Clarifying the identity of marketed edible fungus Huotanjun (Burnt Charcoal Mushroom) in southern China

doi:10.21203/rs.3.rs-3107016/v1

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Research Article

Clarifying the identity of marketed edible fungus Huotanjun (Burnt Charcoal Mushroom) in southern China

https://doi.org/10.21203/rs.3.rs-3107016/v1

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published 09 Nov, 2023

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Huotanjun (Burnt Charcoal Mushroom) is a group of edible mushrooms widely hunted and eaten in southern China. Taxonomically they belong to Russula sect. Nigricantinae. Confusion with the fatal R. subnigricans cause many poisoning incidents in Asia. For a long time, European names were used in Asia but the Asian genotypes are not the same as European species. We studied specimens collected from markets and the field in southern China. Analysis of four DNA loci (ITS, nc LSU, TEF1-α, RPB2) showed that except for a specimen of R. adusta from subalpine Abies forest in southwestern China, all the specimens represent species different from European and North American ones, suggesting prevalent continental endemism in this group. Four new species were described. Among them R. sinoadusta and R. zhuzuijun are Asian counterparts of R. adusta; R. brunneocystidia and R. huotanjun are closely related with European R. densifolia and R. fuliginosa. These species are highly similar to their European siblings and the morphological differences are very subtle. Using multi-locus data, for the first time, we determined the phylogenetic position of R. subnigricans. It was nested in R. sect. Nigricantinae and sister to R. latolamellata, a species also having distant lamellae and only reddening context. Geographically except for R. sinoadusta known only from China, all species are shared by China and Japan. We analyzed presence/absence of the diagnostic toxin of R. subnigricans cycloprop-2-ene carboxylic acid for 12 species and did not detect the polymeric products in any of them but R. subnigricans. Nevertheless, considering several potentially edible species also have spaced lamellae and only reddening discolouration which can be easily confused with R. subnigricans, we strongly recommend to only eat species with decidedly blackening context and gills.

edibility

molecular phylogeny

4 new species

Russula

Russula subgen. Compactae

Russula is one of the most widespread and commonly eaten genera and more than 100 species of the genus were used as edible mushrooms in 28 countries, both in the northern and southern Hemispheres (Boa 2004). The most notable species include those in the R. virescens, R. cyanoxantha, R. delica, R. densifolia, R. edulis, R. griseocarnosa, R. rosacea, and R. xerampelina complexes (Arora 1986; Buyck 1994; Härkönen et al. 2003; Boa 2004; Manassila et al. 2005; Wang et al. 2009; Yang et al. 2021, 2022). These species taxonomically cover five of the eight subgenera recognized by Buyck et al. (2018, 2020). In China, people in the south have a keener interest in hunting and eating Russula than those in the north. The top three favoured mushrooms in southern China are “Qingtoujun”(Green Head Mushroom), “Dahongjun” (Big Red Mushroom) and “Huotanjun” (Burnt Charcoal Mushroom) (Li et al. 2010; Deng et al. 2020; Wang 2020; investigation on Chinese markets by the authors).

The main problem concerning the use of wild edible mushroom in developing countries is naming and recognizing species (Boa 2004). In Asia, European and American names are often applied to their look-alikes, but these Asian look-alikes, especially those occurring in the subtropical and tropical parts, often turn out to be different when DNA sequence data are used and morphology is studied in details. Most DNA sequences published by Manassila et al. (2005) have low similarity to those from the European materials despite the same names. Wang et al. (2009) and Li et al. (2010), using DNA sequence data found that none in the “Dahongjun” complex is contaxic with European R. vinosa, a name that had been used for Chinese species for nearly half a century. Naming most terminal clades that lack matching names of R. subgen. Compactae in Shimono et al. (2014) is still pending. Although Park et al. (2014) named one of the clades of Shimono et al. (2014) (“B-3”) as American R. eccentrica, the clear genetic diversification from authentic R. eccentrica suggests a different species, most likely sister to R. ochrobrunnea described by Zhou et al. (2020) very recently.

Among the three most famous edible Russula groups, correct species recognition in “Huotanjun” has particular importance. “Huotanjun” is a group of mushrooms that are characterized by the context, lamellae and stipe changing to blackish after bruised or cut (thus comes the local name), blackish brown pileus, and presence of lamellulae, taxonomically corresponding to R. sect. Nigricantinae of R. subgen. Compactae. Although quite some species of Russula are poisonous (Moser 1983; Arora 1986; Boa 2004), one of the look-alikes of “Huotanjun”, R. subnigricans is the only lethal species known in the genus, causing deaths from rhabdomyolysis in East Asia (Hongo 1955; Chen et al. 2016). Poisoning incidents by R. subnigricans have happened quite many times in Asia due to confusion between the edible species in the field (Li et al. 2020, 2021, 2022). During a few investigations on poisoning incidents by one of us (HJL), a species that is similar to R. adusta and R. subnigricans was found but the edibility needs to be determined. To solve the naming problem and answering the edibility of species under this local name, this study gathered samples both from markets and the field and use DNA sequence data and morphological characters to re-assess the real identifies of the species in this edible category. Presence of the diagnostic toxin of R. subnigricans, i.e. cycloprop-2-ene carboxylic acid identified by Matsuura et al. (2009) was detected for each phylogenetic species involved using mass spectrometry to evaluate their edibility.

Sampling

Specimens were collected from six provincial regions in central and southern China, i.e. Guizhou, Hubei, Hunan, Sichuan, Tibet and Yunnan. 20 specimens were gathered from markets under the name “Huotanjun” (partly marked in Fig. 1). Seven specimens were obtained from the patients when retrieving possible material responsible for poisoning incidents (partly marked in Fig. 1). Specimens are deposited at the Cryptogamic section (HKAS), Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and Sichuan Academy of Agricultural Sciences, China (SAAS).

ITS sequences of all specimens were amplified and analyzed. Sequences with ≥ 97% similarity and query cover ≥ 90% (covering ITS1, 5.8S, and ITS-2 regions) to our sequences in GenBank were retrieved by BLASTn, including those both from fruiting bodies and environmental samples. In the multi-locus data, for the four new species, we filtered our specimens to a minimum sampling that was able to cover geographical origins and genetic variations in the ITS region. We basically followed the sampling of De Lange et al. (2021) to assemble our multi-locus dataset. To minimize the negative effect of missing data in phylogenetic analysis, we excluded three samples with only ITS in the multi-locus dataset of De Lange et al. (2021), i.e. cantharellicola UC1999420, R. cascadensis OSC 1064009 and R. ingwa MEL2101936. The multi-locus data of R. dissimulans BPL704 was supplemented with sequences retrieved from JGI (http://genome.jgi.doe.gov). The TEF1-α of R. ambusta holotype SAV F-3358 (MW273313), which was not listed in Table 1 of De Lange et al. (2021), was added. We added R. ochrobrunnea, R. nigrocarpa and the famous lethal mushroom R. subnigricans to enrich the sampling and infer the relationships between edible and poisonous species. To test the phylogenetic position of the Chinese sample HMAS 265004 that was represented by an ITS sequence KX441086 in De Lange et al. (2021), we amplified the multigene of this sample and included a contaxic Chinese sample XHW5563. Sequence data were mostly borrowed from De Crop et al. (2017), Buyck et al. (2018), Adamčík et al. (2019), Zhou et al. (2020) and De Lange et al. (2021).

Morphological analysis

Basic morphological methods followed Wang et al. (2021). Specifically, 20 randomly selected spores and cystidia were measured for each individual. Colour changes of cystidium contents were tested using sulfovanillin (SV, Caboň et al. 2017). For photographing of the pileipellis, the section was mounted with 10% KOH and photographed with Digital Camera DFC450 installed in Leica DM 2500 microscope. Microscopic descriptions and terminology followed Adamčík et al. (2019).

