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Article

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

by
Xiu-Hong Xu
1,2,3,†,
A-Min Chen
4,†,
Nan Yao
5,
Ting-Chi Wen
3,6,
Yun Pei
1,2 and
Wan-Ping Zhang
1,2,*
1
College of Agriculture, Guizhou University, Guiyang 550025, China
2
Institute of Vegetable Industry Technology Research, Guizhou University, Guiyang 550025, China
3
The Mushroom Research Centre, Guizhou University, Guiyang 550025, China
4
Guiyang Vegetable Technology Extension Station, Guiyang 550025, China
5
Guiyang Rural Revitalization Service Center, Guiyang 550025, China
6
The Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang 550025, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
J. Fungi 2023, 9(1), 122; https://doi.org/10.3390/jof9010122
Submission received: 17 December 2022 / Revised: 4 January 2023 / Accepted: 5 January 2023 / Published: 15 January 2023
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Abstract

:
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.

1. Introduction

Lactifluus (Pers.) Roussel belongs to the Russulaceae (Russulales) and is a genus of milkcaps, which is predominantly represented in subtropical and tropical regions [1,2,3,4,5]. The genus contains approximately 389 taxa (www.indexfungorum.org, accessed 31 October 2022), although De Crop (2016) estimated that there might be up to 530 Lactifluus species on Earth [6]. A significant number of new Lactifluus species have been discovered in the past ten years [7,8,9,10]. These species are mainly distinguished by their velvety pileus and stipe and a pleurotoid milkcap [1,11,12]. The hymenophoral trama of Lactifluus species has spherical cells (sphaerocytes), and the pileipellis structure and hymenium frequently contain cells with thicker cell walls [7,8].
Lactifluus is well known for the existence of several species complexes [12,13,14]. For example, Lactifluus volemus was discovered to have about 45 different clades [15,16,17], whereas Lactifluus piperatus is estimated to contain over 30 clades [18,19]. At the species level, the similarity of DNA data of Lactifluus species with similar or even the same morphology is low [11,15,16,17]. Groups with large differences in morphological characteristics show close kinship [7,20], and there are many cryptic and pseudocryptic species [12,14,15,19,21]. Furthermore, with the extensive use of DNA data, the sequences of the internal transcribed spacer (ITS), the large subunit 28S rRNA region (nrLSU), and the region between conserved domains 6 and 7 of the second largest subunit of RNA polymerase II (RPB2) are often used to identify Lactifluus species [20,22,23,24,25,26,27].
The greatest diversity of the genus is known from the Afrotropics, with 78 described species, although Lactifluus is also well represented in Asia, with 58 described species [8]. The genus is found in a wide range of vegetation types, including tropical and subtropical rainforests, subtropical dry forests, monsoon forests, tree savannahs, Mediterranean woodlands, temperate broadleaf and coniferous forests, and montane forests [23,28,29,30]. Host plants for Lactifluus species are members of the Betulaceae, Dipterocarpaceae, and Fagaceae families [16,31,32,33,34]. Lactifluus are commonly found in soil [27]. In order to adapt to the environment, some Lactifluus species with smaller basidiocarps and pleurotoid milkcaps are discovered on the stems and epigeous roots of trees, such as Lactifluus brunellus, Lf. multiceps [23], and Lf. raspei [35].
As a famously ectomycorrhizal fungus, Lactifluus contains a large number of wild edible mushrooms that are widely consumed worldwide [8,36]. Since it has a milk-like exudate, Lactifluus is easy to identify, except for some species of Lactifluus piperatus and Lactifluus vellereus complex groups that exude a spicy milk, which may cause discomfort after eating [37]. The scene of selling Lactifluus is more common in Europe, Central America, North America, and Asia [38,39,40,41,42,43], and they have a considerable socioeconomic value. A very recent account of edible mushroom species at the global scale lists some 100 edible milkcap species [44]. In particular, the Lactifluus sect. Lactifluus, such as Lactifluus tenuicystidiatus (X.H. Wang and Verbeken) X.H. Wang and Lactifluus volemus (Fr.) Kuntze have been eaten in Guizhou and Yunnan for many years and are locally called “naijiangjun” or “red naijiangjun”, being common, local, wild, edible fungi [16,39,45,46,47,48,49,50]. Several Lactifluus species, including Lf. bertillonii, Lf. rugatus, Lf. volemus, and Lf. vellereus, have also been shown to contain bioactive secondary metabolites, primarily sterols, sesquiterpenes, and sugar alcohols [51,52,53,54,55,56].
Southwest China includes Sichuan, Yunnan, and Guizhou Provinces, together with Chongqing City [57]. Geographically, southwest China is divided into the southeast of the Qinghai Tibet Plateau, the Sichuan Basin, and most of the Yunnan–Guizhou Plateau. The sub-region is divided into east and west sub-regions from Yalong River in the north to Nanpan River in the south of Kunming and the Hengduanshan sub-region in the northwest. The terrain structure is mainly plateaus and mountains with complex natural environmental conditions and diverse climates [58], which make the area rich in biological resources [59,60,61,62,63]. During numerous macrofungal surveys in the coniferous forests in the Wumeng Mountains of Suiyang County, Guizhou Province, three new Lactifluus species were discovered, based on detailed macro- and micro-morphological observations with descriptions, color photographs, and the sequence analyses of the ITS, LSU, and RPB2 regions. The objective of this research was to provide new evidence for understanding the distribution ranges and species diversity of Lactifluus species in China.

