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Research Article
Four new species of Russula subsect. Cyanoxanthinae from China (Russulales, Russulaceae)
expand article infoYanliu Chen‡, Bin Chen‡§, Ruoxi Liang|, Shengkun Wang‡, Mengya An‡, Jinhua Zhang‡, Jingying Liang, Yaxin Wang‡, Xuelian Gao‡, Junfeng Liang‡
‡ Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China
§ Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China
| Northwestern Polytechnical University, Xi’an, China
¶ Longyandong Forest Farm of Guangdong Province, Guangzhou, China

Corresponding author: Junfeng Liang ( jfliang2000@163.com )

Academic editor: Olivier Raspé
© 2024 Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang, Xuelian Gao, Junfeng Liang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Chen Y, Chen B, Liang R, Wang S, An M, Zhang J, Liang J, Wang Y, Gao X, Liang J (2024) Four new species of Russula subsect. Cyanoxanthinae from China (Russulales, Russulaceae). MycoKeys 107: 21-50. https://doi.org/10.3897/mycokeys.107.123304
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Abstract

Four new species of Russula subsect. Cyanoxanthinae, viz. Russula atrochermesina Y.L. Chen & J.F. Liang, R. lavandula Y.L. Chen, B. Chen & J.F. Liang, R. lilaceofusca Y.L. Chen & J.F. Liang and R. perviridis Y.L. Chen, B. Chen & J.F. Liang, from China are proposed, based on morphological and molecular evidence. Russula atrochermesina can be distinguished by its violet pileus with tuberculate-striate margin, distant lamellae that stain greyish-yellow when bruised, basidiospores ornamented by isolated warts, wide hymenial cystidia on lamellae edges, cystidia content negative reaction in sulphovanillin and branched subterminal cells in pileipellis. Russula lavandula has a purplish-white to violet red pileus with a yellow centre, frequently present lamellulae and furcations, stipe often with pale yellow near the base, isolated basidiospores ornamentation and unbranched cuticular hyphal terminations, while R. lilaceofusca is characterised by its lilac brown to dark brown pileus, crowded lamellae with lamellulae and furcations, stipe often turning reddish-yellow when bruised, subreticulate basidiospores ornamentation and clavate hymenial cystidia often with capitate appendage whose contents that change to reddish-black in sulphovanillin. Russula perviridis is characterised by its large basidiomata, smooth pileus surface, frequently present lamellulae and furcations, stipe with yellow-brown tinge, globose to broadly ellipsoid basidiospores with subreticulate ornamentation, long hymenial cystidia that turn greyish-black in sulphovanillin and symbiotic with Quercus semecarpifolia. Phylogenetic analyses, based on multi-gene ITS+LSU+mtSSU+rpb2, indicate that R. atrochermesina, R. lavandula, R. lilaceofusca and R. perviridis are closely related to R. pallidirosea and R. purpureorosea, R. banwatchanensis, R. lakhanpalii and R. nigrovirens, respectively.

Key words

Ectomycorrhiza, edible fungi, morphology, new species, phylogeny

Introduction

Russula Per. is amongst the most ecologically and economically important groups of macrofungi (Looney et al. 2018; Caboň et al. 2019). More than 800 valid species in the genus have been described, but this figure is far below the 2,000 estimated species number (Adamčík et al. 2019). Russula is currently divided into nine subgenera, viz. Russula subg. Archaeae Buyck and V. Hofst., R. subg. Brevipedum Buyck & V. Hofst., R. subg. Compactae (Fr.) Bon, R. subg. Crassotunicatae Buyck & V. Hofst., R. subg. Cremeoochraceae Buyck & X.H. Wang, R. subg. Glutinosae Buyck & X.H. Wang, R. subg. Heterophyllidiae Romagn., R. subg. Malodorae Buyck & V. Hofst. and R. subg. Russula Pers. (Buyck et al. 2018; Buyck et al. 2020; Buyck et al. 2024). The subsect. Cyanoxanthinae Singer belongs to R. subg. Heterophyllidiae and is characterised by the following set of characters: medium to large basidiomata, lamellulae and furcations mostly present, mild to acrid context taste, white to cream spore print, basidiospores with inamyloid suprahilar spots, metachromatic pileipellis in cresyl blue and mainly one-celled pileocystidia. In the wild mushroom markets in south-western China, mushrooms known locally as “Mitangjun” have always been named as R. cyanoxantha (Schaeff.) Fr. However, they comprise several species that differ from R. cyanoxantha. The species diversity in this subsection has remained unresolved for a long time on the Asian continent, including in China. In the past decade, the number of new species described within this subsection from East and Southeast Asia has increased due to the use of DNA sequence information (Zhao et al. 2015; Zhang et al. 2017; Song et al. 2019; Ghosh et al. 2020; Khatua et al. 2021; Crous et al. 2022; Song 2022).

We aim to clarify the species diversity and geographical distribution of the genus Russula in China. From 2012 to 2023, several large-scale fungal surveys were conducted in different regions of China and some interesting specimens were collected. We conducted ITS analyses and found that they belong to the Cyanoxanthinae and are distinct from described species. Subsequently, through morphological comparisons and further multi-gene tree analyses, it was confirmed that they are new species. Here, we propose four new species and provide their morphological descriptions, related illustrations and molecular phylogenetic positions.

Materials and methods

Sampling and morphological studies

The specimens studied in this paper were collected and photographed from five subtropical provinces in China during 2012–2023. Samples were dried at 50 °C and deposited in the Herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF).

Macro-morphological features were described using detailed field notes and photographs of fresh basidiomata. Colour designations followed those in the Methuen Handbook of Color (Kornerup and Wanscher 1981). The description of microscopic morphological features followed the template of Adamčík et al. (2019). All micromorphological features were observed using a ZEISS Imager M2 microscope (Carl Zeiss AG, Oberkochen, Germany) with oil immersion lenses at a magnification of 1000×. Basidiospores were observed and measured in Melzer’s Reagent in side view excluding ornamentation. Other microscopic structures were pretreated in 5% potassium hydroxide (KOH) and then stained with 1% Congo red solution. The pileipellis was examined in cresyl blue for the presence of ortho- or metachromatic reactions (Buyck 1989). Sulphovanillin (SV) was used to determine variation in the cystidia content (Caboň et al. 2017). Measurements of basidiospores are expressed as (Min–)AV-SD–AV–AV+SD(–Max), where Min = the minimum value, Max = the maximum value, AV = average, SD = standard deviation and Q stands for the length/width ratio of the basidiospores. A scanning electron microscope (SEM. JEOL JSM-SU8020) was used to illustrate the structure and ornamentation of the basidiospores.

DNA extraction, amplified and sequenced

A rapid extraction kit for fungal DNA (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to extract total DNA from dried specimens. A total of four nuclear loci including ITS (internal transcribed spacer region of ribosomal DNA), LSU (ribosomal nuclear large subunit), mtSSU (ribosomal mitochondrial small subunit) and rpb2 (second largest subunit of RNA polymerase II) were amplified and sequenced. The ITS regions were amplified with the ITS1/ITS4 primer pairs (White et al. 1990). The LSU regions were amplified with the LR0R/LR5 primer pairs (Vilgalys and Hester 1990). The rpb2 regions were amplified with the fRPB2-6F/fRPB2-7cR primer pairs (Matheny 2005). The mtSSU regions were amplified with the MS1/MS2 primer pairs (White et al. 1990). Amplifications were performed in 25 µl reactions containing 1.5 µl of Taq DNA polymerase (5U/µl, Thermostable DNA polymerase, BBI), 5 µl of 5× PCR Buffer, 2.5 µl of dNTP (200 µmol/l), 3.5 µl MgCl2, 1 µl of each primer (5 µmol/l), 2 µl of DNA template and 8.5 µl of ddH2O.

