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Morphological Studies and Phylogenetic Analyses Unveil Two Notable New Species of Russula Subg. Heterophyllinae from China

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12 October 2024

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14 October 2024

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Abstract
Russula, a prominent genus of ectomycorrhizal fungi, is notably abundant and diverse in China. We present here the findings from studies on various Chinese Russula collections. Two notable species within subg. Heterophyllinae, namely Russula leucoviridis and R. subswatica, were described and illustrated based on comprehensive morphological characteristics and molecular evidence. Morphologically, Russula leucoviridis is characterized by its small to medium-sized basidiomata with a cuticle that cracks and breaks into small green patches near the center after mature, a pastel green to green pileus center with light yellow spots, and a white to greenish white pileus margin; whereas R. subswatica is distinguished by an infundibuliform pileus with a deeply depressed center after mature, a light orange to grayish orange pileus center, and a yellowish white to light orange margin with a purplish to purplish-black hue, and a white to yellowish white stipe that exhibits a purplish hue at the junction with the pileus. The phylogenetic analyses were constructed using a combined dataset of ITS, nrLSU, RPB2 and mtSSU. Relying on both morphological characteristics and multigene phylogeny, the former species is classified within subsect. Virescentinae, while the latter is affiliated with subsect. Griseinae. In this study, we contribute new data on infrageneric phylogenetic relations and enrich the species diversity of Subg. Heterophyllinae in China.
Keywords: 
Subject: Biology and Life Sciences  -   Ecology, Evolution, Behavior and Systematics

1. Introduction

The genus Russula Pers., a type genus of the family Russulaceae, is recognized as the largest genus within the phylum Basidiomycota [1,2]. It comprises approximately 1,100 described species, with estimates suggesting the existence of at least 3,000 species [3,4]. Species of this genus typically form ectomycorrhizal symbiotic associations with a wide range of host trees primarily including Pinus spp., Castanopsis spp., Picea spp., Larix spp., Fagus spp. and Castanea spp., as well as with various herbs, sedges, and shrubs [5,6,7]. These fungi inhabit a diverse array of ecosystems, ranging from Arctic tundra to tropical forests, and play a crucial role in forest development and maintaining ecological balance [8,9]. Numerous species of Russula are renowned as wild edible mushrooms in various regions around the world and are commonly traded commercially [9,10,11]. Investigations and statistics on the resource diversity of Chinese macrofungi indicate that approximately 78 species of Russula are edible in China, with the Russula griseocarnosa complex and the R. virescens complex being particularly popular [10,12,13]. However, the genus Russula also includes several toxic species, making precise identification essential for the safe utilization of these fungi. The rapid advancements in molecular biology techniques have greatly advanced the study of the systematics and phylogeny of Russula. Recent molecular phylogenetic studies have indicated that the taxa within the genus Russula are divided into nine subgenera, namely: R. subg. Glutinosae Buyck & X.H. Wang, R. subg. Archaeae Buyck & V. Hofst., R. subg. Compactae (Fr.) Bon, R. subg. Brevipedum Buyck & V. Hofst., R. subg. Crassotunicatae Buyck & V. Hofst., R. subg. Heterophyllidiae Romagnesi, R. subg. Cremeoochraceae Buyck & X.H. Wang, R. subg. Malodorae Buyck & V. Hofst., and R. subg. Russula Per. [14,15,16]. The infrageneric classification system in this study followed Buyck et al. [14,15,16].
Subgenus Heterophyllidia is one of the most complex and diverse groups within the genus Russula. The subgenus is mainly characterized by the mostly medium to large basidiomata, rarely very small basidiomata, almost all possible pileus colors, equal lamellae, mild to strongly acrid taste, mostly white or cream basidiospore print, basidiospores with inamyloid or partly amyloid suprahilar spot, mostly abundant gloeocystidia that are typically mucronate to obtuse-rounded, and pileipellis without primordial hyphae [14]. In China, numerous new species of subg. Heterophyllidia have been continuously described and reported in recent studies [17,18,19,20,21,22], underscoring the region as a hotspot for the discovery of previously unknown species. During surveys on the species diversity and geographic distribution of the subgenus Heterophyllidia, the first author of this paper discovered that many species, including the R. virescens complex, R. cyanoxantha complex, R. heterophylla complex, and several undescribed species, were being consumed and sold. Therefore, it is essential to clarify the species diversity within this subgenus to ensure the safe and sustainable consumption of these wild fungi. In this study, several new collections of subg. Heterophyllidia from China were examined using morphological characteristics and phylogenetic analyses, resulting in detailed descriptions and illustrations of two notable new species, along with an updated phylogenetic tree to confirm their distinct placements. This study significantly enhances our understanding of species diversity within the subg. Heterophyllidia in China.