DNA extraction and sequencing

DNA extractions and PCR amplification followed Wang et al. (2009, 2012). The four nuclear markers used by De Lange et al. (2021), i.e. ITS, nc LSU, TEF1-α and RPB2 were amplified. General primers published by White et al. (1990), Liu et al. (1999), Moncalvo et al. (2000) and Morehouse et al. (2003) and specific primers by Cao et al. (2023) were used. For sequences having heterozygous INDELs or ambiguous sites, PCR products were sequenced bidirectionally to make contigs of the amplified regions or verify the ambiguous sites. Flot et al.’s (2006) method was used to determine the INDEL heterozygosity and phase the sequences into two copies. In a few cases, when amplification products were either faint or direct phasing was impossible, PCR products were cloned with the Takara^® pMD^TM18T cloning kit (Dalian, China) following the manufacturer’s instructions. Colonies were screened for the presence of the desired products with primers M13F and M13R. At least two clones with desired PCR products were sequenced. Raw sequences were assembled with Sequencher v4.1.4 (Gene Codes Corporation, Ann Arbor). PCR amplification and Sanger sequencing of four specimens of R. nigrocarpa and R. ochrobrunnea partly failed. Therefore, lower coverage whole genome sequencing (“genome skimming”), using the next generation sequencing by Illumina NovoSeq-5500 platform, was conducted to recover the target loci for these samples. Genomic DNAs were extracted using a CTAB protocol and library preparation followed the protocol described by Zeng et al. (2018). The raw data were de novo assembled using GetOrganelle toolkit (Jin et al. 2020). The target regions were extracted from assemblies using the reference sequences of their sister species. All sequences generated were deposited in NCBI GenBank, with accessions OP673407–OP673489, OP680804–OP680928, OP723358–OP723361, OP723363–OP723366, OP723477–OP723480, OP846960–OP846963, OR117141–OR117151, OR123712–OR123715, OR123717–OR123719, OR144417–OR144418, OR178753–OR178756 (partly shown in Table 1).

Table 1

Sequence data used for multi-locus phylogenetic analysis in this study. Sequences generated in this study are in bold. Samples marked with “*” were sequenced using next generation sequencing. HT: holotype, ET: epitype
Species	Voucher specimen	Origin	GenBank accession
Species	Voucher specimen	Origin	ITS	nc LSU		TEF1-α	RPB2
R. acrifolia	LD 16–022 (GENT)	Sweden	MW172319	MW182479		MW273325	MW306683
R. acrifolia	RDL 18 − 012 (GENT)	Sweden	MW172320	MW182480		MW273326	MW306684
R. adusta	LD 16–025 (GENT)	Sweden	MW172316	–		MW273322	MW306682
R. adusta	RDL 18–020 (GENT)	Sweden	MW172317	MW182477		MW273323	–
R. adusta	XHW10592 (HKAS 125828)	Tibet, China	OP680885	OP680818		OP673422	OP673464
R. albonigra	F-3465(SAV)	Slovakia	MW172293	MW182460		MW273309	MW306669
R. albonigra	F-20197 (SAV) ET	Slovakia	MW172299	MW182464		MW273312	MW306673
R. ambusta	FH 2008 ST01 (F. Hampe herbarium)	Germany	MW172300	MW182465		–	–
R. ambusta	F-3358 (SAV) HT	Slovakia	MW172301	MW182466		MW273313	–
R. cf. anthracina	RDL 16–031 (GENT)	Italy	MW172313	MW182474		MW273319	MW306679
R. cf. anthracina	RDL 18–026 (GENT)	Sweden	MW172315	MW182476		MW273321	MW306681
R. archaeosuberis	BB 12.085 (PC)	Italy	KY800355	KU237593		KU238019	KU237878
R. atramentosa	FH 2011-002R (F. Hampe herbarium)	Belgium	MW172322	MW182482		MW273328	MW306685
R. atramentosa	FH 0170824-02 (F. Hampe herbarium)	Germany	MW172324	MW182483		MW273329	MW306687
R. ashihoi	XHW6550 (HKAS 117128)	Sichuan, China	OP680916	OP680835		OP673439	OP673481
R. ashihoi	XJW6 (HKAS 89701)	Sichuan, China	OP680917	OP680836		OP673440	OP673482
R. ashihoi	XHW5762 (HKAS 116489)	Tibet, China	OP680918	OP680837		OP673441	OP673483
R. ashihoi	JW641 (HKAS 111395)	Yunna, China	OP680919	OP680838		OP673442	OP673484
R. brunneocystidia	XHW3875 (HKAS 96464)	Yunnan, China	OP680872 OP680873	OP680812		OP673415	OP673458
R. brunneocystidia	SHLDS2 (HKAS 125830) HT	Yunnan, China	OP680874 OP680875	OP680813		OP673416	OP673459
R. brunneocystidia	XHW3811 (HKAS 95394)	Yunnan, China	OP680876 OP680877	OP680814		OP673417	OP673460
R. brunneocystidia	XHW3817 (HKAS 95388)	Yunnan, China	OP680878 OP680879	OP680815		OP673418	OP673461
R. brunneocystidia	XHW3807 (HKAS 95390)	Yunnan, China	OP680880	OP680816		OP673419 OP673420	OP673462
R. brunneocystidia	XHW4117 (HKAS 96706)	Yunnan, China	OP680881 OP680882	OP680817		OP673421	OP673463
R. camarophylla	MPG11-7-09 (PC)	Spain	KY800353	KU237579		KU238006	KU237865
R. cortinarioides	BB 07.133 (PC) HT	USA	KP033485	KP033496		–	KP033507
R. cortinarioides	BB 07.103 (PC)	USA	KP033480	KP033491		KU237985	KP033502
R. densifolia	RDL 16 − 001/2 (GENT)	Belgium	MW172325	MW182484		MW273330	MW306688
R. densifolia	RDL 17–024 (GENT)	Belgium	MW172327	MW182486		MW273332	MW306690
R. densissima	FH 2014 ST04 (F. Hampe herbarium)	Germany	MW172328	–		–	MW306691
R. densissima	FH 2010 ST02 (F. Hampe herbarium)	Germany	MW172329	–		–	MW306692
R. dissimulans	BPL704 (TENN)	USA	KY848513		JGI project 110523
R. aff. earlei	XHW4597 (HKAS 104722)	Yunnan, China	OP680928	OP680844		OP673447	OP673489
R. cf. eccentrica	BB 07.044 (PC)	USA	KP033479	KP033490		KU237937	KP033501
R. cf. eccentrica	BB 07.132 (PC)	USA	KP033478	KP033489		KU237926	KP033500
R.cf. fuliginosa	FH RUS 14091201 (F. Hampe herbarium)	Slovakia	MW172331	MW182488		MW273334	MW306694
R. cf. fuliginosa	FH RUS 14091001 (F. Hampe herbarium)	Slovakia	MW172330	MW182487		MW273333	MW306693
R. gossypina	BB 06.002 (PC)	Madagascar	KY800350	KU237450		KU237886	KU237736
R. huotanjun	SPW523 (HKAS 94938)	Yunnan, China	OP680845 OP680846	OP680804		OP673407	OP673448
R. huotanjun	XHW4576 (HKAS 104882)	Yunnan, China	OP680847 OP680848	OP680805		OP673408	OP673449
R. huotanjun	XHW3809 (HKAS 95392)	Yunnan, China	OP680849 OP680850	OP680806		OP673409	OP673450
R. huotanjun	XHW3829 HT (HKAS 96418)	Yunnan, China	OP680851 OP680852	OP680807		OP673410	OP673451 OP673452
R. huotanjun	XHW3808 (HKAS 95391)	Yunnan, China	OP680853 OP680854	OP680808		OP673411	OP673453
R. huotanjun	XHW4104 (HKAS 96693)	Yunnan, China	OP680855 OP680856	OP680809		OP673412	OP673454 OP673455
R. huotanjun	XHW4954 (HKAS 104899)	Yunnan, China	OP680857 OP680858	OP680810		OP673413	OP673456
R. huotanjun	HF1127 (HKAS 125834)	Hubei, China	OP680859 OP680860	OP680811		OP673414	OP673457
R. kangchenjungae	KVP 09-106 (GENT) HT	India	KR364129	JN389004		–	JN375607
R. kangchenjungae	XHW5641 (HKAS 116346)	Tibet, China	OP680927	OP680843		OP673446	OP673488
R. lateriticola	BB 06.031 (PC)	Madagascar	KP033476	KP033487		KU237888	KP033498
R. latolamellata	GDGM 79563	Guangdong, China	MN275545	MN839557		MT085604	MT085632
R. latolamellata	GDGM 79561 HT	Guangdong, China	MN275543	MK881933		MT085574	MK880660
R. latolamellata	FL912 (HKAS 78216)	Guangdong, China	OP680923 OP680924	OP680839 OP680840		OP673443	OP673485
R. nigrocarpa*	GDGM 79720 HT	Guangdong, China	OP723363	OP723358		OP723477	OP846960
R. nigrocarpa*	FL1002 (HKAS 78247)	Guangdong, China	OP723364	OP723359		OP723478	OP846961
R. nigricans	RDL 17 − 004 (GENT)	Belgium	MW172332	MW182489		MW273335	MW306695
R. nigricans	RDL 17 − 005 (GENT)	Belgium	MW172333	MW182490		MW273336	MW306696
R. nigrifacta	RDL 16–044 (GENT) HT	Italy	MW172308	MW182470		MW273317	MW306677
R. nigrifacta	F-1501 (SAV)	Slovakia	MW172302	MW182467		MW273314	MW306674
R. ochrobrunnea*	GDGM 79718 HT	Guangdong, China	OP723365	OP723360		OP723479	OP846962
R. ochrobrunnea*	GDGM 79719	Guangdong, China	OP723366	OP723361		OP723480	OP846963
R. polyphylla	BB 07.134 (PC)	USA	KP033486	KP033497		KU238023	KP033508
R. polyphylla	BB 07.023 (PC)	USA	KP033481	KP033492		KU237986	KP033503
R. reticulofolia	GDGM 79559 HT	Guangdong, China	MN275541	MK881932		MT085573	MK880659
R.roseonigra	FH 2014 ST01 (F. Hampe Herbarium)	Germany	MW172336	–		MW273338	MW306698
R.roseonigra	RDL 16–024 (GENT)	Italy	MW172337	MW182492		MW273339	MW306699
R. sinoadusta	XHW4301 (HKAS 104865) HT	Yunnan, China	OP680886	OP680819		OP673423	OP673465
R. sinoadusta	XHW4114 (HKAS 96703)	Yunnan, China	OP680887	OP680820		OP673424	OP673466
R. sinoadusta	XHW4302 (HKAS 104864)	Yunnan, China	OP680888	OP680821		OP673425	OP673467
R. sinoadusta	XHW4786 (HKAS 104874)	Yunnan, China	OP680889	OP680822		OP673426	OP673468
R. sinoadusta	YNCN20220724-02-03 (HKAS 125833)	Yunnan, China	OP680896	OP680825		OP673429	OP673471
R. sinoadusta	TCGD-002 (HKAS 125839)	Yunnan, China	OP680894	OP680823		OP673427	OP673469
R. sinoadusta	Li 150914-03 (HKAS 125836)	Guzhou, China	OP680895	OP680824		OP673428	OP673470
Russula sp1	XHW5563 (HKAS 112498)	Tibet, China	OP680925	OP680841		OP673444	OP673486
Russula sp1	HMAS 265004	Tibet, China	OP680926	OP680842		OP673445	OP673487
Russula sp2	rpr-663 (HKAS 122413)	Yunnan, China	ON794289	OR144417		OR178753	OR178755
Russula sp2	rml-902 (HKAS 122565)	Yunnan, China	ON794426	OR144418		OR178754	OR178756
Russula sp	RW 1975 (GENT)	Italy	MW172309	MW182471		–	---
Russula sp	FH 12–064 (GENT)	Thailand	MN130076	–		–	MN380517
R. subnigricans	XHW4724 (HKAS 104742)	Yunnan, China	OR117145 OR117146	OR117141 OR117142		OR123712 OR123713	OR123717
R. subnigricans	YNYS20220815-01 (HKAS 128523)	Sichuan, China	OR117147 OR117148	OR117143		OR123714	OR123718
R. subnigricans	YNSP20220906-01 (HKAS 128524)	Yunnan, China	OR117149 OR117150	OR117144		OR123715	OR123719
R. ustulata	SAV F-2610 (SAV)	Italy	MW172310	MW182472		–	–
R. ustulata	AV 16–019 (GENT) HT	Norway	MW172312	MW182473		MW273318	MW306678
R. zhuzuijun	QWQ R121 (HKAS 121154)	Hunan, China	OP680897 OP680898	OP680826		OP673430	OP673472
R. zhuzuijun	XHW3813 (HKAS 95384)	Yunnan, China	OP680899	OP680827		OP673431	OP673473
R. zhuzuijun	XHW3810 (HKAS 95393) HT	Yunnan, China	OP680900	OP680828		OP673432	OP673474
R. zhuzuijun	XHW3885 (HKAS 96474)	Yunnan, China	OP680901	OP680829		OP673433	OP673475
R. zhuzuijun	XHW4115 (HKAS 96704)	Yunnan, China	OP680902	OP680830		OP673434	OP673476
R. zhuzuijun	XHW3814 (HKAS 95385)	Yunnan, China	OP680903	OP680831		OP673435	OP673477
R. zhuzuijun	SAAS 3568	Yunnan, China	OP680904	OP680832		OP673436	OP673478
R. zhuzuijun	XHW3876 (HKAS 96465)	Yunnan, China	OP680905	OP680833		OP673437	OP673479
R. zhuzuijun	XHW4768 (HKAS 104747)	Yunnan, China	OP680906	OP680834		OP673438	OP673480