The thermal cycling conditions for ITS, LSU, rpb2 and mtSSU included an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s (denaturation); 53 °C for 30 s (ITS), 55 °C for 45 s (LSU), 54 °C for 45 s (mtSSU), 56 °C for 1 min (rpb2) (annealing), 72 °C for 1 min (elongation) and a final extension at 72 °C for 10 min. PCR products were purified using Bioteke’s Purification Kit (Bioteke Corporation, Beijing, China) and sequenced with an ABI 3730 DNA analyser using an 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. All sequences used in this study are shown in Table 1.

Table 1.
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GenBank accession numbers for sequences used in this study. The newly-generated sequences are labelled in bold; holotypes are labelled by letter T in parentheses.

Taxon Voucher Location NCBI. No References
ITS LSU rpb2 mtSSU
R. atrochermesina RITF6878 (T) Yunnan, south-western China OR907106 OR907057 OR914538 OR934536 This study
R. atrochermesina RITF6460 Yunnan, south-western China OR907107 OR907056 OR934535 This study
R. banwatchanensis BBH 49228 (T) Thailand MT940813 MT940823 MT965687 Crous et al. (2022)
R. cyanoxantha UE29.09.2002-2 France DQ422033 DQ422033 DQ421970 Buyck et al. (2008)
R. cyanoxantha FH 12–201 Germany KR364093 KR364225 KR364341 De Crop et al. (2017)
R. dinghuensis K15052704 (T) Guangdong, southern China KU863581 MK881922 MK882050 Zhang et al. (2017)
R. dinghuensis RITF6860 Guangdong, southern China OR907119 OR907061 OR914549 OR934507 This study
R. dinghuensis RITF5885 Guangdong, southern China OR907120 OR914550 OR934503 This study
R. dinghuensis RITF5142 Jiangxi, central China OR907123 OR907058 OR914551 OR934502 This study
R. dinghuensis RITF6855 Guangxi, southern China OR907121 OR907060 OR914552 OR934504 This study
R. dinghuensis RITF6740 Guangxi, southern China OR907122 OR907059 OR914553 OR934506 This study
R. dinghuensis RITF6859 Guangdong, southern China OR907124 OR907062 OR914554 OR934505 This study
R. flavobrunnea var. violaceotincta 71/BB 06.050 Madagascar KU237468 KU237754 KU237312 Buyck et al. (2018)
R. fusiformata K15052703 (T) Guangdong, southern China MK049978 MK881942 MK882070 Song (2022)
R. fusiformata RITF6671 Zhejiang, eastern China OR907108 OR907049 OR914567 OR934501 This study
R. fusiformata RITF6487 Jiangxi, central China OR907109 OR914563 OR934500 This study
R. lakhanpalii CAL 1795 (T) India NR_173867
R. lakhanpalii RITF2600 Hubei, central China OR907090 OR907076 OR934525 This study
R. lakhanpalii RITF6973 Yunnan, south-western China OR907089 OR914542 OR934526 This study
R. lakhanpalii RITF6868 Yunnan, south-western China OR907091 OR907078 OR914543 OR934528 This study
R. lakhanpalii RITF6474 Yunnan, south-western China OR907092 OR907077 OR914544 OR934527 This study
R. lakhanpalii RITF6940 Yunnan, south-western China OR907088 OR907079 OR934529 This study
R. langei 450/BB 07.792 France KU237510 KU237796 KU237356 Buyck et al. (2018)
R. lavandula RITF6329 Yunnan, south-western China OR907083 OR907065 OR914534 OR934513 This study
R. lavandula RITF6340 Yunnan, south-western China OR907085 OR907066 OR914535 OR934515 This study
R. lavandula RITF3196 Yunnan, south-western China OR907086 OR907067 OR934512 This study
R. lavandula RITF3282 (T) Yunnan, southwestern China OR907087 OR907068 OR914537 OR934511 This study
R. lavandula RITF6349 Yunnan, south-western China OR907084 OR907069 OR914536 OR934514 This study
R. lilaceofusca RITF6330 (T) Yunnan, south-western China OR907102 OR907075 OR914539 OR934530 This study
R. lilaceofusca RITF2645 Hubei, central China OR907093 OR907074 OR914540 OR934531 This study
R. lilaceofusca RITF2631 Hubei, central China OR914541 This study
R. lilaceofusca RITF3761 Guizhou, south-western China OR907094 This study
R. lilacina MMCR00191 Thailand MT940809 MT940819 MT965685 Crous et al. (2022)
R. lotus HKAS79209 (T) Guangdong, southern China MG214688 MG214695 Li and Deng (2018)
R. lotus RITF3330 Yunnan, south-western China OR907050 This study
R. maguanensis HKAS 102277 (T) Yunnan, south-western China MH724918 MH714537 MH939989 Wang et al. (2019)
R. nigrovirens HKAS 55222 (T) Yunnan, south-western China KP171173 Zhao et al. (2015)
R. nigrovirens RITF6408 Yunnan, south-western China OR907095 OR907073 OR914568 OR934521 This study
R. pallidirosea UTC 00274382 (T) USA NR_153259 Kropp (2016)
R. perviridis RITF3131 (T) Yunnan, south-western China OR907098 OR907072 OR914548 OR934523 This study
R. perviridis RITF2912 Xizang, western China OR907100 OR907070 OR914547 OR934522 This study
R. perviridis RITF6983 Yunnan, south-western China OR907099 This study
R. perviridis RITF6982 Yunnan, south-western China OR907101 OR907071 OR914545 OR934524 This study
R. perviridis RITF6734 Sichuan, south-western China OR914546 This study
R. perviridis RITF3143 Yunnan, south-western China OR907097 This study
R. perviridis RITF6790 Xizang, western China OR907096 This study
R. phloginea CNX530524068 (T) Yunnan, south-western China MK860701 MK860704 MK860708 Song et al. (2019)
R. phloginea RITF6904 Yunnan, south-western China OR907113 This study
R. phloginea RITF6905 Yunnan, south-western China OR907116 OR907051 OR914555 OR934516 This study
R. phloginea RITF6906 Yunnan, south-western China OR907117 OR907055 OR914558 OR934520 This study
R. phloginea RITF6907 Yunnan, south-western China OR907114 OR907052 OR914556 OR934517 This study
R. phloginea RITF6908 Yunnan, south-western China OR907115 OR907054 OR914557 OR934518 This study
R. phloginea RITF6914 Yunnan, south-western China OR907118 OR907053 OR914559 OR934519 This study
R. pseudocyanoxantha CUH AM177 (T) India NR_173166 Khatua et al. (2021)
R. purpureorosea H17050506 (T) Guangdong, southern China MK049976 MK881941 MK882069 Song (2022)
R. purpureorosea RITF6835 Guangdong, southern China OR907104 OR907082 OR914565 OR934533 This study
R. purpureorosea RITF6834 Guangdong, southern China OR907103 OR907081 OR914566 OR934534 This study
R. purpureorosea RITF5886 Guangdong, southern China OR907105 OR907080 OR914564 OR934532 This study
R. purpureoviridis BBH 49226 (T) Thailand MT940817 MT965684 Crous et al. (2022)
R. subpallidirosea K15052818 (T) Guangdong, southern China KU863582 MK881923 MK882051 Zhang et al. (2017)
R. subpallidirosea RITF3219 Yunnan, south-western China OR907111 OR907063 OR914560 OR934508 This study
R. subpallidirosea RITF3343 Yunnan, south-western China OR907112 OR914562 OR934509 This study
R. subpallidirosea RITF6264 Yunnan, south-western China OR907110 OR907064 OR914561 OR934510 This study
R. substriata HKAS 102278 (T) Yunnan, south-western China MH724921 MH714540 MH939992 Wang et al. (2019)
R. variata BPL241 USA KT933959 KT933818 KT933889 Looney et al. (2016)

DNA sequence alignments and molecular phylogenetic analyses

Representative sequences of species belonging to the Cyanoxanthinae were obtained from the GenBank database, based on recent published studies (Crous et al. 2022; Song 2022). Two species of subsect. Substriatinae, R. maguanensis J. Wang, X.H. Wang, Buyck & T. Bau and R. substriata J. Wang, X.H. Wang, Buyck & T. Bau, were used as outgroups. Sequences of four genes were aligned separately using MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/) and the alignment was manually refined using BioEdit v.7.0.9 (Hall 1999).

