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

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 pastel-green to green pileus center with light-yellow spots, a white to greenish-white pileus margin, and a cuticle that cracks and breaks into small green patches after maturation, whereas R. subswatica is distinguished by an infundibuliform pileus with a deeply depressed center after maturation, 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 conducted 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 provide new scientific data that enhance the comprehension of species diversity within the subgenus Heterophyllinae in China.

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 1100 described species, with estimates suggesting the existence of at least 3000 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. [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 the maintenance of the 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, mild to strongly acrid taste, mostly white or cream basidiospore print, basidiospores with inamyloid or partly amyloid suprahilar spots, mostly abundant gloeocystidia that are typically mucronate to obtuse-rounded, one-celled dermatocystidia, and pileipellis without primordial hyphae [1,14]. 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 the 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. The color codes primarily adhered to the standards set by Kornerup and Wanscher [23]. A microscopic examination of the specimen sections was carried out using a ZEISS Imager M2 microscope (Carl Zeiss AG, Oberkochen, Germany). 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, JEOL Ltd., Tokyo, Japan). 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]; and 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 the phylogenetic analyses, species from the subgenus Heterophyllidia that exhibit high similarity to our new species were selected, along with representative species that were 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, and 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. GenBank accession numbers for the sequences used in the phylogenetic analysis are provided. T denotes holotype.