2. Materials and Methods

2.1. Morphological Studies

Fresh basidiomata were collected and photographed in their natural forest habitat. All specimens were dried at temperatures ranging from 45 to 55 °C and subsequently deposited in the herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF). The terminology used to describe the morphological characteristics follows the guidelines established by Adamčík et al. [3]. Macroscopic characteristics were documented through meticulous notes and high-resolution photographs. Color codes primarily adhere to the standards set by Kornerup and Wanscher [23]. Microscopic examination of the specimen sections was carried out using a ZEISS Imager M2 microscope. Estimates of spore ornamentation density were conducted in accordance with the methodology described by Adamčík and Marhold [24]. The estimates of hymenial cystidia density were performed referring to Buyck [25]. The basidiospores were observed and measured in Melzer’s reagent from a lateral perspective, with ornamentation excluded. Following pretreatment of dried specimens with 5% potassium hydroxide (KOH), additional micromorphological characteristics were identified and measured in Congo red. The coloration of the cystidia contents was observed in a sulfovanillin (SV) solution [26]. The pileipellis were examined using cresyl blue to verify the presence of ortho- or metachromatic reactions [27]. The structure and ornamentation of the basidiospores were illustrated using a JEOL JSM-6510 scanning electron microscope (SEM). Basidiospore measurements are presented in the format (Min–)AV-SD–AV–AV+SD(–Max), where Min denotes the minimum value, Max denotes the maximum value, AV represents the average value, SD stands for the standard deviation, and Q indicates the length-to-width ratio of the basidiospores.

2.2. Molecular Study

Total genomic DNA was extracted from dry specimens using an optimized CTAB protocol [28]. The following four regions were amplified and sequenced using standard primer sets for enhanced accuracy and fluency: 600 base pairs of the ITS region of rDNA with the primers ITS1 and ITS4 [29]; 900 base pairs of the nuclear ribosome large subunit (nrLSU) with the primers LR0R and LR5 [30]; 700 base pairs of the second largest subunit of RNA polymerase II (RPB2) using the primers bRPB2-6f and fRPB2-7cr [31,32]; 600 base pairs of the ribosomal mitochondrial small subunit (mtSSU) using primers MS1 and MS2 [29]. The successful PCR products were sequenced using an ABI 3730 DNA analyzer with the ABI BigDye 3.1 terminator cycle sequencing kit (Shanghai Sangon Biological Engineering Technology and Services Co., Ltd, Shanghai, China). The newly generated sequences have been submitted to the GenBank database.

2.3. Phylogenetic Analyses

For phylogenetic analyses, species from the subgenus Heterophyllidia that exhibit high similarity to our new species were selected, along with representative species closely related to subsect. Griseinae Jul. Schäff. and subsect. Virescentinae Singer. Russula maguanensis J. Wang, X.H. Wang, Buyck & T. Bau and R. substriata J. Wang, X.H. Wang, Buyck & T. Bau were utilized as the outgroup. The NCBI accession numbers for the sequences employed in the phylogenetic tree are detailed in Table 1. Initial sequence alignment was conducted using the online version MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/). To ensure reliable and accurate results, manual evaluations and adjustments were made in BioEdit as necessary [33]. Maximum likelihood (ML) and Bayesian analysis (BA) methods were employed for conducting the phylogenetic analyses. Maximum likelihood (ML) analysis was performed using RAxML-HPC2 on XSEDE (8.2.12) via the CIPRES Science Gateway (www.phylo.org). The ML analysis utilized the rapid bootstrap algorithm with 1000 replicates to ensure the robustness of the results under the GAMMA model. Bootstrap support (BS) values of 70% or higher on the final tree were considered significant. Bayesian analysis (BA) was conducted using MrBayes 3.2.7a on XSEDE through the CIPRES Science Gateway (www.phylo.org) under the GTR model. A total of 50 million generations were executed across four independent Markov chains, with tree sampling occurring every 100 generations. For each analysis, the initial 25% of the trees were excluded as part of the burn-in phase. The 50% majority-rule consensus trees were used to obtain Bayesian posterior probability (PP) values, and nodes with PP values of 0.95 or higher were deemed to have significant support.

3. Results

3.1. Phylogeny

Both the Maximum Likelihood (ML) and Bayesian Analysis (BA) of the combined ITS, nrLSU, RPB2 and mtSSU sequence dataset produced consistent tree topologies, with only the ML tree depicted in Figure 1. Posterior probabilities from the Bayesian analysis (BA) are also indicated along the branches. The samples of our two new species each formed a strongly supported clade, nested within subsect. Virescentinae and Griseinae. They are distinctly separate from any known and sequenced species within subg. Heterophyllidia. Phylogenetic analyses also confirmed that both subsections Virescentinae and Griseinae are monophyletic groups, each with robust support indicated by bootstrap values (BS) of 100% and posterior probabilities (PP) of 1, while the monophyly of the remaining four subsections within subgenus Heterophyllidia was also significantly supported.
Russula leucoviridis is phylogenetically positioned as the sister taxon to a clade consisting of two Chinese species R. prasina G.J. Li & R.L. Zhao and R. xanthovirens Y. Song & L.H. Qiu, with 100% bootstrap support and 1.00 posterior probabilities. Our second species, R. subswatica clustered with Pakistani species R. swatica Sarwar & Hanif, demonstrating robust phylogenetic support with 100% bootstrap support and 1.00 posterior probabilities.