2. Material and Methods

2.1. Study Site and Specimen Collection

Eight specimens of the three new species were collected from Zunyi City, Suiyang County, Guizhou Province, China. Morphological descriptions were based on detailed field notes. Color names and codes were referred to HTML color codes (http://www.htmlcolorcode.org/ accessed on 1 November, 2022) [24]. The collections were dried with an electrical dryer at 50~60 °C until fully dry. Voucher specimens were deposited in the Cryptogamic Herbarium, Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), and Herbarium Mycology, Institute of Microbiology, Chinese Academy of Sciences (HMAS). The herbarium codes follow the Index Herbariorum.

2.2. 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].

2.3. DNA Extraction, Amplification, and Sequencing

Dried specimens were used to extract genomic DNA using an EZgeneTM 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).

2.4. 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].

2.5. Phylogenetic Analyses

All DNA datasets were aligned using the online version of MAFFT v.7 [69] (http://mafft.cbrc.jp/alignment/server/ accessed on 18 November, 2022) using the L-INS-i algorithm, then trimmed and edited in MEGA7.0 [70]. All phylogenetic analyses were performed in the PhyloSuite_v1.2.2 [71]. Phylogenetic analyses were conducted using the maximum likelihood (ML) strategy in IQ-TREE [72] and Bayesian inference (BI) in MrBayes v3.2.6 [73]. ML phylogenies were inferred using IQ-TREE under an edge-linked partition model for 5000 ultrafast [74] bootstraps, as well as the Shimodaira–Hasegawa–like approximate likelihood ratio test [75]. ModelFinder [76] was used to select the best-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].