Phylogenetic analyses, based on a multi-gene matrix, were conducted using both Randomised Accelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI). A partition homogeneity test (PHT) was performed using heuristic searches with PAUP* 4.0a (Swofford 2003) to evaluate incongruence amongst individual genes. The sequences were then concatenated using Phyutility v.2.2 (Smith and Dunn 2008) for multi-gene analyses as no supported conflicts (P < 0.5). The final aligned dataset was submitted to TreeBASE (31001). A mixed-model (partitioned) scheme was used for ML and BI analyses, ITS, LSU, mtSSU and rpb2. The best-fit model of sequence evolution for each gene was estimated using MrModelTest 2.3 (Nylander 2004), based on the Akaike Information Criterion. The best model for the BI analysis of the ITS and LSU datasets was the GTR+I+G model; the SYM+G model was optimal for rpb2; and the GTR+I model was optimal for mtSSU. RaxML 7.0.3 (Stamatakis 2006) and MrBayes v.3.2 (Ronquist et al. 2012) were used to perform the RAxML and BI analyses, respectively. Maximum Likelihood analysis was conducted using non-parametric bootstrapping with 1,000 replicates. Bootstrap support (BS) above 70% (Nuhn et al. 2013) was considered significant. BI analysis was performed using the Metropolis-coupled Markov Chain Monte Carlo method. Four chains were run for 1,500,000 generations and trees were sampled every 100 generations. Other parameters were kept at their default settings. The analysis was terminated when the average standard deviation of the splitting frequency was below 0.01 and the PSRF values for all parameters were near 1. Bayesian posterior probabilities (BPP) were calculated after discarding the first 25% of the samples as burn-in and branches with BPP over 0.95 were considered significantly supported.

Results

Phylogeny

The four-gene dataset (ITS+LSU+mtSSU+rpb2) included 65 samples representing 22 species. The matrix contains a total of 2,786 nucleotide sites, including 652 ITS sites, 896 LSU sites, 516 mtSSU sites and 722 rpb2 sites. The tree topologies of both the RAxML and BI analyses were similar and only the tree inferred by the ML analysis is displayed, but with both BS and BPP values (Fig. 1).

Figure 1. 

The RAxML likelihood tree based on the four-gene dataset (ITS+LSU+mtSSU+rpb2). Support values (BS > 70%, BPP > 0.95) are displayed above or below the branches, with the maximum values of BS (= 100%) and BPP (= 1.0) represented by asterisks (*). The four new species are marked with different colours.

Our molecular phylogenetic analyses show that Cyanoxanthinae can be divided into two major supported lineages and one species from Madagascar. All samples from China fell into major clades and represent 12 species, of which four are new species. The four new species proposed each formed a well-supported branch and were distinct from other related taxa. Russula atrochermesina Y.L. Chen & J.F. Liang, sp. nov. (BS = 99%, BPP = 1.00) was closely related to R. pallidirosea Kropp and R. purpureorosea Y. Song. Russula lavandula Y.L. Chen, B. Chen & J.F. Liang, sp. nov. (100% BS and 1.00 BPP) was sister to R. banwatchanensis Sommai, Pinruan, Somrith. & Luangsa-ard (80% BS and 0.97 BPP). Russula lilaceofusca Y.L. Chen & J.F. Liang, sp. nov. (BS = 96%, BPP = 1.00) was allied to R. lakhanpalii (90% BS and 1.00 BPP). Russula perviridis Y.L. Chen, B. Chen & J.F. Liang, sp. nov. (BS = 97%, BPP < 0.95) was close to R. nigrovirens Q. Zhao, Y.K. Li & J.F. Liang (BS = 83%, BPP < 0.95).

Taxonomy

Russula atrochermesina Y.L. Chen & J.F. Liang, sp. nov.

MycoBank No: 851267
Figs 2A–C, 3A–D, 4, 5

Diagnosis

Russula atrochermesina can be distinguished by its violet pileus with tuberculate-striate margin, distant lamellae that stain greyish-yellow when bruised, wide hymenial cystidia on lamellae edges, cystidia content negative reaction in sulphovanillin, branched subterminal cells in pileipellis.

Holotype

China, Yunnan Province, Chuxiong Yi Autonomous Prefecture, Lufeng City, Tuoan Township, 25°14'35"N, 101°46'23"E, alt. 2250 m, 7 Sep 2023, Y.L. Chen (RITF6878).

Etymology

‘atrochermesina’ refers to the dark purple colour of pileus.

Description

Basidiomata medium to large-sized; pileus 75–115 mm in diameter, initially hemispherical when young, convex to applanate when mature, margin tuberculate-striate, not cracked; surface dry, glabrous, peeling readily, violet white (16A2) to pale violet (16A3), dark violet (16F6) in the centre. Lamellae adnate, 5–8 per cm near pileus margin, cream, staining greyish-yellow (2B5) when bruised; lamellulae present and irregular in length; furcations frequently present near the stipe; edge entire and concolorous. Stipe 80–170 × 25–40 mm, cylindrical to subcylindrical, white (1A1), often with yellowish-white (1A2) tinge, solid. Context white (1A1), unchanging when bruised, compact, 5 mm thick in half of the pileus radius; taste mild; odour inconspicuous. Spore print not observed.

Figure 2. 

SEM photos of basidiospores of the four new species A–C Russula atrochermesina (RITF6878, holotype) D–F Russula lavandula (RITF6329) G–I Russula lilaceofusca (RITF6330, holotype) J–L Russula perviridis (RITF3131, holotype). Scale bars: 20 µm (G); 10 µm (A, B, D, E, H, J, K); 5 µm (C, F, I, L).