3.2. Taxonomy

Russulaleucoviridis B. Chen & J. F. Liang, sp. nov. (Figure 2, Figure 3 and Figure 4)
MycoBank: MB855842
Diagnosis: Pileus small to medium-sized; cuticle adhering many small frosty particles near the center when young, cracking and broken into small green patches near the center after mature, patches crowded towards the center; pastel green to green towards the center with light yellow spots, white to greenish white near the margin.
Holotype: China, Hainan Province, Wuzhishan City, Shuiman Town, Wuzhi Mountain National Reserve;18°51′59.50″ N, 109°40′30.59″ E, 800m asl., in evergreen broad-leaved forest; 23 June 2016, leg. JXM105 (RITF3405).
Etymology: Leuco (Latin) = white; viridis (Latin) = green; the species name refers to white pileus margin and green center.
Description: Pileus small to medium-sized, 30–55 mm in diameter; initially hemispheric when young, expanding to planeconvex with a depressed center after mature; margin incurved, not cracked, striation up to the 1/3 of the radius; cuticle dry, glabrous, peeling to 1/4 of the radius, many small adhering frosty particles near the center when young, cracking and broken into small green patches near the center after mature, patches crowded towards the center; pastel green (27A4) to green (27A7) towards the center with light yellow (3A5) spots, white (1A1) to greenish white (28A2) near the margin. Lamellae adnate to almost free, 3–5 mm deep, 8–10 at 1 cm near the pileus margin, white (1A1) to cream; lamellulae sometimes present and irregular in length; furcations occasional near the stipe; edge entire and concolor. Stipe 20–55 × 7–16 mm, cylindrical, slightly inflated towards the base, white (1A1) to almond, medulla initially stuffed when young, becoming hollow after mature. Context 2–3 mm thick in half of the pileus radius, white (1A1), unchanging when bruised, taste mild, odor inconspicuous. Spore print white (1A1) to cream.
Basidiospores (5.7–)6.0–6.9–7.8(–8.8) × (4.8–)5.3–5.9–6.5(–7.0) μm, Q = 1.03–1.15–1.27(–1.36), subglobose to broadly ellipsoid; ornamentation of medium-sized, moderately distant [5–6(–8) in a 3 μm diameter circle] amyloid warts or spines, 0.3–0.5 μm high, locally reticulate, occasionally fused in short chains [0–2(–3) in the circle], occasionally to frequently connected by line connections [2–3(–4) in the circle]; suprahilar spot small, amyloid. Basidia (35.0–)39.0–46.0–53.0(–56.2) × (8.4–)9.3–10.3–11.4 μm, mostly 4-spored, sometimes 2- and 3-spored, clavate; basidiola subcylindrical or clavate, ca. 5.5–9.5 μm wide. Hymenial gloeocystidia on lamellae sides dispersed to moderately numerous, (46.0)48.6–57.7–66.8(76.0) × (7.4–)8.4–10.1–11.9(12.7) μm, typically fusiform, occasionally clavate, apically mainly obtuse, occasionally acute, sometimes with 4–8 μm long appendage, thin-walled; contents granulose or heteromorphous, turning reddish black in SV. Hymenial gloeocystidia on lamellae edges often narrower, (38.7–)44.8–55.7–66.5(–75.2) × (5.6–)6.3–7.8–9.2(–9.8) μm, typically clavate, sometimes fusiform, apically mainly obtuse, rarely mucronate, sometimes with 4–8 μm long appendage, thin-walled; contents heteromorphous, turning reddish black in SV. Marginal cells (16.7–)18.4–22.8–27.2(–30.6) × (3.2–)3.8–4.5–5.1 μm, clavate or lageniform, sometimes flexuous. Pileipellis orthochromatic in cresyl blue, not sharply delimited from the underlying context, 240–320 μm deep, two-layered; suprapellis 180–200 μm deep, composed of loose, ascending or erect hyphal terminations; subpellis 80–140 μm deep, composed of intricate, dense, 3–6 μm wide hyphae. Hyphal terminations near the pileus margin occasionally unbranched, sometimes flexuous, thin-walled; terminal cells (10.8–)13.2–22.3–31.4(–37.4) × (4.0–)4.5–5.0–5.7(–6.2) μm, mainly narrowly lageniform, occasionally cylindrical, apically attenuated or constricted, sometimes obtuse; subterminal cells often shorter and wider, ca. 5–8 μm wide, rarely branched. Hyphal terminations near the pileus center similar; terminal cells (8.0–)10.0–14.1–18.2(–25.0) × (2.6–)3.1–3.7–4.3(–4.7) μm, mainly lageniform or subcylindrical, apically attenuated or constricted; subterminal cells often shorter and wider, rarely branched, ca. 4–6 μm wide. Pileocystidia near the pileus margin always one-celled, (30.9–)32.3–37.5–42.8(–47.5) × (2.8–)3.8–5.0–6.2(–6.8) μm, mainly clavate, occasionally fusiform or lanceolate, apically typically obtuse, occasionally acute, sometimes with 2–5 μm long, round or ellipsoid appendage, thin-walled; contents granulose or heteromorphous, turning reddish black in SV. Pileocystidia near the pileus center similar, always one-celled, (29.6–)31.4–36.5–41.6(–44.6) × (3.3–)3.7–4.4–5.2(–5.4) μm, thin-walled, mainly clavate or cylindrical, apically obtuse, contents granulose or heteromorphous, turning reddish black in SV.