Phylogenetic analysis

We used two datasets to solve the identities of our specimens and relationships among the species: ITS and the ITS-nc LSU-TEF1-α-RPB2 combined datasets. For the ITS dataset, besides the specimens collected by us, three additional sets of data were included: 1) matches with similarity ≥ 97% and coverage ≥ 90% by BLASTn in GenBank using our queries (with the nc SSU rDNA and nc LSU rDNA ends trimmed). These sequences were filtered to keep the biggest genetic variation of each terminal clade; 2) species with morphological similarities, i.e. R. adusta, R. albonigra, R. densifolia and R. nigricans; (3) available sequences of species in R. sect. Nigricantinae originally described from Asia, if not included in 1). These species are R. ashihoi, R. indonigra, R. latolamellata, R. nigrocarpa and R. subnigricans. Alignment was made by the online version of the multiple sequence alignment program MAFFT v7 (Katoh & Toh 2008), using the L-INS-I strategy and manually adjusted in BioEdit v.7.1.3.0 (Hall 1999). No partitioning was set for the ITS dataset, considering relatively low variation among the species investigated. Neighbor-Joining method implemented in MEGA X (Kumar et al. 2018) was used to group the samples, using the following settings: bootstrap method (with 1,000 replicates) as test of phylogeny, p-distance as substitution model, transitions and transversions both included for substitutions, uniform rates among sites, homogenous pattern among lineages and pairwise deletion for gaps treatment. A NJ bootstrap (NJ-BS) ≥ 70% was considered as significant support for a node to be monophyletic. The ITS tree was rooted with midpoint.

For the ITS-nc LSU-TEF1-α-RPB2 combined datasets, to be able to compare with the results of De Lange et al. (2021), we used the same partitioning except that the short non-informative 18S region was removed, i.e. ITS-1, 5.8S, ITS-2, nc LSU, the TEF1-α introns, the RPB2 intron, the first, second and third codon positions of the exons of the two protein-coding genes. We performed Maximum likelihood (ML) analysis for the concatenated multi-locus datasets (Stamatakis 2014). Incongruences among the equivalent datasets were examined by comparing the ML bootstrap proportions (ML-bs) for the same set of taxa. For the ML analyses a GTR-GAMMA model was assigned and all free model parameters were estimated by the program. An ML bootstrap analysis was performed using 1,000 rapid bootstrapping replicates from random starting trees, followed by a subsequent ML search similarly using 1,000 replicates. An ML-bs ≥ 70% was considered as significant support for a node to be monophyletic. A conflict was assumed to be significant when two different relationships (one monophyletic and the other non-monophyletic) for the same set of taxa were both supported with ML-bs 70%. Trees were viewed and exported in FigTree v1.3.1 and further processed with Adobe Illustrator.

Mass spectrometry analysis

We examined the presence/absence of the diagnostic toxin cycloprop-2-ene carboxylic acid (C₄H₄O₂) for 14 Chinese specimens of the 12 phylogenetic species in our multi-locus phylogeny (Fig. 2). Among them, YNYS20220815-01, YNSP20220906-01 and XH4724 of R. subnigricans were used as positive control. The theoretical molecular mass [M]⁺ by HREI-mass (m/z) of the toxin is 84.0211 (Matsuura et al. 2009). We followed this value and the theoretical mass of the polymeric products given by Matsuura et al. (2009) as reference values.