Basidiospores (6.3–)6.7–7.2–7.6(–8.7) × (5.3–)5.8–6.2–6.5(–7.4) µm, Q = (1.05–)1.11–1.15–1.20(–1.25), subglobose to broadly ellipsoid, hyaline in 5% KOH; ornamentation of small to medium, dense (5–9 in a 3 μm diam. circle) amyloid warts, less than 0.6 μm high, mostly isolated or fused in pairs, occasionally fused by short lines; suprahilar plage inamyloid. Basidia (33.0–)37.0–43.0–48.5(–52.0) × (9.0–)10.0–11.5–12.5(–14.0) µm, clavate, 2- to 4-spored, thin-walled; basidiola clavate, ca. 8.0–12.5 µm wide. Hymenial cystidia on lamellae sides moderately numerous, (52.5–)55.0–60.5–65.5(–69.5) × (7.0–)8.0–8.5–9.5(–10.0) µm, fusiform, apically acute, usually with an appendage, thin-walled; contents heteromorphous, yellow in sulphovanillin. Hymenial cystidia on lamellae edges longer and wider than those on lamellae sides, (54.5–)58.0–65.0–71.5(–75.5) × (8.0–)9.0–10.5–11.5(–13.0) µm, fusiform, apically mostly acute, usually with an appendage, thin-walled; contents crystalline, yellow in sulphovanillin. Marginal cells undifferentiated. Pileipellis hyphae of all tissues metachromatic in cresyl blue, sharply delimited from the underlying context, two-layered, gelatinised; suprapellis 260–400 µm deep, composed of densely arranged and erect hyphal terminations; subpellis 150–200 µm deep, composed of horizontally orientated and 2–9 μm wide hyphae. Hyphal terminations near the pileus margin branched, thin-walled; terminal cells (9.0–)13.5–18.0–22.5(–27.5) × (2.5–)3.5–4.0–5.0 µm, mainly clavate, occasionally cylindrical, apically mostly obtuse; subterminal cells usually shorter, ca. 3.0–5.5 µm wide, branched. Hyphal terminations near the pileus centre slightly narrower than those near the pileus margin; terminal cells (10.0–)13.5–17.5–21.5(–26.5) × (2.5–)3.0–3.5–4.0(–5.0) µm, cylindrical, slightly attenuated. Pileocystidia near the pileus margin always 1-celled, (22.5–)28.5–36.0–44.0(–50.0) × (4.0–)4.5–5.0–6.0(–6.5) µm, dispersed, clavate, occasionally fusiform, apically usually obtuse, sometimes with a round or elliptical appendage, thin-walled; contents crystalline, no reaction in sulphovanillin. Pileocystidia near the pileus centre slightly narrower than those near the pileus margin, (30.0–)32.0–38.0–44.0(–51.5) × (3.5–)4.0–4.5–5.0(–5.5) µm.

Figure 3. 

Fruiting bodies photos of four new species A–D Russula atrochermesina (A-C-RITF6878, holotype, D-RITF6460) E–I Russula lavandula (E-F-RITF6329, G-H-RITF6340, I-RITF3282, holotype) J–L Russula lilaceofusca (J-K-RITF6330, holotype, L-RITF2631) M–R Russula perviridis (M-N-RITF3131, holotype, O-P-RITF6982, Q-R-RITF6983). Scale bars: 40 mm (A, B, D–G, I, J, M–O); 5 mm (C, L); 20 mm (H, K, P, Q, R).

Habitat

On the ground under mixed forests dominated by Castanopsis sclerophylla and Pinus yunnanensis.

Known distribution

South-western China (Yunnan Province).

Additional specimens examined

China, Yunnan Province, Chuxiong Yi Autonomous Prefecture, Lufeng City, Tuoan Township, 25°14'34"N, 101°45'39"E, alt. 2300 m, 28 Jul 2022, X.L. Gao (RITF6460).

Figure 4. 

Russula atrochermesina (RITF6878, holotype) A basidia B basidiola C hymenial cystidia on lamellae sides D hymenial cystidia on lamellae edges. Scale bar: 10 µm.

Notes

Phylogenetic analyses showed that R. atrochermesina is related to R. pallidirosea, R. purpureorosea and R. purpureoviridis Khamsuntorn, Lueangjaroenkit, Sommai & Pinruan. However, R. pallidirosea from American Samoa has a pallid to pinkish pileus and smaller hymenial cystidia on the lamellae edges (40.0–55.0 × 5.0–7.0 µm) and is associated with tropical trees (Kropp 2016). Russula purpureorosea differs in the following set of characters: pale pinkish-purple pileus, lamellae furcations absence, narrower hymenial cystidia on lamellae edges (54.0–60.0–95.0 × 7.0–8.5–10.0 µm) and unbranched hyphal terminations in the pileipellis (Song 2022). Russula purpureoviridis, originally described from Thailand, differs in a greyish-green pileus, narrower hymenial cystidia on lamellae edges (45.0–77.5 × 7.5–10.0 µm), constricted terminal cells in the pileipellis and larger pileocystidia (52.5–87.5 × 5.0–8.5 µm) (Crous et al. 2022). The morphology of R. atrochermesina is similar to R. fusiformata. However, R. fusiformata lacks lamellulae and furcations and has narrower hymenial cystidia on lamellae edges (35.5–52.0–78.0 × 5.0–9.0–11.0 µm) and shorter and wider terminal cells near the pileal centre (5.5–14.0 × 3.0–8.0 µm) (Song 2022).

Figure 5. 

Russula atrochermesina (RITF6878, holotype) A hyphal terminations near the pileus margin B hyphal terminations near the pileus centre C pileocystidia near the pileus margin D pileocystidia near the pileus centre. Scale bar: 10 µm.

Russula lavandula Y.L. Chen, B. Chen & J.F. Liang, sp. nov.

MycoBank No: 851264
Figs 2D–F, 3E–I, 6, 7

Diagnosis

Russula lavandula is characterised by its purplish-white to violet red pileus with a yellow centre, frequently present lamellulae and furcations, stipe often with pale yellow near the base, isolated basidiospores ornamentation and unbranched cuticular hyphal terminations. It is mainly distinguished from R. lotus Fang Li by its frequently present lamellulae and furcations, subglobose to broadly ellipsoid basidiospores, moderately numerous and narrower hymenial cystidia on lamellae sides and shorter cuticular terminal cells.

Etymology

‘lavandula’ refers to the colour of its pileus similar to lavender.

Holotype

China, Yunnan Province, Kunming City, Wild Duck Lake, 25°07'34"N, 102°51'42"E, alt. 2100 m, 27 Jul 2014, H.J. Li (RITF3282).

Description

Basidiomata medium-sized; pileus 40–80 mm in diameter, initially hemispherical when young, convex to applanate with a depressed centre after maturity; margin incurved, striation short or inconspicuous, cracked after maturity; surface dry, glabrous, peeling readily, locally cracking into pale yellow (2A3), purplish-white (14A2) to greyish-magenta (13D5 or 14E6) patches, rose (13B3), purplish-white (14A2) to violet red (14B6), sometimes white (1A1) at the margin, yellowish-white (1A2) to golden yellow (4C7) in the centre. Lamellae adnate to slightly adnexed, 10–13 per cm near pileus margin, white (1A1), unchanging when bruised, 3–4 mm wide; lamellulae usually present and irregular in length; furcations frequently present throughout the lamellae; edge entire and concolorous. Stipe 40–60 × 16–25 mm, cylindrical, flexuous and tapering towards the base, white (1A1), often with pale yellow tinge near the base, solid. Context white (1A1), unchanging when bruised, 3–4 mm thick in half of the pileus radius; taste mild; odour inconspicuous. Spore print not observed.

Figure 6. 

Russula lavandula (RITF3282, holotype) A basidia B hymenial cystidia on lamellae sides C hymenial cystidia on lamellae edges D basidiola. Scale bar: 10 µm.