Additional specimens examined: China, Hainan Province, Wuzhishan City, Shuiman Town, Wuzhi Mountain National Reserve; 18°51′59.50″ N, 109°40′30.59″ E, 800m asl., in evergreen broad-leaved forest; 23 June 2016, leg. JXM106 (RITF3406).
Notes: Russula leucoviridis is positioned within the subsection Virescentinae based on a combination of its morphological characteristics and phylogenetic analysis. In phylogenetic analysis, R. leucoviridis is the sister taxon to a clade consisting of R. prasina and R. xanthovirens, both originating from China, with strong support evidenced by 100% bootstrap values and 1.00 posterior probability. However, R. prasina often has the larger basidiomata (43–130 mm), a grass green pileus center and pale yellowish green margin, pale ocher lamellae and ochraceous yellow stipe base [34]; R. xanthovirens differs from R. leucoviridis in having the larger basidiomata (50–75 mm), a yellowish green to deep green pileus center with cracked yellowish margin when age, whitish lamellae with reddish hue, white stipe with green tinge, often shorter and wider basidia [(29–)30–51.5(–60) × (9.5–)10–9.5–14.5(–15) μm] and often longer pileocystidia (40–69.5 × 4–9 μm) [35].
Given cracking pileus surface, R. virescens (Schaeff.) Fr., R. parvovirescens Buyck, D. Mitch. & Parrent and R. viridirubrolimbata J. Z. Ying and R. crustosa Peck of subsect. Virescentinae resemble R. leucoviridis. However, R. virescens, originally described from Europe, can be distinguished by usually larger basidiomata [40–90(125) mm], a green to yellowish green pileus and shorter basidia (40–52 × 6–9.2 μm) [36]. The American species R. parvovirescens has a greenish brown to metallic bluish green pileus with green patches, often larger basidiospores (6.7–9.1 × 5.7–7.2 μm) and smaller Basidia (38–45 × 8–9 mm) [37]. R. viridirubrolimbata, (originally reported from China) is distinct in a light yellowish olive to yellowish olive pileus center with a pinkish red to light jasper red margin, smaller basidia (31−43 × 7−10μm), larger hymenial gloeocystidia on lamellae sides (78–107 × 8–10 μm) and absence of hymenial gloeocystidia on lamellae edges [13,38]. Russula crustosa, initially described in North America, possesses a brownish-yellow, greenish or subolivaceous pileus, shorter basidia [(29–)30–32–33.5(–35) × (7.5–)8–9.5–10.5(–11) μm], longer hymenial gloeocystidia on lamellae sides [(59–)63–69–75(–80) × (7–)8–9.5–11(–13) μm] and absence of hymenial gloeocystidia on the lamellar edges [39].
Russula subswatica B. Chen & J. F. Liang, sp. nov. (Figure 5, Figure 6 and Figure 7)
MycoBank: MB855843
Diagnosis: Pileus medium-sized to large; initially flat hemispheric when young, expanding and infundibuliform with a deeply depressed center after mature; light orange to grayish orange near the center, yellowish white to light orange towards the margin, partially with a purplish to purplish-black hue. Stipe white to yellowish white, exhibiting a purplish hue at the junction with the pileus.
Holotype: China. Xizang Autonomous Region, Nyingchi City, Motuo County; 29°22′13.43″ N, 95°26′58.75″ E, 900m asl., in mixed forest dominated by Quercus aquifolioides Rehder & E. H. Wilson; 15 July 2014, leg. T25530 (RITF2994).
Etymology: The species name refers to its morphological resemblance to Russula swatica.
Discription: Pileus medium-sized to large, 55–70 mm in diameter; initially flat hemispheric when young, soon expanding and infundibuliform with a deeply depressed center after mature; margin incurved, sometimes cracked after mature, striation short and inconspicuous; cuticle smooth, dry, glabrous, peeling to 1/3 of the radius; light orange (5A4) to grayish orange (6B5) near the center, yellowish white (2A2) to light orange (5A4) towards the margin, partially with a purplish to purplish-black hue. Lamellae deccurent, 2–4 mm deep, 8–11 at 1 cm near the pileus margin, white (1A1) to cream; lamellulae present and irregular in length; furcations occasional near the stipe; edge entire and concolor. Stipe 60–70 × 12–15 mm, cylindrical, slightly inflated towards the base, white (1A1) to yellowish white (2A2), exhibiting a purplish hue at the junction with the pileus, medulla initially stuffed when young, becoming hollow after mature. Context 3–5 mm thick in half of the pileus radius, white (1A1), unchanging when bruised, taste mild, odor inconspicuous. Spore print white (1A1) to yellowish white (2A2).