3. 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 (#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). Basidia are 24.37–41.24 × 6.29–13.02 μm, Q = 2.29–2.99–3.98, with four sterigmata, which form four spores; and the sterigmata are 2.14–7.37 μm long. Pleuromacrocystidia are moderate to abundant, 51.07–63.83 × 4.44–7.89 µm, emergent up 30 µm, fusiform to subfusiform with fusoid, acuminate to subobtuse apices, originating from the subhymenial region. Pleuropseudocystidia are 1.68–4.11 µm wide. Cheilolamprocystidia are 31.53–58.66 × 6.37–6.04 µm, subcylindric to subfusiform with acuminate to subobtuse apices. Marginal cells are (15.35–)15.93–26.4(–29.03) × (1.64–)2.56–5.58(–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, Suiyang, Guizhou Province, China. The examined material is in China in Guizhou Province, Zunyi City, Suiyang (N 28°24′8″ E 107°5′31″, 1013.64 m), growing in groups on soil in association with Castanopsis spp., examined on 23 July 2020 by Xiuhong Xu (holotype is HKAS 122860, and isotype is HMAS 351908). (ITS = OL423562-OL423564, LSU = OL423575-OL423577, and RPB2 = OM030352-OM030354.)
Lactifluus qinggangtangensis W.P. Zhang, A.M. Chen, and X.H. Xu, sp. nov., is shown in 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 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 (2.71–)3.24–8.45(–8.54) × (2.89–)3.04–8.11(–8.41) μm, Q = (0.86–)0.86–1.15(–1.33), Q = 1.05 ± 0.13 μm, and they are subglobose and hyaline, with a strongly amyloid ornamentation composed of interconnected warts forming a complete reticulum up to 1.42 µm high (Figure 2). Basidia are 22.88–44.9 × 5.83–11.00 μm, Q = 2.93–3.84–4.90, with four sterigmata; they form four spores; and sterigmata are 2.21–6.78 μm long. Pleuromacrocystidia are moderate to abundant, 48.89–90.84 × 7.63–17.79 µm, fusiform to subfusiform with fusoid, acuminate to subobtuse apices, originating from the sub-hymenial region. Pleuropseudocystidia are 0.94–5.27 µm wide. Cheilolamprocystidia are 31.14–57.75 × 4.46–9.79 µm and transparent. Marginal cells are 18.84–67.41 × 2.24–10.71 µm, sublageniform, tortuous, tapering toward the apex, hyaline, fusoid, sometimes flexuous, thin-walled, and hyaline. Lactifers are 3.23–9.50 µm broad. The pileipellis is broken hyphoepithelium to epithelium, often with round cells separated and scattered, forming a cutis between piles of round cells, rarely of globose cells, forming a continuous layer. The stipitipellis is a cutis of densely interwoven hyphae mostly parallel with the stipe length.
The known distribution is Qinggangtang, Suiyang, Guizhou Province, China. The examined material is in China, Guizhou Province, Zunyi City, Suiyang (N 28°20′50″ E 107°10′11″, 943.27 m), and is growing in groups on soil in association with Castanopsis spp., examined on 23 July 2020 by Xiuhong Xu (holotype is HKAS 122862, and isotype is HMAS 351909). (ITS = OL423568-OL423569, LSU = OL423581, OL655455, and RPB2 = OM030358.)
Lactifluus jianbaensis W.P. Zhang, A.M. Chen, and X.H. Xu, sp. nov., is shown in Figure 4. The MycoBank ID is 842969. The etymology refers to the collection site “Jianba”, and the holotype is HKAS 122862.
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 × 8.59–13.75 mm, central, solid, dry, smooth, dark orange (W3C), uneven in color, white at the base, cylindrical, and slightly curved. The latex is abundant and watery. Basidiospores are (5.09–)5.25–7.52(–7.68) × (2.85–)3.75–7.24(–8.00) μm, Q = (0.80–)0.86–1.40(–1.79), Q = 1.06 ± 0.13 μm, subglobose, and hyaline, with a strongly amyloid ornamentation composed of interconnected warts forming a complete reticulum up to 2.17 µm high. Basidia are 31.32–44.77 × 11.59–16.06 μm, with four sterigmata, and form four spores. Pleuromacrocystidia are moderate to abundant, 65.38–102.98 × 5.19–11.67 µm, fusiform to subfusiform with fusoid, acuminate to subobtuse apices, originating from the sub-hymenial region. Marginal cells are 18.13–28.24 × 5.19–11.67 µm, sublageniform, tortuous, tapering toward the apex, hyaline, fusoid, sometimes flexuous, thin-walled, and hyaline. Lactifers are 2.55–6.77 µm broad. The pileipellis is subcylindric to subfusiform to fusiform with rounded to acuminate apex; the margin is wavy, composed of rounded to elongated to somewhat irregularly shaped cells. The stipitipellis is composed of elements.
The known distribution is Jianba, Suiyang, Guizhou Province, China. The examined material is in China, Guizhou Province, Zunyi City, Suiyang (N 29°0′24″ E 107°43′50″, 1044.54 m), growing in groups on soil in association with Pinus sp., examined on 12 October 2020 by Xiuhong Xu (holotype is HKAS 122862, and isotype is HMAS 351910). (ITS = OL423565-OL423567, LSU = OL423578-OL423580, RPB2 = OM030355-OM030355.)

4. 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. 40–80 mm), smaller basidiospores (2.71–8.54 × 2.89–8.41 μm vs. 8.0–9.0–9.1–10.2(–10.5) × 7.4–8.3–8.4–9.4(–9.6) μm), smaller ridges (1.42 µm vs. 2.0 µm), and smaller basidia (22.88–44.9 × 5.83–11.00 μm vs. 40–65 × 11–14 μm). Lf. jianbaensis can be distinguished from Lf. acicularis by the height of the spore ornamentation, which can be up to 2.17 µm in Lf. jianbaensis but only up to 1.1–1.4 μm in Lf. acicularis.
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.