For each sample, 10 mg dry weight of pileus of each sample was ground in liquid nitrogen and mixed with 100 µL extraction buffer (methanol:water:0.01M hydrochloric acid = 5:4:1, vol/vol/vol). The mixture was transferred to a centrifuge tube, followed by a centrifugtation (13000 rpm) for 2 min. Finally the supernatant was filtered with a 0.22 µm polyethersulfone syringer filter (BS-PES-22, Biosharp).

Liquid chromatography-mass spectrometry (LC-MS) analyses were conducted through Agilent 1290 Infinity II HPLC system with column (4.6×100 mm I.D., particle size 2.7 µm, Agilent Technologies). To detect the toxin, the filtered supernatant was applied on Agilent 1290UPLC/6540 UHD precision mass quadrupole time-of-flight (Q-TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA). The HPLC elution solution A was 0.02 M ammonium acetate:acetonitrile (90:10,vol/vol) adjusted to pH 5 with glacial acetic acid, and solution B was 0.02 M aqueous ammonium acetate:acetonitrile (76:24,vol/vol), pH 5 (Luo et al. 2022). The ESI mass spectrum was viewed using MassHunter software (v.8.0).

ITS data analysis

Among the 69 specimens sequenced by this study, 26 have length variation in the ITS region, resulting in 52 copies after phasing (Table 1). For two copies having only length variation, (i.e. without degenerated bases), only one copy was included in the ITS analysis. The ITS alignment has 98 sequences and 674 characters, among which 54 sequences were generated by this study and 44 from GenBank. ITS-1 region has 191−224 bp, 5.8S 148 bp and ITS-2 237−246 bp.

All our specimens from markets with a local name “Huotanjun”, together with specimens with a “Huotanjun” morphology collected in the field formed 9 terminal monophyletic clades in R. sect. Nigricantinae (Fig. 1). Except that a Tibetan specimen XHW10592 has identical ITS type as two samples from Sweden, none of the Asian clades are the same as European or American ones. Specimens with dense lamellae formed six monophyletic terminal clades. Among them, R. huotanjun clade (NJ-bs 79%) comprised specimens collected from southwestern and central China and samples of “A-4” from Japan by Shimono et al. (2014). The R. brunneocystidia clade (NJ-bs 100%) was composed of samples from Yunnan and “A-5” and “A-6” clades in Shimono et al. (2014). The other clades were Eurasian R. adusta (NJ-bs 96%), Russula sp1 (NJ-bs 98%) with two specimens from Tibet, China, R. nigrocarpa (NJ-bs 100%) from South China, Russula sp2 (NJ-bs 100%) with two specimens from Yunnan, China and one from Japan. All our specimens of the above terminal clades have reddening then blackening context.

Our specimens with spaced lamellae and blackening context fell into three terminal clades. The first clade (NJ-bs 98%), R. sinoadusta included samples from markets and those obtained from investigation on poisoning incidents by Chinese Center for Disease Control and Prevention. Samples of this clade were exclusively from China. Sister to R. sinoadusta (NJ-bs 84%) were two environmental samples from USA (KX899066 and JQ616801). The second clade R. zhuzuijun (NJ-bs 100%) had the widest geographical distribution among all the four new species. The specimens are from four provinces of China and Japan (represented by three environmental sequences AB807970, LC029801 and MZ338122). The third terminal clade was R. ashihoi (NJ-bs 100%), with four specimens from subalpine southwestern China and India Himalayas. All our specimens in these three clades have context reddening first then grayish blackening.

Multi-locus phylogenetic analyses

Different genealogies inferred conflicting phylogenetic relationships among R. acrifolia, R. cf. anthracina and R. roseonigra. In the ITS genealogy, R. cf. anthracina formed a monophyletic clade with R. roseonigra with ML-bs 71%, with R. acrifolia being sister to them. In the RPB2 genealogy R. roseonigra was basal to the clade formed by R. cf. anthracina and R. acrifolia (ML-bs 99%). The TEF1-α genealogy gave the third relationships, i.e. R. acrifolia first grouped with R. roseonigra with ML-bs 79%, leaving R. cf. anthracina basal to them. These samples have identical nc LSU sequences and formed a well-supported clade in the nc LSU genealogy. Analysis of the four-locus combined data supported the relationship given by the RPB2 genealogy.

The topology of the tree generated by the four-locus data was basically congruent with that of De Lange et al. (2021). Two major clades were retrieved, i.e., clade I corresponding to R. sect. Nigricantinae and clade II to R. sect. Polyphyllae. All specimens from markets and the field with a local name “Huotanjun” were deeply nested in R. sect. Nigricantinae. Russula zhuzuijun and R. sinoadusta, the two species with spaced lamellae formed a well-supported evolutionary lineage with European R. adusta with ML-bs 82%. The third species with distant lamellae R. ashihoi was basal to the clade formed by European R. nigricans and North American R. dissimulans with ML-bs 100%. This ashihoi-nigricans-dissimulans lineage was basal to all north temperate species. Russula brunneocystidia and R. huotanjun, the two new species with crowded lamellae grouped with European R. densifolia, R. fuliginosa, R. densissima, R. atramentosa and Asian R. reticulofolia with full support. Samples of R. nigrocarpa, which were not included by De Lange et al. (2021), formed a terminal clade basal to the clade formed by R. acrifolia, R. cf. anthracina and R. roseonigra with ML-bs 91%. The lethal species R. subnigricans formed a monophyletic clade with Chinese R. latolamellata with ML-bs 90%, nested in R. sect. Nigricantinae. The Chinese specimen of R. adusta XHW10592, together with two Swedish samples formed a fully supported evolutionary lineage. HMAS 265004, as “KX441086 China” in De Lange et al. (2021) and our sample XHW5563, also from Tibet, China formed a sister lineage of European R. albonigra. In R. sect. Polyphyllae (clade II in Fig. 2), Chinese R. ochrobrunnea was sister to North American R. eccentrica and the successive sibling was Asian R. kangchenjungae (ML-bs 100%) from subalpine southwestern China and Indian Himalayas.

Mass spectrometry analysis

For the 14 specimens of 12 phylogenetic species used for mass spectrometry analysis of cycloprop-2-ene carboxylic acid, we detected the 443 [5M + Na]⁺ and 527 [6M + Na]⁺ spectra from all the three specimens of R. subnigricans (Fig. 3). The measured mass values were: XHW4724 (HKAS 104742): 443.0953 for [5M + Na]⁺ and 527.1170 for [6M + Na]⁺; YNYS20220815-01 (HKAS 128523): 443.0948 for [5M + Na]⁺ and 527.1162 for [6M + Na]⁺; YNYS20220601-01 (HKAS 128524): 443.0952 for [5M + Na]⁺ and 527.1174 for [6M + Na]⁺. The mass discrepancy ranged from 23.02 ppm to 24.15 ppm for [5M + Na]⁺ and 19.73 ppm to 17.45 ppm, all lower than the allowable deviation cutoff of 50 ppm. Except for these three samples, we did not detect polymers of this toxin in any of the samples analyzed.

TAXONOMY

Russula sinoadusta X.H. Wang, L.H. Huang & H.J. Li, sp. nov. (Fig. 4).

MycoBank no.: XXXXX

Holotype: China, Yunnan Prov., Maguan Co., Mabai Town, Caiyuanzi village, 23°05'.00.7" N 104°26'56.3" E, 1,258 m asl., in fagaceous forest, 5 Aug. 2017, leg. X.H. Wang, no. 4301 (HKAS 104865, KUN!)

Etymology: sino- = Chinese, adusta = R. adusta, a Chinese species closely related with and similar to R. adusta

Diagnosis: differing from R. adusta by the decidedly reddening context, more spaced lamellae and smaller spores with higher spore ornamentation

Pileus medium-sized, 60–70 mm diam, hemisphaerical when young, then planoconvex to applanate, centrally depressed; margin involute when young, smooth, not striate; surface smooth, slightly viscid when wet, brown at center, orange gray to light brown at the center, much paler at the margin, with whitish or paler patches; cuticle not peeling. Context 5–6 mm thick at mid-radius, white, cream, discolouring pinkish brown, then slowly changing to grayish black, taste mild. Lamellae 5–6 mm deep, subdistant to distant, moderately thick, fragile, segmentiform to subventricose, adnate to subdecurrent, white, later yellowish white, blackening or browning with age; with numerous lamellulae of different lengths; edges even, concolourous, blackening when bruised. Stipe 30–60 × 18–20 mm, central, cylindrical, firm and fleshy, smooth, white, the lower part light gray-brown. Odor indistinctive. Spore print white.