Basidiospores (5.7–)6.0–7.0–8.0(–8.5) × (4.7–)5.1–6.2–7.2(–8.3) μm, Q = 1.03–1.15–1.27(–1.36), subglobose to broadly ellipsoid; ornamentation of medium-sized, dense [9–11(–12) in a 3 μm diameter circle] amyloid warts, 0.2–0.8 μm high, occasionally to frequently fused in short or long chains [2–3(–4) in the circle], occasionally connected by line connections [0–1(–2) in the circle]; suprahilar spot medium-sized, amyloid. Basidia (36.6–)39.8–44.0–48.0(–54.0) × (6.4–)8.5–9.6–10.7(–11.0) μm, mostly 4-spored, sometimes 2- and 3-spored, clavate; basidiola clavate or subcylindrical, ca. 6–9.5 μm wide. Hymenial gloeocystidia on lamellae sides dispersed to moderately numerous, (55.3)60.5–72.4–84.3(97.0) × (10.2)7.7–8.8–9.9(10.5) μm, typically clavate or fusiform, occasionally lanceolate, apically mainly mucronate, occasionally obtuse, often with 4–10 μm long appendage, thin-walled; contents heteromorphous or granulose, turning yellowish brown in SV. Hymenial gloeocystidia on lamellae edges often shorter, (44.8–)52.4–68.3–84.2(–95.8) × (5.5–)7.8–9.6–11.4 μm, typically clavate or subcylindrical, occasionally fusiform, apically mainly obtuse, occasionally mucronate, usually with 3–6 μm long appendage thin-walled; contents heteromorphous, turning reddish black in SV. Marginal cells (15.8–)19.6–24.2–28.8(–35.5) × (3.6–)3.8–5.3–6.7(–9.0) μm, subcylindrical or clavate, sometimes flexuous. Pileipellis orthochromatic in cresyl blue, not sharply delimited from the underlying context, 200–260 μm deep, two-layered; suprapellis 140–180 μm deep, composed of loose, ascending or erect hyphal terminations; subpellis 80–100 μm deep, composed of relatively dense, intricate, 3–5 μm wide hyphae. Hyphal terminations near the pileus margin rarely unbranched, occasionally flexuous, thin-walled; terminal cells (9.2–)12.4–19.3–26.3(–35.2) × (4.3–)4.9–5.6–6.3(–7.0) μm, typically lageniform, occasionally clavate or subcylindrical, apically attenuated or constricted, sometimes obtuse; subterminal cells frequently shorter and wider, ca. 5–7 μm wide, typically unbranched. Hyphal terminations near the pileus center similar; terminal cells (13.7–)16.8–20.7–25.3(–27.7) × (3.7–)4–4.5–5(–6.7) μm, mainly lageniform or subcylindrical, apically attenuated or constricted; subterminal cells often shorter and wider, unbranched, ca. 4–6.5 μm wide. Pileocystidia near the pileus margin always one-celled, (45.5–)49.4–59.5–69.6(–77.4) × (5.8–)6.0–6.8–7.6(–10.0) μm, mainly clavate, occasionally subcylindrical, apically obtuse, occasionally mucronate, sometimes with 3–5 μm long, round or ellipsoid appendage, thin-walled; contents heteromorphous, turning yellowish brown in SV. Pileocystidia near the pileus center often shorter and narrower, always one-celled, (38.3–)42.6–52.6–62.5(–70.8) × (4.8–)5.5–6.3–7.0 μm, thin-walled, mainly clavate or fusiform, apically obtuse or mucronate, sometimes with 3–6 μm long appendage, contents heteromorphous or granulose, turning r yellowish brown in SV.
Additional specimens examined: China, Xizang Autonomous Region, Nyingchi City, Motuo County; 29°22′13.43″ N, 95°26′58.75″ E, 900m asl., in mixed forest dominated by Quercus aquifolioides Rehder & E. H. Wilson; 15 July, 2014, leg. T25531 (RITF2995).
Notes: The integration of morphological characteristics and phylogenetic analysis positions R. subswatica within the subsection Griseinae. In the phylogenetic tree, R. subswatica is clustered with Pakistani species R. swatica, exhibiting strong phylogenetic support with 100% bootstrap support and 1.00 posterior probabilities. However, R. swatica is distinguished from R. subswatica by its light brown to gray pileus, narrower basidiospores (6–8 × 4–6 μm), and often shorter and wider hymenial gloeocystidia (59–70 × 10–15 μm) [40].
Morphologically, R. subswatica exhibits certain similarities with R. medullata Romagn., and R. shawarensis Kiran & Khalid, both of which belong to subsect. Griseinae. However, R. medullata, originally report from Europe, can be distinguished by a lilac, flesh-pink, pinkish brown, and pinkish grey pileus, ochraceous lamellae after mature and ochre spore print [36,41]. The Pakistani species R. shawarensis has a light pinkish brown to grey buff pileus, very rare to absent lamellulae, shorter basidia [(31.5–)34–39.5(–41) × (7–)9–10.5(–11) μm], often larger hymenial gloeocystidia on lamellae sides [(66–)72–92.5(–105) × (9.5–)10–13(–14) μm], longer the terminal cells of the hyphae near both the pileus margin (av. 48.2 × 3.8 μm) and the pileus center (av. 41.5 × 4.2 μm), and longer pileocystidia near the pileus center (av. 58 × 7 μm) [42].