Basidiospores (6.5–)7.1–7.7–8.3(–9.2) × (5.9–)6.4–6.9–7.5(–8.0) µm, Q = (1.01–)1.05–1.11–1.17(–1.24), subglobose to broadly ellipsoid, hyaline in 5% KOH; ornamentation of small, moderately distant to dense (6–8 in a 3 μm diam. circle) amyloid warts, less than 0.5 μm high, mostly isolated, occasionally connected by short line connections or ridges, not forming a reticulum; suprahilar plage indistinct, inamyloid. Basidia (24.0–)28.5–33.5–39.0(–43.5) × (6.0–)7.0–8.5–9.5(–10.5) µm, clavate, 2- to 4-spored, thin-walled; basidiola clavate or subcylindrical, ca. 5–10 µm wide. Hymenial cystidia on lamellae sides moderately numerous, (42.5–)47.0–54.5–62.5(–64.5) × (5.0–)6.5–7.5–8.5 µm, clavate or fusiform, apically mostly obtuse, partially acute, sometimes with a 4–6 µm and papillate appendage, thin-walled; contents granulose or heteromorphous, reddish-black in sulphovanillin. Hymenial cystidia on lamellae edges shorter, but wider than those on lamellae sides, (35.0–)39.0–46.0–52.5(–56.0) × (6.0–)7.0–8.1–9.2(–10.0) µm, mostly clavate or subcylindrical, apically mostly obtuse, occasionally with 2–5 µm long, papillate or moniliform appendage. Marginal cells undifferentiated. Pileipellis only hyphae of suprapellis metachromatic in cresyl blue, sharply delimited from the underlying context, 300–450 µm deep, two-layered, gelatinised; suprapellis 180–200 µm deep, composed of densely arranged and prostrate to erect hyphal terminations; subpellis 140–260 µm deep, composed of horizontally orientated, intricate and 3–5 μm wide hyphae. Hyphal terminations near the pileus margin unbranched, thin-walled, occasionally flexuous; terminal cells (12.5–)14.5–19.5–24.5(–26.0) × 4.5–5.0–5.5(–6.5) µm, mainly cylindrical, occasionally lageniform, apically mostly obtuse; subterminal cells usually shorter and slightly wider, ca. 4–7 µm wide, unbranched. Hyphal terminations near the pileus centre shorter and narrower than those near the pileus margin; terminal cells (11.0–)13.5–17.0–20.5(–21.5) × (3.5–)4.0–4.2–4.5 µm, clavate or cylindrical, apically obtuse; subterminal cells usually wider, ca. 4–6 µm, unbranched. Pileocystidia near the pileus margin always 1-celled, (20.0–)27.0–36.0–45.0(–48.0) × 4.5–5.5–6.0(–6.5) µm, clavate, occasionally fusiform, apically usually obtuse, sometimes with 2–6 µm long, round or elliptical appendage, thin-walled; contents crystalline, reddish-black in sulphovanillin. Pileocystidia near the pileus centre similar to those near the pileus margin, (20.0–)26.0–36.0–45.5(–52.5) × 4.0–5.0–5.5(–6.0) µm.

Figure 7. 

Russula lavandula (RITF3282, holotype) A pileocystidia near the pileus margin B pileocystidia near the pileus centre C hyphal terminations near the pileus margin D hyphal terminations near the pileus centre. Scale bar: 10 µm.

Habitat

On the ground under mixed forests of Pinus yunnanensis, Lithocarpus dealbatus and Quercus spp.

Known distribution

South-western China (Yunnan Province).

Additional specimens examined

China, Yunnan Province, Chuxiong Yi Autonomous Prefecture, Lufeng City, Guangtong Town, 25°14'43"N, 101°45'53"E, 18 Sep 2022, X.L. Gao (RITF6329); ibid, 25°14'48"N, 101°45'28"E, alt. 2400 m, 24 Sep 2022, X.L. Gao (RITF6340); ibid, 25 Sep 2022, X.L. Gao (RITF6349); Kunming City, Wuhua District, Qiongzhu Temple, 25°3'58"N, 102°37'29"E, alt. 2150 m, 28 Jul 2012, Y.J. Hao (RITF3196).

Notes

Russula lavandula is phylogenetically related to Thai R. banwatchanensis and Indian R. pseudocyanoxantha Paloi, K. Acharya & S. Khatua (Fig. 1). However, R. lavandula can be easily distinguished from them by its cracked pileus. Moreover, R. banwatchanensis differs in its darker coloured pileus, lack of lamellulae, thick-walled basidia and often longer pileocystidia of 42.5–127.0 × 2.5–5.0 µm (Crous et al. 2022) and R. pseudocyanoxantha differs in its darker coloured pileus, lack of lamellae furcations and association with Shorea robusta (Khatua et al. 2021). Russula lavandula can be easily confused with four Chinese species, via. R. lotus, R. phloginea J. Song & J.F. Liang, R. purpureorosea and R. subpallidirosea J.B. Zhang and L.H. Qiu in the field. However, R. lotus, originally described from southern China, can be easily distinguished by its absence of lamellae furcations, broadly ellipsoid to ellipsoid basidiospores, dispersed and wider hymenial cystidia on lamellae sides (52.0–70.0 × 10.0–16.0 μm) and longer cuticular terminal cells (10.0–40.0 × 4.0–8.0 μm) (Li and Deng 2018). Russula phloginea, which occurs in subalpine areas, differs in having lamellae furcations only present near the stipe, smaller basidiospores of (6.0–)6.5–8.0 × 5.0–6.5 μm, longer hymenial cystidia of (48.0–)60.0–78.5(–79.5) × 7.5–9.5(–10.0) μm and pileocystidia with a moniliform apex (Song et al. 2019). Russula purpureorosea and R. subpallidirosea, can be distinguished from R. lavandula by their rosy brown, pale pinkish-purple or pale greyish-pink pileus centre and occurrence at low altitudes (Zhang et al. 2017; Song 2022). Besides, R. purpureorosea lacks lamellae furcations and has shorter terminal cells of the pileipellis (6.5–15.5 × 2–5.5 μm) and wider pileocystidia (17.0–53.0 × 4.5–9.0 μm) (Song 2022). In addition, R. cyanoxantha, can be confused with R. lavandula. However, R. cyanoxantha can be distinguished by its uncracked pileus cuticle, longer hymenial cystidia up to 100 µm and slender cuticular hyphal end cells of 2–3 µm (Bon 1988; Sarnari 1998).

Russula lilaceofusca Y.L. Chen & J.F. Liang, sp. nov.

MycoBank No: 851265
Figs 2G–I, 3J–L, 8, 9

Diagnosis

Russula lilaceofusca is mainly characterised by its lilac brown to dark brown pileus, crowded lamellae with the presence of lamellulae and furcations, stipe often turning reddish-yellow when bruised, subreticulate basidiospores ornamentation and clavate hymenial cystidia often with capitate appendage whose contents change to reddish black in sulphovanillin. It can differ from R. cyanoxantha in shorter hymenial cystidia and wider cuticular terminal cells and differ from R. fusiformata in frequently present lamellulae and furcations, clavate hymenial cystidia on lamellae edges that have no reaction in sulphovanillin and occasionally branched cuticular hyphal terminations.

Holotype

China, Yunnan Province, Chuxiong Yi Autonomous Prefecture, Lufeng City, G30).

Etymology

‘lilaceofusca’ refers to a lilac brown pileus.

Description

Basidiomata medium-sized; pileus 40–60 mm in diameter, initially hemispherical when young, convex to applanate after mature; margin incurved, no striation, not cracked; surface dry, glabrous, reddish-white (8A2), lilac (15B4), brown (7E5) to dark brown (7F5). Lamellae adnate, very crowded, 20–24 per cm near pileus margin, cream, unchanging when bruised; lamellulae present and irregular in length; furcations frequently present; edge entire and concolorous. Stipe 35–50 × 8–17 mm, cylindrical to subcylindrical, slightly expanded towards the base, white (1A1), staining reddish-yellow (4A6) when touched, solid. Context white (1A1), unchanging when bruised, soft; taste mild; odour inconspicuous. Spore print not observed.