Basidiospores (120/6/6) (6.5) 7.0–7.5–8.5 (9.5) × (5) 5.5–6.0–7.0 (8.0) µm, broadly ellipsoid, Q = (1.07) 1.12–1.21–1.33 (1.40); ornamentation of low, moderately dense amyloid warts and short ridges, 0.2–0.5 µm high, connected by short, fine line connections, subreticulate; suprahilar spot medium-sized, not amyloid. Basidia 43–59 × 8–13 µm, narrowly clavate, 4-spored, with granular contents.

Pleurocystidia common to abundant, 45–93 × 5–7.5 µm, narrowly fusiform to narrowly clavate, often flexuous, apcially obtuse, or slightly moniliform with small appendage to even slightly mucronate, thin-walled, with granular contents; without reaction in SV. Cheilocystidia common to abundant, 50–80 × 5–7 µm, similar to pleurocystidia in shape, flexuous, apically obtuse or tapering towards the top in a moniliform way, sometimes with a small appendage, thin-walled, granular contents mostly at the middle and apex, without reaction in SV. Pileipellis 75–220 µm thick, embedded in gluten, composed of ascending to erect hyphae, clearly delimited from trama, subpellis not delimited from suprapellis; hyphae 3–7 µm wide, thin-walled, with yellowish brown pigmentation, mostly cylindrical, some inflated in the middle or tapering towards apex; terminal cells 23–45 × 4–5.5 µm, flexuous, clavate to cylindrical with obtuse apices, with massy oily brown pigments; pileocystidia absent. Hyphae of stipitipellis 3.5–5 µm wide, cylindrical; caulocystidia not observed. Clamp connections absent from all tissues.

Habitat and distribution: growing with Pinus yunnanensis, P. massoniana and fagaceous trees. Known from southwestern China (Guizhou and Yunnan).

Additional specimens examined: China, Guizhou Prov., Meitan Co., 27°47′26″ N, 107°27′23″ E, 965 m asl., 14 Sept. 2015, leg. H.J. Li, no. Li 150914-03 (HKAS 125836); Wuchuan Co., Shichao Town, Langshui village, 23 Sept. 2014, leg. H.J. Li, no. Li 140923-20 (HKAS 125837). Yunnan Prov., Changning Co., Dashaba village, 24°28′43″ N, 99°24′24″ E, 1,428 m asl., 24 Jul. 2022, leg. Y.C. Wang, no. YNCN20220724-02-01 (HKAS 125831), no. YNCN20220724-02 (HKAS 125832), no. YNCN20220724-02-03 (HKAS 125833); Maguan Co., Mabai Town, Caiyuanzi village, 23°05'.00.7" N, 104°26'56.3" E, 1,258 m asl., in fagaceous forest, 5 Aug. 2017, leg. X.H. Wang & B. Buyck, no. 4302 (HKAS 104864); Maguan Co., Mabai Town, Yupo village, 23°00′48″ N, 104°21′08″ E, 1,365 m asl., 15 Oct. 2017, leg. X.H. Wang, no. 4786 (HKAS 104874); Shidian Co., Dianyang Town, Wuyi village, 24°42′35″ N, 99°10′39″ E, 1,627 m asl., 5 Jun. 2022, leg. T.H. Li, no. SD202203-02 (HKAS 125838); Shilin Co., wild mushroom market, 15 Aug. 2016, leg. X.H. Wang & S.H. Li, no. 4114 (HKAS 96703); Tengchong Co., Gudong Town, Lingtaisi, 25°17′28″ N, 98°28′15″ E, 1,824 m asl., 23 Jul. 2016, leg. H.J. Li, no. TCGD002 (HKAS 125839).

Notes: This new species and the species described below are two Asian counterparts of European R. adusta. All of them have grayish brown pileus, subdistant lamellae and eventually blackening context and gills. The two new species have more spaced lamellae than R. adusta. The reddening discolouration, which is decided in the two Chinese species, is very faint or even absent in R. adusta (Shaffer 1962; Moser 1983; Knudsen & Vesterholt 2008; De Lange et al. 2021). The spores of R. adusta, as described by Knudsen & Vesterholt (2008) are bigger (7.5–9 × 6–7.5 µm) and the ornamentation does not exceed 0.2 µm (De Lange et al. 2021). The odor of “empty wine casks” of typical R. adusta is not detected in the two Chinese species. We have a specimen of R. adusta from Tibet, China (XHW10592). The spores are 8.0–8.8–9.5 × 6.0–6.8–7.5 µm with ornamentation only 0.1–0.2 µm high. This confirmed the difference between the two Chinese species and R. adusta. In the ITS tree, the closest relative to R. sinoadusta is a small terminal clade formed by two North American environmental samples represented by KX899066 and JQ616801. More North American samples grouped with R. adusta but the sequences are not identical (Fig. 1). Shaffer (1962) commented that the diagnostic “empty wine casks” odor of R. adusta was not found from North American specimens. The diversity of “R. adusta” in North America also needs clarifying.

Russula zhuzuijun S.H. Li & X.H. Wang, sp. nov. (Fig. 5)

MycoBank no.: XXXXX

Holotype: China, Yunnan Prov., Songming Co., Junmachang market, 8 July 2016, S.H. Li, no. XHW3810 (HKAS 95393, KUN!)

Etymology: a local name, meaning that the pileus looks like a snout when growing from the earth

Diagnosis: differing from R. sinoadusta by the more remarkable blackening, pileipellis with hyphae more horizontally arranged and fewer hymenial cystidia

Pileus medium-sized to big, 70–80 mm diam, hemispherical when young, becoming applanate with a depressed center when mature, shallowly infundibuliform; surface dry, viscid when wet; center sometimes cracking into small yellowish brown scales, tan, grayish, yellowish brown to blackish brown, locally paler, turning blackish brown when aging or drying; cuticle not easy to peel. Context 4–6 mm thick at mid-radius, white to cream whitish, slightly reddening before blackening, taste mild. Lamellae 5–8 mm deep, subdistant, moderately thick, fragile, segmentiform to subventricose, cream white, later yellowish white, blackening with age or when bruised; with different lengths of gills; edges even, concolorous or marginate. Stipe 30–40 × 15–25 mm, cylindrical, stout, slightly hollow with a big cavity; surface smooth, white to grayish brown, becoming brownish or black with age and bruised. Odor indistinctive. Spore print white.

Basidiospores (280/14/14) 6.0–7.0–8.0 (9.0) × 5.0–6.0–7.0 (7.5) µm, broadly ellipsoid to ellipsoid, Q = (1.08) 1.14–1.22–1.35 (1.40); ornamentation of not very low, distant amyloid warts and short ridges, 0.3–0.5 µm high, some fused into chains and connected by short, fine line connections, subreticulate; suprahilar spot medium-sized, not amyloid. Basidia 25–58 × 7–14 µm, clavate, 4-spored, with granular contents. Pleurocystidia rare, 30–85 × 4–8 µm, cylindrical, narrowly fusiform to narrowly clavate, some slightly flexuous, apically obtuse or with small appendage, rarely with 2–3 knobs, thin-walled, with granular or needle-like contents; without reaction in SV. Cheilocystidia uncommon, 30–70 × 4–8 µm, similar to pleurocystidia in shape but smaller, cylindrical to narrowly clavate, flexuous, thin-walled, with granular or needle-like contents. Pileipellis 90–380 µm thick, embedded in gluten, not sharply delimited from trama, gradually passing, subpellis not delimited from suprapellis; hyphae more or less horizontally irregularly oriented, often more compact near surface, locally aggregate into projection 30–100 µm high; hyphae cylindrical, mostly 4–6 µm wide, some inflated to 7–10 µm, subfusiform, mostly with oily brown pigmentation; terminal cells 14–43 × 4–6.5 µm, clavate, cylindrical, with obtuse apices and brown pigments; subterminal cells often wider; Pileocystidia extremely rare, 4–5 µm wide, cylindrical, with granular contents, thin-walled, without reaction in SV. Hyphae of stipitipellis 4–5 µm wide, cylindrical; caulocystidia not observed. Clamp connections absent from all tissues.