4. Discussion

In this study, we have described and named two new species of Russula subg. Heterophyllinae: R. leucoviridis and R. subswatica. The identification and characterization of these species enhance our understanding of the biodiversity and phylogenetic relationships within the subg. Heterophyllinae in China. These findings highlight the richness and diversity of ectomycorrhizal fungi in the region, significantly contributing to both taxonomic and ecological studies. Russula leucoviridis is notable for its small to medium-sized basidiomata with a cuticle that cracks and breaks into small green patches near the center after mature, a pastel green to green pileus center with light yellow spots, and a white to greenish white pileus margin. In contrast, R. subswatica is characterized by its infundibuliform pileus, a light orange to grayish orange pileus center, and a yellowish white to light orange margin with a purplish to purplish-black hue, and a white to yellowish white stipe that exhibits a purplish hue at the junction with the pileus. These unique morphological characteristics are crucial for differentiating these new species from closely related ones, and their distinctiveness is further confirmed by multi-gene phylogenetic analysis. Phylogenetic studies based on ITS, nrLSU, RPB2, and mtSSU regions, offer robust support for the classification of these species. The assignment of R. leucoviridis to subsection Virescentinae and R. subswatica to subsection Griseinae suggests distinct evolutionary pathways and adaptation strategies within the subg. Heterophyllinae. In conclusion, the combination of molecular evidence and morphological characteristics provides strong support for the recognition and delineation of these new species. The addition of R. leucoviridis and R. subswatica not only enriches the taxonomic diversity of the subgenus but also highlights the ongoing need for meticulous taxonomic research to uncover the hidden diversity of fungi.

Author Contributions

Conceptualization, B.C. and J.L.; methodology, B.C.; software, B.C. and F.Y.; validation, B.C. and J.L.; formal analysis, B.C.; investigation, B.C. and F.Y.; resources, J.L.; data curation, J.L.; writing—original draft preparation, B.C.; writing—review and editing, B.C. and J.L.; visualization, B.C.; supervision, J.L.; project administration, B.C., J.L. and F.Y.; funding acquisition, B.C., J.L. and F.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (No. 32300010, 32370018 and 32101499) and the Guangzhou Science and Technology Plan Project (2024A04J3421).

Data Availability Statement

All data obtained and analyzed in this study have been included in
this article.

Acknowledgments

We are grateful to Qi Zhao (Chinese Academy of Sciences) and Xumeng Jiang (Northwest A&F University) for providing the samples.

Conflicts of Interest

The authors declare no conflict of interests.