Taxon	Voucher	Location	ITS	LSU	RPB2	mtSSU	Reference
R. aeruginea	AT2003017	Sweden	DQ421999	DQ421999	DQ421946	–	[34]
R. aeruginea	MSCZ2017100110	China	MH422576	MK881944	–	MK882072	Unpublished
R. albidogrisea	TLH18053005	China	MN275581	MN839562	MT085637	MN839610	[19]
R. albidogrisea	RITF1871	China	MW397095	MW397128	–	MW403841	[35]
R. albolutea	RITF2653 (T)	China	MT672478	MW397120	MW411340	MW403833	[35]
R. albolutea	RITF4460	China	MT672475	MW397121	MW411341	MW403834	[35]
R. atroglauca	SAV F-3019	Slovakia	MT738270	MT738248	MT732156	–	[36]
R. aureoviridis	H17071311	China	MN275564	MN839561	MT085636	MN839609	Unpublished
R. aureoviridis	H16082612 (T)	China	KY767809	MK881920	–	MK882048	[37]
R. chlorina	HBAU 15024 (T)	China	MT505888	–	–	–	[38]
R. crustosa	BPL265	USA	KT933966	KT933826	KT933898	–	[39]
R. cyanoxantha	FH 12-201	Germany	KR364093	KR364225	KR364341	–	[40]
R. cyanoxantha	UE29.09.2002-2	France	DQ422033	DQ422033	DQ421970	–	[34]
R. faustiana	FH 2011 BT314	Germany	MT738277	MT738253	MT732162	–	[36]
R. grisea	FH12234	Germany	KT934006	KT933867	KT933938	–	[39]
R. grisea	UE2005.08.16-01	Germany	DQ422030	DQ422030	DQ421968	–	[34]
R. heterophylla	hue103 (TUB)	Europe	AF418609	AF325309	–	–	[41]
R. heterophylla	UE20.08.2004-2	Sweden	DQ422006	DQ422006	DQ421951	–	[34]
R. icterina	RITF6686 (T)	China	PP700452	PP700462	PP707790	PP700443	[22]
R. icterina	RITF6773	China	PP700453	PP700463	PP707792	PP700445	[22]
R. ilicis	563IC52	Europe	AY061682	–	–	–	[42]
R. indoilicis	UZ-04-18 (T)	India	MW547505	–	–	–	[43]
R. junzifengensis	HMAS298101 (T)	China	OR826832	OR826833	OR915864	OR941507	[44]
R. junzifengensis	HMAS298102	China	OR880061	OR880054	OR915865	OR941508	[44]
R. leucoviridis	RITF3405 (T)	China	PQ373872	PQ373868	PQ381291	PQ373876	This work
R. leucoviridis	RITF3406	China	PQ373873	PQ373869	PQ381292	PQ373877	This work
R. luofuensis	RITF4708 (T)	China	MW646975	MW646987	–	MW646998	[18]
R. luofuensis	RITF4714	China	MW646977	MW646989	–	MW647000	[18]
R. maguanensis	XHW4765	China	MH724918	MH714537	MH939989	OQ586179	[45]
R. mariae	HCCN19111	South Korea	KF361762	KF361812	KF361712	–	[46]
R. medullata	FH 2011 BT001	Germany	MT738280	MT738256	MT732166	–	[36]
R. mustelina	FH12226	Germany	KT934005	KT933866	KT933937	–	[39]
R. niveopicta	FHMU958 (T)	China	OP837461	OP837453	–	–	[20]
R. niveopicta	FHMU941	China	OP837462	OP837454	–	–	[20]
R. orientipurpurea	SFC20170819-08	South Korea	MT017550	–	MT199638	MT196926	[47]
R. orientipurpurea	SFC20170725-37 (T)	South Korea	MT017548	–	MT199639	MT196927	[47]
R. pallidula	RITF2613	China	MH027958	MH027960	MH091698	MW403845	[48]
R. pallidula	RITF3331	China	MH027959	MH027961	MH091699	MW403846	[48]
R. parazurea	MF01.10.2003	Europe	DQ422007	DQ422007	DQ421945	–	[34]
R. parazurea	TUB nl1370	Europe	AF418611	–	–	–	[41]
R. parvovirescens	SDRM 6280	USA	MK532789	–	–	–	Unpublished
R. perviridis	RITF3131	China	OR907098	OR907072	OR914548	OR934523	[21]
R. perviridis	RITF2912	China	OR907100	OR907070	OR914547	OR934522	[21]
R. phloginea	RITF6905	China	OR907116	OR907051	OR914555	OR934516	[21]
R. phloginea	RITF6906	China	OR907117	OR907055	OR914558	OR934520	[21]
R. prasina	HMAS 281232 (T)	China	MH454351	–	–	–	[49]
R. pseudocrustosa	HBAU15015 (T)	China	T337520	–	–	–	[13]
R. pseudomodesta	CMUB40005 (T)	Thailand	OQ957419	OQ968300	–	–	[50]
R. pseudomodesta	SDBR-CMUSOU33	Thailand	OQ957482	OQ968301	–	–	[50]
R. pseudopunicea	BJTC ZH1392 (T)	China	OP133164	OP133204	OP156853	OP143937	[51]
R. pseudopunicea	BJTC ZH1389	China	OP133163	OP133203	OP156852	OP143936	[51]
R. shawarensis	LAH36425	Pakistan	MT738293	MT738267	MT732176	–	[36]
R. shawarensis	LAH36426	Pakistan	MT738292	MT738268	MT732177	–	[36]
R. subalpinogrisea	KD 18-33 (T)	India	MK253444	–	–	–	[52]
R. subbubalina	RITF4710 (T)	China	MW646978	MW646990	–	MW647001	[18]
R. subbubalina	RITF4715	China	MW646979	MW646991	–	MW647002	[18]
R. subpunicea	RITF3715 (T)	China	MN833635	MW397124	MW411344	MW403837	[35]
R. subpunicea	RITF2648	China	MN833638	MW397125	MW411345	MW403838	[35]
R. substriata	XHW4766	China	MH724921	MH714540	MH939992	OQ371394	[45]
R. subswatica	RITF2994 (T)	China	PQ373870	PQ373866	–	PQ373874	This work
R. subswatica	RITF2995	China	PQ373871	PQ373867	–	PQ373875	This work
R. swatica	MH142272016 (T)	Pakistan	MK389374	–	–	–	[53]
R. variata	BPL241	USA	KT933959	KT933818	KT933889	–	[39]
R. vesca	AT2002091	Sweden	DQ422018	DQ422018	DQ421959	–	[34]
R. vesca	BPL284	USA	KT933978	KT933839	KT933910	–	[39]
R. violeipes	SFC20121010-06	South Korea	KF361808	KF361858	KF361758	–	[46]
R. violeipes	SFC20121010-09	South Korea	KF361807	KF361857	KF361757	–	[46]
R. virescens	HJB9989	Belgium	DQ422014	DQ422014	DQ421955	–	[34]
R. viridicinnamomea	RITF3324	China	MW397098	MW397130	MW411348	MW403847	[54]
R. viridicinnamomea	K15091418 (T)	China	MK049972	MK881938	–	MK882066	[19]
R. viridirubrolimbata	HBAU15011	China	MT337526	–	–	–	[13]
R. werneri	IB1997/0786	Sweden	DQ422021	–	–	–	[34]
R. wulingshanensis	BTJC C403 (T)	China	OP133166	OP133206	OP156855	OP143939	[51]
R. wulingshanensis	BTJC C399	China	OP133165	OP133205	OP156854	OP143938	[51]
R. xanthovirens	H15060611 (T)	China	MG786056	MN839560	MT085635	MN839608	[55]
R. xanthovirens	B17091630	China	MG786055	MK881928	–	MK882056	[55]

. Initial sequence alignment was conducted using the online version of MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/) (accessed on 15 September 2024). To ensure reliable and accurate results, manual evaluations and adjustments were carried out in BioEdit as necessary [33]. The maximum likelihood (ML) and Bayesian analysis (BA) methods were employed for 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 datasets produced consistent tree topologies, with only the ML tree depicted in Figure 1. The 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 (PPs) of 1, while the monophyly of the remaining five 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 the Pakistani species R. swatica Sarwar & Hanif, demonstrating robust phylogenetic support with 100% bootstrap support and 1.00 posterior probabilities.

3.2. Taxonomy

Russula leucoviridis B. Chen & J. F. Liang, sp. nov. (Figure 2, Figure 3, Figure 4 and Figure 5).

MycoBank: MB855842.

Diagnosis: Pastel-green to green pileus center with light-yellow spots, white to greenish-white pileus margin, a cracking cuticle forming small green patches near the center after maturation, crowded towards the center, and distinct ITS data among all known species of subsect. Virescentinae.