Figure 8. 

Russula lilaceofusca (RITF6330, holotype) A hymenial cystidia on lamellae sides B basidiola C marginal cells D basidia E hymenial cystidia on lamellae edges. Scale bar: 10 µm.

Basidiospores (6.5–)7.0–7.8–8.7(–10.5) × (5.5–)6.1–6.9–7.7(–9.4) µm, Q = (1.05–)1.09–1.13–1.17(–1.26), subglobose to broadly ellipsoid; ornamentation of small to medium, dense (5–9 in a 3 μm diam. circle) amyloid warts, less than 0.5 μm high, subreticulate, connected by short line connections or ridges; suprahilar plage large, inamyloid. Basidia (26.5–)30.5–38.5–47.0(–56.0) × (9.5–)10.5–12.5–14.0(–15.5) µm, clavate or ellipsoid, 1- to 4-spored, thin-walled; basidiola clavate or ellipsoid, ca. 8.0–13.0 µm wide. Hymenial cystidia on lamellae sides dispersed to moderately numerous, (39.5–)43.5–48.5–53.5(–60.0) × (8.0–)9.5–10.5–11.5(–12.0) µm, mostly clavate, apically mostly obtuse, often with a 2.0–4.5 µm round appendage, thin-walled; contents granulose or heteromorphous, reddish-black in sulphovanillin. Hymenial cystidia on lamellae edges shorter and narrower than those on lamellae sides, (31.0–)36.0–41.5–47.0(–54.0) × (7.0–)7.5–8.5–10.0(–10.5) µm, mostly clavate, apically mostly obtuse, occasionally with a 2–3 µm long round or elliptical appendage, thin-walled; contents heteromorphous, reddish-black in sulphovanillin. Marginal cells (13.5–)15.0–18.5–22.0(–26.0) × 4.0–5.0–5.5(–6.5) µm, lageniform, clavate or subcylindrical. Pileipellis only hyphae of suprapellis metachromatic in cresyl blue, sharply delimited from the underlying context, two-layered, gelatinised; suprapellis 130–200 µm deep, composed of loosely arranged and erect hyphal terminations; subpellis 90–200 µm deep, composed of horizontally orientated and intricate hyphae. Hyphal terminations near the pileus margin occasionally branched, thin-walled, often flexuous; terminal cells (11.0–)12.5–18.5–24.0(–32.0) × (3.0–)3.5–4.0–5.0(–5.5) µm, mainly lageniform or cylindrical, apically obtuse, sometimes attenuated or constricted; subterminal cells usually longer and slightly wider, ca. 3.5–7.0 µm wide, occasionally branched. Hyphal terminations near the pileus centre slightly shorter than those near the pileus margin; terminal cells (9.5–)11.5–16.0–20.0(–25.5) × (3.0–)3.5–4.0–5.0(–5.5) µm, lageniform or cylindrical, apically obtuse, sometimes attenuated or constricted; subterminal cells usually equal or slightly wider, ca. 2.8–6.6 µm, occasionally branched. Pileocystidia near the pileus margin always 1-celled, (23.5–)26.5–35.0–43.5(–51.0) × (3.5–)4.0–4.5–5.5 µm, subfusiform or cylindrical, apically usually obtuse, always with 2–3 µm long, round or elliptical appendage, thin-walled; contents heteromorphous, reddish-black in sulphovanillin. Pileocystidia near the pileus centre similar to those near the pileus margin, (30.5–)34.5–39.5–44.5(–53.0) × 3.5–4.0–4.5(–5.5) µm.

Figure 9. 

Russula lilaceofusca (RITF6330, holotype) A hyphal terminations near the pileus margin B hyphal terminations near the pileus centre C pileocystidia near the pileus margin D pileocystidia near the pileus centre. Scale bar: 10 µm.

Habitat

On the ground of broad-leaved forests dominated by Quercus spp. or mixed forests with Pinus spp.

Known distribution

Central (Hubei Province) and south-western China (Guizhou and Yunnan Provinces).

Additional specimens examined

China, Guizhou Province, Zunyi City, Kuankuoshui National Nature Reserve, 28°14'29"N, 107°11'59"E, alt. 1750 m, 27 Sep 2014, H.J. Li (RITF3761); Hubei Province, Shennongjia Forest District, Jianglongping, 31°25'46"N, 110°20'18"E, alt. 1500 m, 9 Aug 2015, Y.K. Li (RITF2645); ibid, 11 Aug 2015, Y.K. Li (RITF2631).

Note

Phylogenetic analyses showed that R. lilaceofusca was closely related to the Indian species R. lakhanpalii and R. variata from the United States. However, R. lakhanpalii differs in having a yellowish-white to pale yellow areolate pileus with orange-brown centre, cystidia that show no change in sulphovanillin and longer and wider pileocystidia of (27.0–)40.0–63.0–86.5(–123.0) × (4.0–)4.5–5.5–6.5(–7.0) μm near the pileus margin (Ghosh et al. 2020). Russula variata differs in having a green pileus and basidiospores with higher warts of 0.4–1.0 µm (Hessler 1960; Burge 1979). In morphology, Chinese R. fusiformata Y. Song can be easily confused with this species. However, R. fusiformata can be distinguished by its striate pileus margin, absence of lamellulae and furcations, fusiform hymenial cystidia on the lamellae edges, cystidia that show a negative reaction in sulphovanillin and unbranched terminal cells in the pileipellis (Song 2022). Russula lilaceofusca is similar to R. cyanoxantha, but R. cyanoxantha has a greenish-violet pileus, longer hymenial cystidia up to 100 μm and slender cuticular hyphal end cells of 2–3 µm (Romagnesi 1967; Bon 1988; Sarnari 1998).

Russula perviridis Y.L. Chen, B. Chen & J.F. Liang, sp. nov.

MycoBank No: 851266
Figs 2J–L, 3M–R, 10, 11

Diagnosis

Russula perviridis is characterised by its large basidiomata, smooth pileus surface, frequently present lamellulae and furcations, a coarser stipe with yellow-brown tinge, globose to broadly ellipsoid basidiospores with locally reticulate ornamentation, long hymenial cystidia that turn greyish-black in sulphovanillin and is symbiotic with Quercus semecarpifolia. It differs from R. dinghuensis in longer and wider hymenial cystidia on lamellae edges, subreticulate basidiospores ornamentation and is associated with Quercus semecarpifolia. It differs from R. nigrovirens in frequently present furcations, subreticulate basidiospores ornamentation, longer and wider hymenial cystidia and related host plants.

Holotype

China, Yunnan Province, Diqing Tibetan Autonomous Prefecture, Shangri La County, Bitahai Nature Reserve, 27°49'42"N, 99°59'30"E, alt. 3600 m, 20 Aug 2014, Q. Zhao (RITF3131).

Etymology

‘perviridis’ refers to a dark green pileus.

Description

Basidiomata large-sized; pileus 90–150 mm in diameter, initially hemispherical to when young, applanate with a depressed centre when mature; margin usually incurved, striation short or inconspicuous, cracked when mature; surface dry, smooth and glabrous, peeling readily, greyish-green (27E5–30C5) to dark green (30F5), sometimes yellow-brownish (2C5) near the centre, greyish-yellow (2C3) or greenish-white (26A2) at the margin. Lamellae adnate to subfree, 9–12 per cm near pileus margin, white to cream, staining yellowish-brown (5E6) when bruised, 6–8 mm wide; lamellulae present and irregular in length; furcations frequently present near the stipe to half of the lamellae; edge entire and concolorous. Stipe 70–90 × 20–40 mm, cylindrical, white (1A1) with yellow-brown (2C5) tinge, solid. Context white (1A1), unchanging when bruised, 4–9 mm thick in half of the pileus radius, relatively soft; taste mild; odour inconspicuous. Spore print not observed.