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Figure 1. The phylogenetic tree based on the ITS-nrLSU-RPB2-mtSSU dataset. Species within the subgenus Heterophyllidia that exhibit high similarity to our new species, as well as representative species closely related to subsections Griseinae and Virescentinae, were selected. Support values are shown with Bootstrap support (BS) ≥70% and Bayesian Posterior Probabilities (PP) ≥0.95.
Figure 1. The phylogenetic tree based on the ITS-nrLSU-RPB2-mtSSU dataset. Species within the subgenus Heterophyllidia that exhibit high similarity to our new species, as well as representative species closely related to subsections Griseinae and Virescentinae, were selected. Support values are shown with Bootstrap support (BS) ≥70% and Bayesian Posterior Probabilities (PP) ≥0.95.
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Figure 2. Figure 2. Basidiomata and microscopic structures of Russula leucoviridis. (A–D) Basidiomata. (E,F) Basidiospores in SEM (JSM-6510LV). Scale bars: 10mm (A–D), 2 μm (E), 1μm (F).
Figure 2. Figure 2. Basidiomata and microscopic structures of Russula leucoviridis. (A–D) Basidiomata. (E,F) Basidiospores in SEM (JSM-6510LV). Scale bars: 10mm (A–D), 2 μm (E), 1μm (F).
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Figure 3. Microscopic structures of Russula leucoviridis. (A) Basidia. (B) Basidiola. (C) Marginal cells. (D) Hymenial gloeocystidia on lamellae sides. (E) Hymenial gloeocystidia on lamellae edges. Scale bar: 10 μm.
Figure 3. Microscopic structures of Russula leucoviridis. (A) Basidia. (B) Basidiola. (C) Marginal cells. (D) Hymenial gloeocystidia on lamellae sides. (E) Hymenial gloeocystidia on lamellae edges. Scale bar: 10 μm.
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Figure 4. Microscopic structures of Russula leucoviridis. (A) Pileocystidia near the pileus margin. (B) Pileocystidia near the pileus center. (C) Hyphal terminations near the pileus margin. (D) Hyphal terminations near the pileus center. Scale bar: 10 μm.
Figure 4. Microscopic structures of Russula leucoviridis. (A) Pileocystidia near the pileus margin. (B) Pileocystidia near the pileus center. (C) Hyphal terminations near the pileus margin. (D) Hyphal terminations near the pileus center. Scale bar: 10 μm.
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Figure 5. Basidiomata and microscopic structures of Russula subswatica. (A) Basidiomata. (B–D) Basidiospores in SEM (JSM-6510LV). Scale bars: 15mm (A), 1μm (B–D).
Figure 5. Basidiomata and microscopic structures of Russula subswatica. (A) Basidiomata. (B–D) Basidiospores in SEM (JSM-6510LV). Scale bars: 15mm (A), 1μm (B–D).
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Figure 6. Microscopic structures of Russula subswatica. (A) Basidia. (B) Basidiola. (C) Marginal cells. (D) Hymenial gloeocystidia on lamellae sides. (E) Hymenial gloeocystidia on lamellae edges. Scale bar: 10 μm.
Figure 6. Microscopic structures of Russula subswatica. (A) Basidia. (B) Basidiola. (C) Marginal cells. (D) Hymenial gloeocystidia on lamellae sides. (E) Hymenial gloeocystidia on lamellae edges. Scale bar: 10 μm.
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Figure 7. Russula subswatica of Russula subswatica. (A) Pileocystidia near the pileus margin. (B) Pileocystidia near the pileus center. (C) Hyphal terminations near the pileus margin. (D) Hyphal terminations near the pileus center. Scale bar: 10 μm.
Figure 7. Russula subswatica of Russula subswatica. (A) Pileocystidia near the pileus margin. (B) Pileocystidia near the pileus center. (C) Hyphal terminations near the pileus margin. (D) Hyphal terminations near the pileus center. Scale bar: 10 μm.
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Table 1. GenBank accession numbers for the sequences used in the phylogenetic analysis are provided, with newly generated sequences highlighted in bold.
Table 1. GenBank accession numbers for the sequences used in the phylogenetic analysis are provided, with newly generated sequences highlighted in bold.
Taxon Voucher Location ITS LSU RPB2 mtSSU
R. aeruginea AT2003017 Sweden DQ421999 DQ421999 DQ421946 –
R. aeruginea MSCZ2017100110 China MH422576 MK881944 – MK882072