Habitat and distribution: growing in fagaceous forests (Quercus engleriana and Cyclobalanopsis multinervis) and pine forest (Pinus massoniana and P. yunnanensis). Southwestern (Guizhou, Sichuan and Yunnan) and central-southern China (Hunan), Japan.

Additional specimens studied: China, Hunan Prov., Zhangjiajie, Yongding District, Tianmen Mt., in forests dominated with Q. engleriana and C. multinervis, 12 Sept. 2021, W.Q. Qin, no. R159 (HKAS 122230); Zhangjiajie, Yongding District, Zhangjiajie Campus of Jishou University, 230 m asl., 17 Sept. 2020, leg. W.Q. Qin, in forest of P. massoniana, no. R030 (HKAS 110845); ibid., 1 Dec. 2019, leg. W.Q. Qin, no. R097 (HKAS 111353); ibid., 28 Oct. 2020, leg. W.Q. Qin, no. R121 (HKAS 121154); Sichuan Prov., Huili Co., 18 Jul. 2020, leg. X.L. He, SAAS 3568. Yunnan Prov., Chuxiong City, Zixi Mt., Zidingsi, 25°00'24" N, 101°25'12" E, 2,485 m asl., 8 Aug. 2016, leg. X.H. Wang, no. 3833 (HKAS 96422); Maguan Co., Dalishu Town, Xiangchang village, 23°04'00" N, 104°12'42" E, 1,592 m asl., 14 Oct. 2017, leg. X.H. Wang, no. 4768 (HKAS 104747); Shilin Co., wild mushroom market, 15 Aug. 2016, leg. X.H. Wang, no. 4113 (HKAS 96702), no. 4115 (HKAS 96704); Songming Co., Junmachang market, 8 Jul. 2016, leg. S.H. Li, no. 3813 (HKAS 95384), no. 3814 (HKAS 95385); Wenshan Co., Yangliujing Town, Yangliu village, wild mushroom market, 23°12'52" N, 104°17'57" E, 1,472 m asl., 10 Aug. 2016, leg. X.H. Wang, no. 3885 (HKAS 96474), ibid., 14 Aug. 2016, leg. X.H. Wang, no. 4103 (HKAS 96692); Yanshan Co., wild mushroom market, 10 Aug. 2016, leg. X.H. Wang, no. 3876 (HKAS 96465).

Notes: This is another close relative of R. adusta in Asia. It is often sold in local markets and can be easily confused with R. sinoadusta. Compared with R. sinoadusta, this new species have darker pileus (especially the margin) and more remarkable blackening context. The pileipellis of R. sinoadusta is a regular trichoderm, whereas the pileipellis hyphae of R. zhuzuijun are more interwoven and often locally aggregate into scales. The pileipellis has more inflated cells in R. zhuzuijun than in R. sinoadusta. Pileocystidia, which are absent in R. sinoadusta, are present in R. zhuzuijun in spite of the rarity. Among the specimens examined, two (HKAS 111353 and 122230) have remarkably grayish brown stipe and marginate lamellae.

Russula zhuzuijun has a wide distribution and is a mycorrhizal generalist. Some contaxic environment samples are from Japan (AB807970, AB251827, MZ338122 and LC029801, Fig. 1). The mycorrhizal samples are from Pinus densifolia, P. thunbergii, Picea jezoensis, Pseudotsuga japonica, P. sinensis and Tilia japonica (Wen et al. 2015; Miyamoto et al. 2018a ). Russula adusta” in Wang et al. (2022) is this species. For more notes see under R. sinoadusta.

Russula huotanjun S.H. Li & X.H. Wang, sp. nov. (Fig. 5)

MycoBank no.: XXXXX

Holotype: CHINA. Yunnan Prov., Chuxiong City, Zixi Mt., 25°00'22" N, 101°25'26" E, 2,110 m asl., 8 Aug. 2016, leg. X.H. Wang, no. 3829 (HKAS 96418, KUN!)

Etymology: a local name, “Huotan” = burnt charcoal, meaning that the context changes “burnt charcoal”-coloured when bruised

Diagnosis: differing from R. densifolia in the smaller spores with higher ornamentation and mild taste

Pileus medium-sized to big, 60–90 mm diam, hemisphaerical when young, with a depressed center when mature, shallowly infundibuliform, margin slightly incurved, smooth, not striate; surface smooth, viscid when wet; cracking, grayish, gray-white, tan, yellowish brown to blackish brown, locally paler, turning blackish brown after aging or drying; cuticle not easy to peel. Context 5–7 mm thick at mid-radius, grayish white, discolouring reddish brown, then changing to grayish black, taste mild. Lamellae 1–2 mm deep, very dense, narrow and thin, fragile, segmentiform to subventricose, adnate to subdecurrent, creamy, yellowish white, blackening or browning in age or when bruised; with different lengths of gills; edges even, concolorous. Stipe 40–50 × 15–25 mm, cylindrical, or slightly enlarged towards at the base; surface smooth, white, gray, surface becoming gray-black or black with age and bruised. Odor indistinctive. Spore print white.

Basidiospores (200/10/10) 6.0–7.0–8.0 × 5.0–5.5–6.0 (6.5) µm, broadly ellipsoid to ellipsoid, Q=(1.08) 1.17–1.28–1.40 (1.45); ornamentation of moderately high amyloid warts and short ridges, 0.3–0.7 µm high, some fused into chains and connected by short, fine line connections, rarely forming closed meshes; suprahilar spot medium-sized, not amyloid. Basidia 24–51 × 7–12 µm, clavate, 4-spored, with granular contents. Pleurocystidia common to abundant, 30–102 × 5–10 µm, fusiform to clavate, some slightly flexuous, apically obtuse or with a constriction or small appendage to even slightly mucronate, thin-walled, with sparse contents, granular contents mostly at the middle and apex; without reaction in SV. Cheilocystidia common, 35–84 × 5–9 µm, similar to pleurocystidia in shape, cylindrical to narrowly clavate, thin-walled, granular contents mostly at the middle and apex; without reaction in SV. Pileipellis 90–500 µm thick, embedded in gluten, composed of ascending to erect hyphae, clearly delimited from trama, subpellis not delimited from suprapellis; hyphae mostly 3–5 µm wide, some inflated to 10–13 µm, thin-walled, with yellowish brown pigmentation, mostly cylindrical, some inflated in the middle or tapering towards apex; terminal cells 12–45 × 3–6.5 µm, flexuous, narrowly cylindrical, with obtuse apices and yellowish brown pigmentation; pileocystidia extremely rare, at least 60 × 5 µm, subclavate, with granular contents, moniliform, with inflorescence-like or forked apices, thin-walled, without reaction in SV. Hyphae of stipitipellis 3–5 µm wide, cylindrical; caulocystidia not observed. Clamp connections absent from all tissues.

Habitat and distribution: growing in pine and fagaceous forests. Central and southern China (Hubei, Hunan, Sichuan, Yunnan, Zhejiang) and Japan.

Additional specimen studied: China, Hubei Prov., Hefeng Co., Mulinzi National Nature Reserve, Xiapingzhan, 30°3′20″ N, 110°14′10″ E, 1,300 m asl., 28 Jul. 2021, leg. B.B. Chen et al., no. HF1127 (HKAS 125834), no. HF1157 (HKAS 125835); Sichuan Prov., Dazhou City, Xiliushan, 13 Jun. 2013, leg. X.L. He, SAAS 1211; Jiange Co., Jianmenguan Town, Liangshansi, Liangyazi, 7 Aug. 2015, leg. X.H. Wang, no. 3622 (HKAS 89896); Yunnan Prov., Chuxiong City, Zixi Mt, 25°1'16" N, 101°22'59" E, 2,212 m asl., 28 Aug. 2015, S.P. Wan, no. 523 (HKAS 94938); Jingdong Co., Yinsheng market, 15 Aug. 2017, leg. X.H. Wang & B. Buyck, no. 4576 (HKAS 104882); Lanping Co., Tongdian Town, Longtan village, 26°36'51″ N, 99°27'21″ E, 3,083 m asl., 17 Aug. 2020, leg. X.H. Wang, no. 7966 (HKAS 117593); Maguan Co., Mabai Town, Xinbaozhai village, 23°01'44" N, 104°22'49" E, 1,285 m asl., 14 Aug. 2016, leg. X.H. Wang, no. 4018 (HKAS 96607); Nanhua Co., Shaqiao Town, 11 Oct. 2011, leg. S.H. Li, s.n. (HKAS 85071); Nanhua Co., Shaqiao Town, Tianzimiaopo forest farm, 6 Aug. 2018, leg. X.H. Wang, no. 4954 (HKAS 104899); Songming Co., Junmachang market, 8 Jul. 2016, leg. S.H. li, no. XHW3808 (HKAS 95391), no. XHW3809 (HKAS 95392), no. XHW3816 (HKAS 95387); Wenshan Co., Yangliujing Town, Yangliu village, wild mushroom market, 23°12'52" N, 104°17'57" E, 1,472 m asl., 14 Aug. 2016, leg. X.H. Wang, no. 4104 (HKAS 96693).