Figure 10. 

Russula perviridis (RITF3131, holotype) A basidia B basidiola C marginal cells D hymenial cystidia on lamellae sides E hymenial cystidia on lamellae edges. Scale bar:10 µm.

Basidiospores (5.9–)7.1–7.7–8.3(–9.7) × (5.2–)6.1–6.8–7.4(–8.4) µm, Q = (1.00–)1.07–1.14–1.21(–1.31), globose to broadly ellipsoid, hyaline in 5% KOH; ornamentation of small, dense (5–10 in a 3 μm diam. circle) amyloid warts, less than 0.5 μm high, reticulate, connected by short line connections or ridges; suprahilar plage large, inamyloid. Basidia (26.0–)34.5–42.0–59.0(–55.0) × (8.0–)9.5–11.0–12.0(–13.0) µm, clavate, 2- to 4-spored, thin-walled; basidiola clavate, ca. 6–11 µm wide. Hymenial cystidia on lamellae sides moderately numerous, (56.5–)62.5–69.5–77.0(–81.5) × (6.5–)7.0–7.5–8.0 µm, fusiform or clavate, apically mostly obtuse, sometimes acute, thin-walled; contents heteromorphous, greyish-black in sulphovanillin. Hymenial cystidia on lamellae edges similar to those on lamellae sides, (45.0)54.0–64.5–74.5(–79.5) × 6.0–7.5–8.5(–10.0) µm, mostly clavate, occasionally fusiform, apically mostly obtuse, sometimes with 3–5 µm long appendage, thin-walled; contents granulose or heteromorphous, greyish-black in sulphovanillin. Marginal cells (23.0–)24.5–27.5–33.5 × (4.5–)5.5–6.0–7.0 µm, clavate or subcylindrical, occasionally flexuous. Pileipellis only hyphae of suprapellis weakly metachromatic in cresyl blue, sharply delimited from the underlying context, 300–360 µm deep, two-layered, not gelatinised; suprapellis 180–200 µm deep, composed of erect and densely arranged hyphal terminations; subpellis 140–180 µm deep, composed of horizontally orientated, intricate and 3–5 μm wide hyphae. Hyphal terminations near the pileus margin occasionally branched, thin-walled, sometimes flexuous; terminal cells (17.0–)20.0–25.5–31.5(–38.0) × 3.0–4.0–4.5(–6.0) µm, usually clavate or subcylindrical, apically tapering; subterminal cells usually shorter, ca. 4–6 µm wide, occasionally branched. Hyphal terminations near the pileus centre shorter than those near the pileus margin; terminal cells (13.0–)14.5–20.5–26.5(–32.0) × 3.5–4.0–4.5 µm. Pileocystidia near the pileus margin always 1-celled, (36.5–)40.5–47.5–55.0(–60.0) × (3.0–)3.5–4.0–4.5 µm, fusiform or subcylindrical, apically usually obtuse, sometimes with 2–6 µm long, round or elliptical appendage, thin-walled; contents heteromorphous or granulose, greyish-black in sulphovanillin. Pileocystidia near the pileus centre similar to those near the pileus margin, (26.5–)28.0–36.0–44.0(–50.5) × 3.0–3.5–4.5 µm.

Figure 11. 

Russula perviridis (RITF3131, holotype) A pileocystidia near the pileus margin B pileocystidia near the pileus centre C hyphal terminations near the pileus margin D hyphal terminations near the pileus centre. Scale bar: 10 µm.

Habitat

On the ground under broad-leaved forests dominated by Quercus semecarpifolia.

Known distribution

South-western (Sichuan and Yunnan Provinces) and western China (Xizang Autonomous Region).

Additional specimens examined

China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, Daocheng County, Yading Village, 4 Aug 2022, X.L. He (RITF6734); Yunnan Province, Diqing Tibetan Autonomous Prefecture, Deqin County, Wudi Lake, 27°48'47"N, 99°42'50"E, alt. 3300 m, 11 Sep 2023, Y.L. Chen & J.Y. Liang (RITF6982, 6983); Shangri La City, Bita Sea Scenic Area, 27°49'39"N, 99°59'28"E, alt. 3600 m, 15 Jul 2014, Q. Zhao (RITF3143); Xizang Autonomous Region, Linzhi City, Motuo County, 29°22'13"N, 95°26'59"E, alt. 1800 m, 15 Jul 2014, Q. Zhao (RITF2912); Lhasa City, Mozhugongka County, Zhaxigang Township, 29°42'10"N, 92°4'44"E, alt. 4500 m, 7 Aug 2023, Y. Zhang (RITF6790).

Notes

Phylogenetic analyses showed that R. perviridis is related to two European species, R. langei Bon and R. cyanoxantha and the Chinese species R. nigrovirens. However, R. langei has a violaceous pileus, a white stipe with a lilac tinge and narrower cuticular hyphal terminations (2.0–3.0 μm) (Bon 1988), whereas R. cyanoxantha has non-reticulate basidiospore ornamentation and longer hymenial cystidia up to 100 μm (Bon 1988; Sarnari 1998). Russula nigrovirens differs in having patches on the pileus surface, furcations rarely present near the stipe, basidiospore ornamentation that does not form a reticulum and smaller hymenial cystidia (46.0–55.0 × 6.5–8.5 μm) on the lamellae edges (Zhao et al. 2015). Morphologically, R. perviridis has a green pileus as in Chinese R. dinghuensis J.B. Zhang & L.H. Qiu. However, R. dinghuensis differs in its shorter and narrower hymenial cystidia on lamellae edges (45.0–52.0 × 4.0–6.0 μm) and isolated basidiospore warts (Zhang et al. 2017). Furthermore, R. perviridis can be clearly distinguished from similar Cyanoxanthinae species in its habitat. Russula perviridis is distributed in subalpine areas (over 2,000 m) and is associated with Quercus semecarpifolia. However, R. nigrovirens is associated with Picea spp., Rhododendron spp., Sorbus spp. and Abies spp. and R. dinghuensis is distributed in low-altitude areas (below 1,000 m).

