. The edge bends inward and is integral and brittle in consistency. Lamellae are decurrent, white (W3C) to cream (#FFFFCC), thick and brittle, and 2.00-3.40 mm broad; the edge is concolorous to marginate, furcate, and with different lengths. The attachment to the stipe varies from adnate to adnate with a decurrent tooth to decurrent and is milk white (#FEFCFF) after being bruised, with no discoloration reaction. The stipe is 46.50-81.10 × 9-14 mm, central, solid, dry, lighter colored than that of the pileus, rugulose, white at base, with a lot of white hyphae, cylindrical, and slightly curved; the latex is thick and milky white (#FEFCFF). The context is white (W3C) (#FFFFFF), and the taste is mild. Latex is abundant and sticky and changes from white to brown. Basidiospores are (2.85-)3.96-7.1(-8.6) × (3.19-)3.47-7.93(-8.28) µm, Q = (0.75-)0.83-1.23(-1.26), Q = 1.00 ± 0.13 µm, and they are subglobose and hyaline, with a strongly amyloid ornamentation composed of interconnected warts forming a complete reticulum up to 1.5 µm high (Figure 1) (-5.76) µm, sublageniform, tortuous, tapering toward the apex, hyaline, fusoid, sometimes flexuous, thin-walled, and hyaline. Lactifers are 3.11-7.12 µm wide. The pileipellis is subcylindric to subfusiform to fusiform with rounded to acuminate apex; the margin is wavy; and the subpellis is pseudoparenchymatous, composed of rounded to elongated to somewhat irregularly shaped cells. The stipitipellis is composed of elements.
The known distribution is Taibai Figure 3. The MycoBank ID is 842971. The etymology refers to the collection site "Qinggangtang", and the holotype is HKAS 122861. Pilei are 24.12-52.73 mm diameter and are slightly concave in the center to convex to planoconvex with a broadly depressed center. Velvet is mainly distributed on the edge, and orange (W3C) (#FFA500) is in the center when young, gradually becoming applanate to infundibuliform or concave; the surface is smooth, dry, and velvety, with an uneven distribution. The edge bends inward and is integral and brittle in consistency. Lamellae Pilei are 24.12-52.73 mm diameter and are slightly concave in the center to convex to planoconvex with a broadly depressed center. Velvet is mainly distributed on the edge, and orange (W3C) (#FFA500) is in the center when young, gradually becoming applanate to infundibuliform or concave; the surface is smooth, dry, and velvety, with an uneven distribution. The edge bends inward and is integral and brittle in consistency. Lamellae are decurrent, white (W3C) to cream (#FFFFCC), thick and brittle, and dense; the edge is concolorous to marginate. The attachment to the stipe varies from adnate to adnate with a decurrent tooth to decurrent and is milk white (#FEFCFF) after bruising, with no discoloration reaction. The stipe is 39.12-59.09 × 9.41-11 mm, central, solid, dry, smooth, concolorous with the pileus, white at the base, cylindrical, and slightly curved, and the latex is thick and milky white (#FEFCFF). The context is white (W3C) (#FFFFFF), and the taste is mild. Basidiospores are (2.  Pilei are 42.81-46.25 mm diameter, and are slightly concave in the center to convex to planoconvex with a broadly depressed center, dark orange (W3C) in the center, mango orange (#FF8040) on the edge, and velvet in the center when young, gradually becoming applanate to infundibuliform or concave. The surface is dry and smooth. The edge bends flat and is brittle in consistency. Lamellae are decurrent, white (W3C) to cream (#FFFFCC), Pilei are 42.81-46.25 mm diameter, and are slightly concave in the center to convex to planoconvex with a broadly depressed center, dark orange (W3C) in the center, mango orange (#FF8040) on the edge, and velvet in the center when young, gradually becoming applanate to infundibuliform or concave. The surface is dry and smooth. The edge bends flat and is brittle in consistency. Lamellae are decurrent, white (W3C) to cream (#FFFFCC), thick and brittle, and dense, and the edge is concolorous to marginate. The attachment to the stipe varies from adnate to adnate with a decurrent tooth to decurrent, and the milk is colorless to white (#FEFCFF) and turns brown in a few minutes after being bruised. The stipe is 56. 25-60.94

Discussion
In this study, three new accessions are identified as novel species of Lactifluus sect. Lactifluus in terms of both morphology and phylogeny. Lactifluus taibaiensis, with its sister species Lactifluus rugiformis from South Korea [27]; Lactifluus jianbaensis, with its sister species Lactifluus acicularis from Thailand [15]; and Lactifluus qinggangtangensis, with its sister species Lactifluus pinguis from Thailand [15], form well-separated clades in the resultant phylogram, which indicate the distinct phylogenetic positions of the three new species in Lactifluus sect. Lactifluus.
Lf. taibaiensis is an orange milkcap, with a rugulose stipe, similar to its sister species Lf. rugiformis [27]. There are also many other characteristics to distinguish one another, with Lf. taibaiensis being lighter in the color of the pileus (orange (W3C) (#FFA500) vs. rusty orange (6C8-7C8)) and having a higher ratio of stipe length/pileus diameter (1.3-1.8 vs. 0.7) and different colors of lamellae (cream vs. cream to pale orange). When comparing micromorphologic features between Lf. taibaiensis and Lf. rugiformis, the basidiospores of the former are more subglobose (0.75-1.26 vs. 1.01-1.09) ( Table 2). Lf. jianbaensis differs from its sister species Lf. acicularis in terms of the pileus color (dark orange (W3C) vs. brown (6D5)), the diameter of the pileus (43-46 mm vs. 35-85 mm), and the height of the spore ornamentation of subglobose basidiospores (2.17 µm vs. 1.40 µm); this can also be distinguished from Lf. longipilus. The main difference between Lf. qinggangtangensis and Lf. pinguis is the smaller diameter of the pileus of the former (24.12-52.73 mm vs.   It is noteworthy that three new Lactifluus species were described in Guizhou, southwest China. As a result of the investigations into Lactifluus resources in Guizhou over the years, Guizhou was found to be particularly rich in Lactifluus spp. (Table 2), namely, Lactifluus leoninus Verbeken and E. Horak Verbeken, in Verbeken, Nuytinck, and Buyck [78], Lactifluus bhandaryi Verbeken and De Crop, Lactifluus subpiperatus (Hongo) Verbeken [79], Lactifluus pseudoluteopus X.H. Wang and Verbeken, X.H. Wang [80], and Lactifluus volemus [79,[81][82][83]. Many Lactifluus are considered to be edible mushrooms and are sold at the local markets and along roadsides, fresh, dried, or boiled. In addition to Lactifluus, its related milkcap genus Lactarius is also very rich in subtropical China, and several new species were described recently [84][85][86]. So, the diversity of ectomycorrhizal milkcap mushrooms is rich these areas.