Notes: This new species is an Asian counterpart of European R. densifolia and R. fuliginosa. They share the extremely crowded lamellae, the grayish brown pileus and the context eventually discolouring blackish. Russula huotanjun seems to be intermediate between R. densifolia and R. fuliginosa, as its reddening discolouration is the same as R. densifolia but shares long and slender cells in the pileipellis with R. fuliginosa. The context of R. huotanjun is mild, unlike the acrid context of R. densifolia and R. fuliginosa. The spores of R. huotanjun are smaller than those of the two European species. The newly described R. reticulofolia by Xie et al. (2023) from southern China is also closely related. The frequent and irregular forking lamellae and the “Mickey Mouse” type pileocystidia are not observed in R. huotanjun. The spores of R. reticulofolia are more globose. Russula densifolia in Chen & Zhang (2019) is R. huotanjun.

A Japanese sample of A-4 in Shimono, “R. densifolia 196” (OSA-MY-4277) and two samples labeled as “Russula densifolia” from Southern Yunnan in Wang et al. (2022) (HKAS 122413 and 122565, wrongly cited as HKAS 122430 and HKAS 122669 respectively) formed the closest clade to R. huotanjun in the ITS tree. We sequenced the other genes of the two Yunnan samples. In the multi-locus tree they formed an independent evolutionary lineage, still sister to R. huotanjun. Morphologically this small clade has slightly wider terminal cells in the pileipellis and numerous pileocystidia, some of “Micky Mouse” type. We wait for more samples to determine if this lineage represents another new species.

Although R. huotanjun is here described as a new species, its mycorrhizae have been reported several times in Asia. It is associated with various plants including some heterotrophic ones, such as Pyrola japonica (Uesugi et al. 2016), Cymbidium lancifolium (Ogura-Tsujita et al. 2012), Quercus serrata (Miyamoto et al. 2018b), Pseudotsuga japonica (Murata et al. 2017) and Abies sachalinensis (GenBank accession LC711842) in Japan. This is a typical widely distributed Asian species.

Russula brunneocystidia X.H. Wang & L.H. Huang, sp. nov. (Fig. 7)

MycoBank no.: XXXXX

Holotype: China, Yunnan Prov., Shilin Co., Meiyi Town, Dashao village, 25 Jun. 2016, S.H. Li, no. DS2 (HKAS 125830, KUN!)

Etymology: named after the hymenial cystidia with oily dark brown pigmentation

Diagnosis: differing from R. atramentosa and R. densissima by the dense oily dark brown pigmentation in hymenial cystidia and negative reaction in SV of the cystidia

Pileus medium-sized to big, 50–100 mm diam, hemisphaerical when young, with a depressed center when mature, shallowly infundibuliform; surface smooth, slightly viscid when wet, grayish brown, with white patches, turning blackish brown when aging or drying. Context 4–6 mm thick at mid-radius, firm, white when young, grayish white, dark white, some samples browning before blackening, not reddening or turning pinkish, finally turning black. Lamellae 1–3 mm deep, very dense, narrow and thin, fragile, segmentiform to subventricose, creamy, yellowish-white, blackening with age or when bruised; with different lengths of gills; edges even, concolourous. Stipe 40–50 × 15–25 mm, cylindrical, or slightly tapering towards the base; surface smooth, white when young, gray-black when bruised, finally black. Odor indistinctive. Spore print white.

Basidiospores (120/6/6) 6.0–7.0–8.0 (8.5) × 5.0–5.5–6.5 (7) µm, broadly ellipsoid to ellipsoid, Q=(1.11) 1.17–1.30–1.36 (1.40); ornamentation of low, amyloid warts and short ridges, 0.2–0.4 µm high, some fused into chains and connected by short, fine line connections, subreticulate; suprahilar spot medium-sized, not amyloid. Basidia 30–59 × 7–12 µm, clavate, 4-spored, with granular contents. Pleurocystidia common to abundant, 37–121 × 6–18 µm, fusiform, flask-shaped, apically with double constriction or 1–2 eccentrical appendages, slightly flexuous, thin-walled, filled with oily yellow brown to black brown pigments or partly filled with crystalline or refractive bodies; no reaction in SV. Cheilocystidia common to abundant, 33–103 × 5–14 µm, similar to pleurocystidia in shape, filled with blackish brown pigments. Pileipellis 80–350 µm thick, of irregularly interwoven or ascending hyphae, embedded in gluten, sharply delimited from trama, subpellis not delimited from suprapellis; hyphae 3–7 µm wide, thin-walled, filled with brown pigmentation, mostly cylindrical, some inflated in the middle or tapering towards apex; terminal cells 25–45 × 3.5–7 µm, narrowly cylindrical, with obtuse apices and brown pigmentation; pileocystidia absent. Hyphae of stipitipellis 3–5 µm wide, cylindrical; caulocystidia not observed. Clamp connections absent from all tissues.

Habitat and distribution: not known. Southwestern China (Yunnan) and Japan.

Additional specimen studied: China, Yunnan Prov., Shilin Co., wild mushroom market, 15 Aug. 2016, leg. X.H. Wang, no. 4117 (HKAS 96706); Songming Co., Junmachang market, 8 July 2016, leg. S.H. Li, no. XHW3807 (HKAS 95390), no. XHW3811(HKAS 95394), no. XHW3815 (HKAS 95386), no. XHW3817 (HKAS 95388); Yanshan Co., wild mushroom market, 10 Aug. 2016, leg. X.H. Wang, no. 3875 (HKAS 96464).

Notes: This new species is the Asian counterpart of European R. atramentosa and R. densissima, although the reaction of graying in SV was not observed in our new species. These species share the dark blackish pileus, crowded lamellae and quickly and strongly blackening context that is normally not preceded by reddening. In a few Chinese specimens we observed very faint pinkish discolouration. In markets, R. brunneocystidia looks very much like R. albonigra. Phylogenetic analysis, however, showed that it was not related with the species of the R. albonigra complex.

The most remarkable character of R. brunneocystidia is the oily brown refractive contents in the hymenial cystidia. In the multi-locus tree, the closest relative of R. brunneocystidia is a Thai specimen of “Russula sp. 6”) (FH 12.064). This Thai specimen also has cystidia “filled with brown, irregular, refractive bodies” and its macroscopical morphology is “strongly reminiscent of R. albonigra (Adamčík et al. 2019). Compared with the Thai specimen, R. brunneocystidia has bigger spores. The biggest extremities of the cystidia of R. brunneocystidia are bigger than those of the Thai specimen. Russula brunneocystidia corresponds the clade “A-5” of Shimono et al. (2014). Russula brunneocystia might be confused with the sympatric R. huotanjun in the field. The reddening of R. huotanjun and less blackening could help distinguish them. This species is one of the most common commercialized Russula species in the subtropical-tropical parts of Yunnan.