Holotype: China, Hainan Province, Wuzhishan City, Shuiman Town, Wuzhi Mountain National Reserve; 18°51′59.50″ N, 109°40′30.59″ E, 800 m asl., in evergreen broad-leaved forest; 23 June 2016, leg. JXM105 (RITF3405).

Etymology: Leuco (Latin) = white; viridis (Latin) = green; the species name refers to the 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 maturation; 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 white particles near the center when young, cracking and broken into small green patches near the center after maturation, 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 whole 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 maturation. 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]; small suprahilar spot, 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, 800 m 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. Despite this close relationship, the ITS sequence similarity is 97.30% between R. leucoviridis and R. prasina and 96.27% between R. leucoviridis and R. xanthovirens. Furthermore, 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 [49]; 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 aged, whitish lamellae with a reddish hue, white stipe with a 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) [55].

Given the 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) [56]. 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) [57]. 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 × 7−10 μm), larger hymenial gloeocystidia on the lamellae sides (78–107 × 8–10 μm), and absence of hymenial gloeocystidia on the lamellae edges [13,58]. 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 the lamellae sides [(59–)63–69–75(–80) × (7–)8–9.5–11(–13) μm], and absence of hymenial gloeocystidia on the lamellar edges [59].

Russula subswatica B. Chen & J. F. Liang, sp. nov. (Figure 6, Figure 7, Figure 8 and Figure 9).

MycoBank: MB855843.

Diagnosis: Light-orange to grayish-orange pileus center, yellowish-white to light-orange pileus margin, partially with a purplish to purplish-black hue, white to yellowish-white stipe, exhibiting a purplish hue at the junction with the pileus, and distinct ITS data among all known species of subsect. Griseinae.

Holotype: China. Xizang Autonomous Region, Nyingchi City, Motuo County; 29°22′13.43″ N, 95°26′58.75″ E, 900 m 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.

Description: 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 maturation; margin incurved, sometimes cracked after maturation, 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 decurrent, 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 whole 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 maturation. 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 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, 900 m 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 the Pakistani species R. swatica, exhibiting strong phylogenetic support with 100% bootstrap support and 1.00 posterior probabilities. However, the ITS sequence similarity is 98.73% between R. subswatica and R. swatica. Additionally, R. swatica is distinguished from R. subswatica by its light-brown to gray pileus, whitish stipe with a light-brown base, narrower basidiospores (6–8 × 4–6 μm), and often shorter and wider hymenial gloeocystidia (59–70 × 10–15 μm) [53].

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 reported to be from Europe, can be distinguished by a lilac, flesh-pink, pinkish-brown and gray-olive pileus center, greenish-gray pileus margin, ochraceous lamellae after maturation, ochraceous or dark-ochre spore print, and the association with Populus [56,60]. The Pakistani species R. shawarensis has a light-pinkish-brown to gray 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 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) [36].

Furthermore, several species within the subsection Griseinae, originally described from Asia, are also worthy of attention. The two Chinese species, R. atroaeruginea G.J. Li, Q. Zhao & H.A. Wen and R. chlorina G.J. Li & Chun Y. Deng, are clearly distinct from R. subswatica. Russula atroaeruginea has a dark-green pileus with a radial yellowish tinge and basidiospores with thin, low, and fine ornamentations [61]. Russula chlorina has a pale-yellowish-green pileus, absence of lamellulae, and smaller basidiospores [(5.3–)5.6–6.9(–7.3) × (4.6–)4.9–5.9 μm] [38]. The two species (R. sikkimensis K. Das, Atri & Buyck and R. subalpinogrisea K. Das, I. Bera, A. Ghosh & Buyck) from India are also obviously different from our species. Russula sikkimensis has a parrot-green pileus, absence of lamellulae, subreticulate basidiospores ornamentation, and an association with Abies densa Griff. [62]. Russula subalpinogrisea exhibits larger basidiomata (95–130 mm) and a vivid-green to deep-green pileus [52].

4. Discussion

In this study, we have described and named two new species of Russula subg. Heterophyllinae: R. leucoviridis and R. subswatica. The findings enhance our understanding of the biodiversity and phylogenetic relationships within the subg. Heterophyllinae in China. Russula leucoviridis is notable for its pastel-green to green pileus center with light-yellow spots, a white to greenish-white pileus margin, and a cuticle that cracks and breaks into small green patches after maturation. 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. Based on the morphological characteristics and phylogenetic analyses, R. leucoviridis and R. subswatica are classified into subsect. Virescentinae and subsect. Griseinae, respectively [1,56].

In the phylogenetic analysis, R. leucoviridis is found to be closely related to R. prasina and R. xanthovirens. Nonetheless, there are notable differences in their ITS sequences and morphological characteristics [49,55]. In subsect. Virescentinae, R. virescens, R. parvovirescens, and R. viridirubrolimbata exhibit green pileus and cracked cuticles that are similar to those of R. leucoviridis [57,58]. However, their ITS sequences differ significantly from those of our species, resulting in their separate placement in the phylogenetic tree. Russula subswatica is closely related to R. swatica, but there are some differences in their ITS sequences and morphological characteristics [53]. 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 enriches the taxonomic diversity of the subg. Heterophyllinae.