Author Contributions

Conceptualization, X.-H.X. and A.-M.C.; methodology, X.-H.X., W.-P.Z. and A.-M.C.; validation, X.-H.X., A.-M.C., W.-P.Z., T.-C.W. and Y.P.; formal analysis, A.-M.C.; investigation, N.Y., X.-H.X. and W.-P.Z.; resources, X.-H.X. and A.-M.C.; data curation, A.-M.C.; writing-original draft preparation, A.-M.C.; writing-review and editing, X.-H.X.; visualization, X.-H.X.; supervision, W.-P.Z. and T.-C.W.; project administration, W.-P.Z.; funding acquisition, W.-P.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32160737) and the Science and Technology Foundation of Guizhou Province (Grant nos. (2019) 2451-4 and (2019)3005-2).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogram for Lactifluus sect. Lactifluus generated from maximum likelihood analysis of ITS, LSU, and RPB2 sequence data. Bootstrap support values for maximum likelihood and maximum parsimony greater than 50% and posterior probabilities from Bayesian inference ≥0.95 are given above the branches. The new species are presented in bold type.
Figure 1. Phylogram for Lactifluus sect. Lactifluus generated from maximum likelihood analysis of ITS, LSU, and RPB2 sequence data. Bootstrap support values for maximum likelihood and maximum parsimony greater than 50% and posterior probabilities from Bayesian inference ≥0.95 are given above the branches. The new species are presented in bold type.
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Figure 2. Lactifluus taibaiensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Marginal cells. (E) Pleurocystidia. (F) Cheilocystidia. (G) Basidia. (H) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–H).
Figure 2. Lactifluus taibaiensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Marginal cells. (E) Pleurocystidia. (F) Cheilocystidia. (G) Basidia. (H) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–H).
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Figure 3. Lactifluus qinggangtangensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Marginal cells. (E) Pleurocystidia. (F) Cheilocystidia. (G) Basidia. (H) Stipitipellis. (I) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–I).
Figure 3. Lactifluus qinggangtangensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Marginal cells. (E) Pleurocystidia. (F) Cheilocystidia. (G) Basidia. (H) Stipitipellis. (I) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–I).
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Figure 4. Lactifluus jianbaensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Basidia. (E) Pleurocystidia. (F) Cheilocystidia. (G) Marginal cells. (H) Stipitipellis. (I) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–I).
Figure 4. Lactifluus jianbaensis microscopic characteristics: (A) Fresh basidiomata (holotype). (B) SEM microphotographs. (C) Basidiospores. (D) Basidia. (E) Pleurocystidia. (F) Cheilocystidia. (G) Marginal cells. (H) Stipitipellis. (I) Pileipellis. Scale bars: 4 µm (B), 3 µm (C), and 30 µm (D–I).
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Table
Table 1. Specimen and GenBank accession numbers of DNA sequences used in the molecular analyses. The arrangement of the subgenera and sections in the table follows their position in the concatenated phylogeny of the genus Lactifluus subgenus Lactifluus (Figure 1).