R. albidogrisea TLH18053005 China MN275581 MN839562 MT085637 MN839610
R. albidogrisea RITF1871 China MW397095 MW397128 – MW403841
R. albolutea RITF2653 China MT672478 MW397120 MW411340 MW403833
R. albolutea RITF4460 China MT672475 MW397121 MW411341 MW403834
R. atroglauca SAV F-3019 Slovakia MT738270 MT738248 MT732156 –
R. aureoviridis H17071311 China MN275564 MN839561 MT085636 MN839609
R. aureoviridis H16082612 China KY767809 MK881920 – MK882048
R. crustosa BPL265 USA KT933966 KT933826 KT933898 –
R. cyanoxantha FH 12-201 Germany KR364093 KR364225 KR364341 –
R. cyanoxantha UE29.09.2002-2 France DQ422033 DQ422033 DQ421970 –
R. faustiana FH 2011 BT314 Germany MT738277 MT738253 MT732162 –
R. grisea FH12234 Germany KT934006 KT933867 KT933938 –
R. grisea UE2005.08.16-01 Germany DQ422030 DQ422030 DQ421968 –
R. heterophylla hue103 (TUB) Europe AF418609 AF325309 – –
R. heterophylla UE20.08.2004-2 Sweden DQ422006 DQ422006 DQ421951 –
R. icterina RITF6686 China PP700452 PP700462 PP707790 PP700443
R. icterina RITF6773 China PP700453 PP700463 PP707792 PP700445
R. ilicis 563IC52 Europe AY061682 – – –
R. indoilicis UZ-04-18 India MW547505 – – –
R. junzifengensis HMAS298101 China OR826832 OR826833 OR915864 OR941507
R. junzifengensis HMAS298102 China OR880061 OR880054 OR915865 OR941508
R. leucoviridis RITF3405 China PQ373872 PQ373868 PQ381291 PQ373876
R. leucoviridis RITF3406 China PQ373873 PQ373869 PQ381292 PQ373877
R. luofuensis RITF4708 China MW646975 MW646987 – MW646998
R. luofuensis RITF4714 China MW646977 MW646989 – MW647000
R. maguanensis XHW4765 China MH724918 MH714537 MH939989 OQ586179
R. mariae HCCN19111 South Korea KF361762 KF361812 KF361712 –
R. medullata FH 2011 BT001 Germany MT738280 MT738256 MT732166 –
R. mustelina FH12226 Germany KT934005 KT933866 KT933937 –
R. niveopicta FHMU958 China OP837461 OP837453 – –
R. niveopicta FHMU941 China OP837462 OP837454 – –
R. orientipurpurea SFC20170819-08 South Korea MT017550 – MT199638 MT196926
R. orientipurpurea SFC20170725-37 South Korea MT017548 – MT199639 MT196927
R. pallidula RITF2613 China MH027958 MH027960 MH091698 MW403845
R. pallidula RITF3331 China MH027959 MH027961 MH091699 MW403846
R. parvovirescens SDRM 6280 USA MK532789 – – –
R. perviridis RITF3131 China OR907098 OR907072 OR914548 OR934523
R. perviridis RITF2912 China OR907100 OR907070 OR914547 OR934522
R. phloginea RITF6905 China OR907116 OR907051 OR914555 OR934516
R. phloginea RITF6906 China OR907117 OR907055 OR914558 OR934520
R. prasina HMAS 281232 China MH454351 – – –
R. pseudocrustosa HBAU15015 China MT337520 – – –
R. pseudopunicea BJTC ZH1392 China OP133164 OP133204 OP156853 OP143937
R. pseudopunicea BJTC ZH1389 China OP133163 OP133203 OP156852 OP143936
R. shawarensis LAH36425 Pakistan MT738293 MT738267 MT732176 –
R. shawarensis LAH36426 Pakistan MT738292 MT738268 MT732177 –
R. subbubalina RITF4710 China MW646978 MW646990 – MW647001
R. subbubalina RITF4715 China MW646979 MW646991 – MW647002
R. subpunicea RITF3715 China MN833635 MW397124 MW411344 MW403837
R. subpunicea RITF2648 China MN833638 MW397125 MW411345 MW403838
R. substriata XHW4766 China MH724921 MH714540 MH939992 OQ371394
R. subswatica RITF2994 China PQ373870 PQ373866 – PQ373874
R. subswatica RITF2995 China PQ373871 PQ373867 – PQ373875
R. swatica MH142272016 Pakistan MK389374 – – –
R. variata BPL241 USA KT933959 KT933818 KT933889 –
R. vesca AT2002091 Sweden DQ422018 DQ422018 DQ421959 –
R. vesca BPL284 USA KT933978 KT933839 KT933910 –
R. violeipes SFC20121010-06 South Korea KF361808 KF361858 KF361758 –
R. violeipes SFC20121010-09 South Korea KF361807 KF361857 KF361757 –
R. virescens HJB9989 Belgium DQ422014 DQ422014 DQ421955 –
R. viridicinnamomea RITF3324 China MW397098 MW397130 MW411348 MW403847
R. viridicinnamomea K15091418 China MK049972 MK881938 – MK882066
R. viridirubrolimbata HBAU15011 China MT337526 – – –
R. werneri IB1997/0786 Sweden DQ422021 – – –
R. wulingshanensis BTJC C403 China OP133166 OP133206 OP156855 OP143939
R. wulingshanensis BTJC C399 China OP133165 OP133205 OP156854 OP143938
R. xanthovirens H15060611 China MG786056 MN839560 T085635 MN839608
R. xanthovirens B17091630 China MG786055 MK881928 – MK882056
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