Early in 2014 Shimono et al. and Park et al. showed that none of their Asian samples had identical genotypes with European or North American species. It was until in 2020 that new species with DNA data of R. sect. Nigricantinae were formally described from Asia (Das et al. 2020; Zhou et al. 2020). Together with R. subnigricans described from Japan (Hongo 1955), R. schaefferina and R. kangchenjungae from India (Rawla & Sarwal 1983; Das et al. 2010), in total eight species of R. subgen. Compactae were originally described from Asia. Besides the four new species described here, our data in this paper show that R. ashihoi and R. kangchenjungae are also present in China, with all the samples collected from subalpine habitats in eastern Himalayas. The sample HMAS 265004 (from Tibet, China, GenBank accession KX441086), being a singleton in De Lange et al. (2021), now formed an independent evolutionary lineage with our sample XHW5563 (also from Tibet, China), sister to European R. albonigra. Among all the samples we analyzed, only a sample from Tibet, China (XHW10592) is contaxic with European specimens of R. adusta. Taking the finding of Shimono et al. (2014), Adamčík et al. (2019) and De Lange et al. (2021) we arrived at a similar conclusion to De Lange et al. (2021) that continental endemism is prevalent in R. subgen. Compactae. Only in subalpine and boreal regions (Liu et al. 2017) Asia shares species with Europe. More new species, like here the unnamed “Russula sp1” and “Russula sp2”, need to be formally described when more specimens are available.

Although we delimitate our new species using both DNA data and morphological characters, we have to admit that with the species size getting smaller and smaller, it becomes more and more difficult to recognize species with morphology. Among the new species recently described, except for R. nigrocarpa bearing very small spores (Zhou et al. 2020), all the species are highly similar to the known species or might be taken as morphological variants slightly deviating the typical forms. Our four new species described here are highly similar to their European siblings and this is why in the pre-DNA time the relevant European and North American names were often used in China. The morphology we used to distinguish our species is merely subtle differences in odor, degree of reddening and height of spore ornamentation. More examples like: Zhou et al. (2020) compared R. latolamellata with the morphologically different Indian R. schaefferina. Our multi-locus data showed that it is closely related with R. subnigricans. If we compare R. latolamellata with the description of R. subnigricans given by Hongo (1955), we may conclude that the Chinese species has smaller spores and narrower pleurocystidia. However, after we examined our specimens of R. subnigricans, we found that the Chinese specimens also have smaller spores (6.0–7.0–8.0 × 5.0–6.0–6.5 µm) and narrower pleurocystidia (59–107 × 7.0–10.0 µm); Das et al. (2020) distinguished their R. ashihoi from North American R. dissimulans by the marginate lamellae. Our field notes of R. ashihoi show that the lamellae edge can be concolorus with the sides or blackish brown. In our new species R. zhuzuijun, we also found both concolorous gills and marginate gills (Fig. 5). This observation is contradictory with the statement of Das et al. (2020) that having marginate lamellae is a reliable character of Russula species. In fact, after more specimens of R. ashihoi are observed, we find it is very difficult to separate it from R. nigricans and R. dissimulans. De Lange et al. (2021) called their new species of R. albonigra complex as “pseudocryptic”, meaning if appropriate characters are used, it is possible to distinguish these species. It is true that some of them can be separated, but we need to point out that in most groups researchers are carrying out an un-exhaustive subdivision, i.e. specifically describing some terminal lineages that have relatively clear morphological characters as recognizable species and leaving the others unnamed (Van de Putte et al. 2010; Wang et al. 2015). When DNA data are used for species recognition, it is easy to describe new species based on very few specimens and interpret the variation of individuals as interspecific diversification (Wang et al. 2023). Just as Taylor et al. (2000) said, species can be recognized by gene sequences before changes have occurred in mating behavior or morphology. We may not necessarily expect morphological differences among different phylogenetic species.

Collecting and eating “Huotanjun” is more dangerous than eating other russulas. Every year the fatal R. subnigricans causes several deaths in China (Li et al. 2020, 2021, 2022). This is because hunters assume with confidence that what they have at hand is real edible “Huotanjun”. In Shimono et al. (2014), five clades were labeled as R. subnigricans. Among them, B-1 included the type of R. subnigricans and thus represents authentic R. subnigricans. Using multi-gene data, for the first time we showed that R. subnigricans was nested in R. sect. Nigricantinae, sister to Chinese species R. latolamella, not to R. nigricans as Shimono et al. (2014) showed. Russula latolamellata also has distant lamellae and only reddening discolouration (Zhou et al. 2020). Our analyses of the Japanese samples of B-2−B-5 and our samples show that all of them belong to R. subgen. Polyphyllae (tree not shown), not closely related to R. subnigricans. Among them, B-3 was named as R. eccentrica by Park et al. (2014) but it most likely represents a different Asian species. For the several samples of R. sinoadusta that we obtained in the field during tracing the responsible species leading to rhabdomyolysis (Fig. 2), we noted very slight blackening. We did not detect cycloprop-2-ene carboxylic acid in any of these species, implying that these look-alikes of R. subnigricans are edible. Although using the golden standard “mushroom with distant lamellae + only reddening is poisonous” will miss the potentially edible such as R. aff. eccentrica, R. latolamellata, R. ochrobrunnea, R. kangchenjungae and even R. sinoadusta, it is still valid for all the other edible species with blackening context. At this stage, we strongly recommend to only collect and eat species that have a real “burnt charcoal” colour change. It is not clear why in R. subgen. Compactae only R. subnigricans, a species not sitting on a special phylogenetic position leads to rhabdomyolysis. Tricholoma equestre (Tricholomataceae, Agaricales), a species phylogenetically distant from R. subnigricans also leads to rhabdomyolysis. The poisoning mechanism of T. equestre is still unclear (Saviuc & Danel 2006; Klimaszyk & Rzymski 2018). If this evolves the same type of toxin, horizontal gene transfer, as documented by Luo et al. (2018) cannot be ruled out. The phylogeny provided in this study might be referred for understanding the origin of the toxin.

Acknowledgements

We thank Ms. Shuqin Cao and Weibiao Li, Kunming Institute of Botany (KIB) for their help with lab work, field work and data analysis. Dr. Bart Buyck, Muséum national d'histoire naturelle, and S.F. Shi, Beijing helped with field trips in Yunnan. Fang Li, Sun Yat-Sen University, Dr. Xiaolan He, Sichuan Academy of Agricultural Sciences, China (SAAS) and Mr. S.L. Li, Enshi Tujia and Miao Autonomous Prefecture Academy of Forestry, China provided specimens. Dr. Hong Luo and Yujiao Lvli of KIB helped with the mass spectrometry analysis. Dr. Chun-Xia Zeng and Dr. Zhi-Rong Zhang, Germplasm Bank of Wild Species, KIB helped with the genome skimming sequencing and data analysis. This work is supported by the National Natural Science Foundation of China (No. 32170022 and 31770031), the Biodiversity Survey and Assessment Project (No. 2019HJ2096001006) and “Investigation of Macrofungi of Maguan County” issued by Ministry of Ecology and Environment of the People's Republic of China and the Youth Fund of Guizhou Academy of Sciences [J(2021)16] “Study on isolation, purification and culture characteristics of Lactarius deliciosus strains”.

The sequences generated during the current study are deposited in GenBank. The alignments analyzed during the current study are available from the corresponding author on reasonable request. The specimens examined in this study are deposited at the Cryptogamic section (HKAS), Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and Sichuan Academy of Agricultural Sciences, China (SAAS).

All authors read and approved the final manuscript. The six authors have no relevant financial or non-financial interests to disclose.

This work was supported by the National Natural Science Foundation of China (No. 32170022 and 31770031), the Biodiversity Survey and Assessment Project (No. 2019HJ2096001006) and “Investigation of Macrofungi of Maguan County” issued by Ministry of Ecology and Environment of the People's Republic of China to XHW and the Youth Fund of Guizhou Academy of Sciences [J(2021)16] to JW.

All authors contributed to the study conception and design. XHW, JW, WQQ, SHL and HJL collected specimens. LHH and XHW did lab work. LHH and XHW did data analysis and morphological observation and wrote the first draft of the manuscript. Toxin extraction was conducted by LHH and the results were analyzed by XHW and LHH. All authors commented on previous versions of the manuscript.

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Clarifying the identity of marketed edible fungus Huotanjun (Burnt Charcoal Mushroom) in southern China

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Abstract

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INTRODUCTION

MATERIAL AND METHODS

Sampling

Morphological analysis

DNA extraction and sequencing

Phylogenetic analysis

Mass spectrometry analysis

RESULTS

ITS data analysis

Multi-locus phylogenetic analyses

Mass spectrometry analysis

TAXONOMY

DISCUSSION AND CONCLUSION

Declarations

References

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