Table 1. Specimen and GenBank accession numbers of DNA sequences used in the molecular analyses. The arrangement of the subgenera and sections in the table follows their position in the concatenated phylogeny of the genus Lactifluus subgenus Lactifluus (Figure 1).
SpeciesVoucher Specimen No.LocalityITSLSURPB2
Lactifluus subgenus Lactifluus
Lactifluus sect. Lactifluus
Lf. acicularisH.T. Le 265 (CMU, GENT, MFLU, SFSU)ThailandHQ318277HQ318196HQ328926
Lf. acicularisKVP08002ThailandHQ318226HQ318132HQ328869
Lf. aff. tenuicystidiatusKUN:F75810ChinaKC154105KC154131KC154157
Lf. aff. tenuicystidiatusKUN:F75810ChinaKC154105KC154131KC154157
Lf. aff. volemus var. flavusKVP08023ThailandHQ318227HQ318133HQ328870
Lf. crocatusH.T. Le 268 (CMU, GENT, MFLU, SFSU)ThailandHQ318266HQ318181HQ328917
Lf. dissitusAV-KD-KVP09-134IndiaJN388978JN389026JN375628
Lf. distantifoliusH.T. Le 288 (CMU, GENT, MFLU, SFSU)ThailandHQ318274HQ318193
Lf. distantifoliusDS07-461ThailandHQ318223HQ318124HQ328866
Lf. indovolemusIB 18-013IndiaMN005117
Lf. indovolemusIB 18-003IndiaMN005115
Lf. jianbaensisTB 4ChinaOL423565OL423578OM030355
Lf. jianbaensisTB 5ChinaOL423566OL423579OM030356
Lf. jianbaensisTB 6ChinaOL423567OL423580OM030357
Lf. lamprocystidiatusEH 72-195Papua New GuineaKR364015
Lf. leptomerusAV-KD-KVP 09-131 (GENT)IndiaJN388972JN389023JN375625
Lf. longipilusLTH273ThailandHQ318276HQ318195HQ328925
Lf. longipilusLTH184ThailandHQ318256HQ318169HQ328905
Lf. longipilusAV-RW04-160ThailandHQ318235HQ318143HQ328880
Lf. maenamensisKD 16-008IndiaMF928075
Lf. mexicanusMontoya 5189MexicoMK211179MK211188MK258869
Lf. mexicanusMontoya 5266MexicoMK211180MK211189MK258870
Lf. oedematopusKVP12-001 GENT neotypeGermanyKJ210065KJ210066KJ210068
Lf. oedematopusAF 2386 (BR)BelgiumJQ753876JQ348324JQ348260
Lf. oedematopusAV07-079 GENTBelgiumJQ753835JQ348270JQ348131
Lf. pallidilamellatusM 4716 (XAL)MexicoJQ753824JQ348268
Lf. qinggangtangensisTB 7ChinaOL423568OL423581OM030358
Lf. qinggangtangensisTB 8ChinaOL423569OL655455
Lf. pinguisAV-RW04-162ThailandHQ318221HQ318121HQ328863
Lf. pinguisH.T. Le 117—TypeThailandHQ318211HQ318111HQ328858
Lf. pinguisH.T. Le 255 (CMU, GENT, MFLU, SFSU)ThailandHQ318263HQ318178HQ328914
Lf. rugiformisSFC20150818-14South KoreaMN215387MN215343MN212835
Lf. sect. Tenuicystidiati
Lf. species 17LTH214ThailandHQ318249HQ318158HQ328894
Lf. species 21AV-KD-KVP09-137SikkimJN388958JN389027JN375629
Lf. species 22AV-KD-KVP09-129SikkimJN388957JN389021JN375623
Lf. species 8aKVP08021ThailandHQ318233HQ318140HQ328877
Lf. species 8aLTH170ThailandHQ318252HQ318165HQ328902
Lf. subpruinosusX.H. Wang 3489 (KUN)ChinaKC154110KC154136KC154162
Lf. subvolemusKVP 08-048SloveniaJQ753927JQ348379JQ348241
Lf. taibaiensisTB 1ChinaOL423562OL423575OM030352
Lf. taibaiensisTB 2ChinaOL423563OL423576OM030353
Lf. taibaiensisTB 3ChinaOL423564OL423577OM030354
Lf. versiformisAV-KD-KVP09-002SikkimJN388966JN389030JN375631
Lf. versiformisAV-KD-KVP09-006SikkimJN388965JN389033JN375633
Lf. vitellinusH.T. Le 348 (CMU, GENT, MFLU, SFSU)ThailandHQ318251HQ318164HQ328900
Lf. vitellinusK. Van de Putte 08-024 (GENT, MFLU)ThailandHQ318236HQ318144HQ328881
Lf. volemusL. Pihlik et al.—TAAM095075RussiaJQ753905JQ348357JQ348219
Lf. volemusWalther—STU 406307GermanyJQ753909JQ348361JQ348223
Lf. volemusL. Tedersoo—TAAM182733EstoniaJQ753907JQ348359JQ348221