Key to known species in R. subsect. Cyanoxanthinae in Asia

1 Lamellulae absent or rare 2
– Lamellulae irregularly inserted, but frequent (more than usual in subg. Heterophyllidiae) 4
2 Association with Pinus merkusii 3
– Association with Fagaceae R. fusiformata
3 Lamellae and context changing pale purplish-pink when bruised, hymenial cystidia on lamellae edges numerous and short (35.0–57.5 × 10.0–10.5 μm) R. lilacina
– Lamellae and context unchanging when bruised, hymenial cystidia on lamellae edges dispersed to moderately numerous and long (45.0–77.5 × 7.5–10.0 μm) R. purpureoviridis
4 Basidiospores ornamentation mainly subreticulate to reticulate 5
– Basidiospores ornamentation mainly isolated 7
5 Lamellae instant (9–12/cm at pileus margin), average length of hymenial cystidia on lamellae edges over 50 μm R. perviridis
– Lamellae crowed to very crowed (14–27/cm at pileus margin), average length of hymenial cystidia on lamellae edges less than 50 μm 6
6 Hymenial cystidia on lamellae sites short and wide of (39.5–)43.5–48.5–53.5(–60.0) × (8.0–)9.5–10.5–11.5(–12.0) µm, clavate, pileocystidia near the pileus margin short and narrow [(23.5–)26.5–35.0–43.5(–51.0) × (3.5–)4.0–4.5–5.5] µm R. lilaceofusca
– Hymenial cystidia on lamellae sites short and wide of (45–)47.2–548.5–61.8(–75) × (6–)6.6–7.3–7.9(–8) µm, cylindrical to subclavate to fusiform, pileocystidia near the pileus margin long and wide [(27–)39.8–63.1–86.5(–123) × (4–)4.4–5.4–6.4(–7)] µm R. lakhanpalii
7 Lamellae furcations absent or rare 8
– Lamellae furcations irregularly inserted, but frequent (more than usual in subg. Heterophyllidiae) 12
8 Appearing in subalpine forest dominated by Picea spp., Rhododendron spp., Sorbus spp. and Abies spp. R. nigrovirens
– Appearing in evergreen broad-leaved forest dominated by Shorea robusta, Lithocarpus corneus, Quercus spp. 9
9 Hymenial cystidia on lamellae edges narrow (not exceeding 7 µm wide) R. dinghuensis
– Hymenial cystidia on lamellae edges wide (most cells exceeding 7 µm wide) 9
10 Basidiospores broadly ellipsoid to ellipsoid, occasionally subglobose R. lotus
– Basidiospores subglobose to broadly ellipsoid 11
11 Lamellae adnate, context unchanging when bruising or with FeSO4, hymenial cystidia on lamellae edges short (36–64.5 × 7.2–10.8 µm), pileocystidia short and narrow (18.2–32 × 3.5–5.4 µm) R. pseudocyanoxantha
– Lamellae adnexed to subdecurrent, context changing light yellow when bruising or with FeSO4, hymenial cystidia on lamellae edges long (54–60–95 × 7–8.5–10 µm), pileocystidia short and narrow (17–53 × 4.5–9 µm) R. purpureorosea
12 Basidiospores globose to subglobose (Q < 1.15) R. banwatchanensis
– Basidiospores subglobose to ellipsoid (Q = 1.15–1.45) 13
13 Basidiospores broadly ellipsoid to ellipsoid (Q = 1.30–1.45) R. subpallidirosea
– Basidiospores subglobose to broadly ellipsoid (Q = 1.15–1.30) 14
14 Pileocystidia often with moniliform appendage R. phloginea
– Pileocystidia with capitate appendage 15
15 Pileal margin tuberculate-striate, lamellae distant (5–8/cm near the pileus margin), hymenial cystidia on lamellae edges long and wide [(54.5–)58.0–65.0–71.5(–75.5) × (8.0–)9.0–10.5–11.5(–13.0)] µm, fusiform, cuticular hyphal terminations branched R. atrochermesina
– Pileal margin indistinctly or not striate, lamellae slightly crowded (10–13/cm near the pileus margin), hymenial cystidia on lamellae edges short and narrow [(35.0–)39.1–45.8–52.5(–56.0) × (6.0–)7.0–8.1–9.2(–10.0)] µm, mostly clavate or subcylindrical, cuticular hyphal terminations unbranched R. lavandula

Discussion

Our multi-gene phylogenetic analyses (Fig. 1) indicate that species with white spore print, basidiospores ornamented by warts with inamyloid suprahilar spot, metachromatic pileipellis and one-celled pileocystidia constitute Cyanoxanthinae. The analyses also suggest that Cyanoxanthinae can be divided into two major lineages, which may be distinguishable by the connections of basidiospores ornamentation. Clade 1 represented by R. lilaceofusca and R. perviridis has more connections between the warts. Clade 2 represented by R. lavandula and R. atrochermesina has more isolated warts. Additional studies are need to determine the critical point for the number or density of connections. There are no other morphological differences that can clearly correspond to these two lineages.

We recommend several main characteristics that can be used to distinguish species within Cyanoxanthinae. The first, lamellulae and furcations are crucial characters in our view. Except for R. fusiformata having completely regular lamellae, the remaining species in Cyanoxanthinae have inserted lamellulae or furcations. The density of lamellulae and furcations may require our attention in future research. The second, the species of Cyanoxanthinae have different shape of basidiospores (Q values). Four Chinese species, R. dinghuensis, R. nigrovirens, R. lotus and R. subpallidirosea have ellipsoid basidiospores (Q > 1.3), while other species have broadly ellipsoid basidiospores (Q < 1.3).

The Cyanoxanthinae is a widely distributed group, ranging from tropical forests to temperate regions and have formed ectomycorrhizal relationships with various host plants. All known species come from the Northern Hemisphere, except for one species from Madagascar. Australia is an unexplored continent for studying this group. For the level of species, there seems to be restricted life zones and specific hosts. For example, we found that R. perviridis is only distributed in the subalpine zone and only forms a symbiotic relationship with Quercus semecarpifolia. The European R. cyanoxantha is well-known to many people in China. Despite our team conducting surveys in the southern provinces of China (subtropical regions) for over a decade, we have not found it there. This means it is a strictly temperate species.

Currently, it is known that there are 16 species in the Cyanoxanthinae distributed in Asia, including the four new species described in this article. These species are all found from tropical or subtropical regions and, so far, there have been no new species found from temperate Asia. In view of the wide distribution of members of the subsection in Europe, more attention needs to be paid to temperate Asia.

Amongst the 12 known species in China, we can only confirm that R. phloginea is edible and more support is needed for the edibility of the remaining species.

Acknowledgements

We are very grateful to the following people for providing us with examined specimens: Dr Y.J. Hao (Anhui Agricultural University), Dr X.L. He (Sichuan Academy of Agricultural Sciences), Dr H.J. Li (The Chinese Center for Disease Control and Prevention), Dr Q. Zhao (Kunming Institute of Botany, Chinese Academy of Sciences) and Ms Y. Zhang (China Telecom Corp Ltd).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was supported by the National Natural Science Foundation of China (No. 32370018 and 32300010) and the National Non-profit Institute Research Fund of the Chinese Academy of Forestry (CAFYBB2019ZA001).

Author contributions

Writing the original draft: Yanliu Chen; improving the manuscript: Junfeng Liang; providing fund projects: Junfeng Liang and Bin Chen; collecting studied specimens: Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang and Xuelian Gao. All authors have read and agreed to the published version of the manuscript.

Author ORCIDs

Yanliu Chen https://orcid.org/0000-0002-8054-2517

Bin Chen https://orcid.org/0000-0001-8459-3752

Shengkun Wang https://orcid.org/0000-0002-2487-6344

Mengya An https://orcid.org/0009-0001-6170-5886

Jinhua Zhang https://orcid.org/0000-0001-8523-644X

Jingying Liang https://orcid.org/0000-0003-4111-4859

Yaxin Wang https://orcid.org/0009-0002-1302-5154

Xuelian Gao https://orcid.org/0009-0006-6059-4877

Junfeng Liang https://orcid.org/0000-0002-0433-2577

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

ITS

Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang, Xuelian Gao, Junfeng Liang

Data type: fas

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (39.52 kb)
Supplementary material 2 

LSU

Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang, Xuelian Gao, Junfeng Liang

Data type: fas

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (45.81 kb)
Supplementary material 3 

RPB2

Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang, Xuelian Gao, Junfeng Liang

Data type: fas

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (33.38 kb)
Supplementary material 4 

SSU

Yanliu Chen, Bin Chen, Ruoxi Liang, Shengkun Wang, Mengya An, Jinhua Zhang, Jingying Liang, Yaxin Wang, Xuelian Gao, Junfeng Liang

Data type: fas

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (23.46 kb)
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