Lf. volemusLE 254509RussiaJQ753937JQ348388
Lf. volemusL. Pihlik et al.—TAAM095097RussiaJQ753906JQ348358JQ348220
Lf. volemusKobeke Van de Putte 08-45SloveniaJQ753953
Lf. sect. Allardii
Lf. allardiiJ. Nuytinck 2004-008USAKF220016KF220125KF220217
Lf. sect. Ambicystidiati
Lf. ambicystidiatusKUN:F57008—TypeChinaNR_155311NG_060287KC154148
Lf. sect. Gerardii
Lf. atrovelutinusD. Stubbe 06-003MalaysiaGU258231GU265588GU258325
Lf. bicolorDS06-247MalaysiaJN388955JN388987JN375590
Lf. gerardiiA.Verbeken 05-375USAGU258254GU265616GU258353
Lf. sect. Piperati
Lf. aff. glaucescensAV 05-374North AmericaKF220049KF220150KF220236
Lf. aff. piperatusA.Verbeken 04-202USAKF220021KF220127KF220220
Outgroup
Auriscalpium vulgarePBM_944North AmericaDQ911613DQ911614AY218472
Bondarzewia montanaAFTOL_452No dataDQ200923DQ234539AY218474
Stereum hirsutumAFTOL_492No dataAY854063AF393078AY218520
Table
Table 2. Synopsis of sister species to the new Lactifluus species reported here with respect to distribution and morphological features.
Table 2. Synopsis of sister species to the new Lactifluus species reported here with respect to distribution and morphological features.
SpeciesLf. rugiformisLf. pinguisLf. acicularis
LocationKoreaThailandThailand
Pileus length (mm)50–11035–853.3–4.6
Pileus colorRusty orange (6C8–7C8) tinged with a
more brownish color		Yellowish-orange-brown (5C7–5C8),
brown (6D5)		Yellowish-orange-brown (5C7–5C8),
brown (6D5)
Lamella breadth (mm)Three broad, rarely furcate, with
numerous lamellula of different length		2–4 Narrow to rather broad (1.5–
6 mm)
Lamella colorCream to
pale orange		Whitish to creamCream (4A3–4A4), discoloring to brown (6D5–
6E5) to grayish-brown (5C3–5C4) when damaged
Stipe (mm)30–70 × 15–2040–95 × 10–1545–85 × 5–15
Stipe colorConcolorous
with pileus		Concolorous with pileusYellowish-orange (4A5–5A5),brownish-orange (6C8–6D8) to grayish-brownish-orange
(6C5–5C6–6B5–6B6–6C6), or brown (6D4–6D5)
LatexAbundant, sticky, white turning
dark brown		Copious, sticky, white, unchanging when isolatedWhite
Basidiospores (μm)7.1–8.4–9.6 ×
6.7–7.9–9.2, Q = 1.01–1.05–1.09, globose to subglobose		8.0–9.0–9.1–10.2(–10.5) × 7.4–8.3–8.4–
9.4(–9.6)		7.0–7.9–8.5–9.1(–9.3) × 6.5–7.2–7.8–
8.5, subglobose (Q = 1.01–1.08–1.10–1.21)
Basidia (μm)49.5–60 × 9–12.540–65 × 11–14 40–60 × 9–12
Pileipellis (μm)20–68 × 2.5–4.0, cell wall 0.5–1.5, thick, erect50–14050–120, thick
References[21][15][15]
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MDPI and ACS Style

Xu, X.-H.; Chen, A.-M.; Yao, N.; Wen, T.-C.; Pei, Y.; Zhang, W.-P. Three New Species of Lactifluus (Basidiomycota, Russulaceae) from Guizhou Province, Southwest China. J. Fungi 2023, 9, 122. https://doi.org/10.3390/jof9010122

AMA Style

Xu X-H, Chen A-M, Yao N, Wen T-C, Pei Y, Zhang W-P. Three New Species of Lactifluus (Basidiomycota, Russulaceae) from Guizhou Province, Southwest China. Journal of Fungi. 2023; 9(1):122. https://doi.org/10.3390/jof9010122

Chicago/Turabian Style

Xu, Xiu-Hong, A-Min Chen, Nan Yao, Ting-Chi Wen, Yun Pei, and Wan-Ping Zhang. 2023. "Three New Species of Lactifluus (Basidiomycota, Russulaceae) from Guizhou Province, Southwest China" Journal of Fungi 9, no. 1: 122. https://doi.org/10.3390/jof9010122

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