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. 2024 Jan 15;10(1):70. doi: 10.3390/jof10010070

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China

Sen Liu 1, Mengjia Zhu 1, Nemat O Keyhani 2, Ziyi Wu 1, Huajun Lv 1, Zhiang Heng 1, Ruiya Chen 1, Yuxiao Dang 1, Chenjie Yang 1, Jinhui Chen 1, Pengyu Lai 1, Weibin Zhang 1, Xiayu Guan 3, Yanbin Huang 4, Yuxi Chen 1, Hailan Su 5,*, Junzhi Qiu 1,*
Editors: Ting-Chi Wen, Zhenyuan Zhu, Tao Feng
PMCID: PMC10817631  PMID: 38248979

Abstract

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

Keywords: Basidiomycota, Russulales, Russula, Lactarius, new species, morphological and phylogenetic analyses

1. Introduction

Fungi classified within the order Russulaceae include members of some of the most significant ectomycorrhizal genera found in almost all forest ecosystems, spanning across temperate, subtropical, and tropical regions [1]. The proliferation and ecological relevance of Russulaceae are evident in the lush forests of southern China and various other regions dispersed throughout South Asia. This fungal assemblage plays a pivotal role in mycorrhizal associations, contributing significantly to the vitality and sustainability of forest ecosystems [2,3]. Russulaceae establish intimate associations with various host plants [4,5]. In addition, these fungi have significant medicinal, nutritional, and bioremediation value, including as resources for novel drug discovery [6,7,8]. Fujian, a coastal province in southern China, is surrounded by mountains on three sides and the sea on the other. The subtropical monsoon climate in this region results in relatively warm, short winters and long, rainy summers compared to northern China [9,10]. Botanically, Fujian is positioned at the southernmost end of the Sino-Japanese Floristic Region and faces Taiwan across the sea, the latter of which belongs to the Indo-Malay Region [11]. The main mountain ranges in Fujian include the Wuyi, Shanling, Jiufeng, and Tailao (elevation of ~200–2158 m), which house subtropical evergreen broadleaf forests, mixed coniferous and broadleaf forests, and South Asian tropical rainforests. The major tree species in Fujian comprise Masson’s pine, bamboo groves, willow trees, banyans, and camphor [12], among which the Chinese fir, Chinese yew, Fujian pine, and Chinese swamp cypress are indigenous to Fujian. The diverse and unique local tree species found in Fujian, coupled with the warm and humid climate, are likely important factors conducive to the proliferation of their associated Russulaceae fungi.

The genus Russula Pers. (Russulaceae, Russulales, Basidiomycota) was established by Persoon in 1796 [13]. Members of this genus often constitute crucial components of forest ecosystems via their extensive associations with plants, and also likely as a food source (their fruiting bodies/mushrooms) for a variety of animals [14]. Indeed, a number of Russula species are globally recognized as edible fungi [15] and have displayed promising (biopharmaceutical) properties with respect to possessing anticancer and antioxidative activities [16,17]. The morphological classification system for Russula is characterized by brightly colored fragile caps, brittle context, amyloid warty spores, abundant sphaerocysts in a heteromerous trama, an absence of latex, and simple-septate hyphae [18,19]. The documented number of species cataloged within the genus Russula currently surpasses > 2000, with their fruiting bodies encompassing a vast array of variations in color, morphology, and anatomical characteristics. However, due to the substantial variability exhibited within this taxonomic group, many species still pose considerable challenges in terms of their accurate identification and differentiation. This complexity underscores the likelihood of new species awaiting detection and classification through attempts to compare molecular phylogenetic reconstruction with modern infrageneric classification [20]. Consequently, challenges persist in differentiating and taxonomically categorizing Russula species within fungal surveys and ethnopharmacological investigations [21].

The genus Lactarius also belongs to the family Russulaceae in the order Russulales [22]. In traditional classification, all species that exude latex (or “milk”, hence the term “milkcap” fungi) were grouped under the genus Lactarius. Buyck et al. [22,23] separated Lactifluus and L. furcatus Coker from the genus Lactarius, establishing a new genus named Lactarius sensu novo, which is mainly classified into three subgenera: L. subg. Lactarius, L. subg. Plinthogalus (Burl.) Hesler & A.H. Sm., and L. subg. Russularia (Fr.) Kauffman. Although the exploration of extensive fungal resources has led to the identification of several dozen Russulaceae species across various regions [24,25,26,27], investigations concerning this genus in southern China remain inadequately addressed, with the continual discovery of new species [28,29].

During an exploration aimed at delineating the diversity and geographical distribution of Russula in China, a series of intriguing samples was gathered within Fujian province, China. These isolates displayed characteristics that did not correspond to any known species within the genus. Employing both morphological and molecular phylogenetic analyses, we identify three new species within the Russulaceae family. We present detailed descriptions of these newfound species, complemented by illustrations elucidating their distinctive morphological attributes.

2. Materials and Methods

2.1. Collections and Morphological Analyses

Fresh fruiting bodies of two unknown (putative members of the Russula) mushrooms were collected from the Junzifeng National Nature Reserve, and one from Jiufeng Mountain, Jianyang (putative member of the Lactarius), in the Fujian Province, China, in August 2021. These specimens were collected during field expeditions focused on fungi. Images of the fresh fruiting bodies were captured using a Canon (Tokyo, Japan) EOS 6D Mark II camera. The meticulous documentation of their macroscopic attributes involved the careful examination of fresh samples in their natural diurnal environment. Comprehensive records encompassing macroscopic characteristics and habitat specifics were meticulously collated from collection records and accompanying visual documentation, adhering to the conventions of mycological taxonomic research. To ensure the permanent preservation of specimens, one crucial step was a dehydration process, during which the specimens underwent desiccation within a drying oven set at 45 °C. This meticulous procedure persisted until the moisture content of the fruiting bodies was diligently reduced to below 10%, ensuring their suitability for long-term storage. Microsections of dried specimens were stained with a mixture of 5% potassium hydroxide (KOH) and 1% Congo red. A detailed illustration of the structure and ornamentation of the spores was carried out using a scanning electron microscope (ZEM15C, ZEPTOOLS, Tongling, China). Microscopic features were observed using a Leica microscope (DM2500, Wetzlar, Germany) at magnifications up to 100×. For the description of basidiospores, 20 basidiospores, in profile view, were measured. The basidium length excludes sterigmata. The notation (a-)b–c(-d) was used to represent the dimensions of basidiospores, where the range ‘b–c’ covered 90% or more of the measured values. ‘a’ and ‘d’ represent the extreme values. An average length/width ratio (Q value) was calculated from 20 spores, along with the standard deviation, reflecting the characteristics of the basidiospores. The specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS) at the Institute of Microbiology, Chinese Academy of Sciences, with the specimen numbers HMAS 298099, HMAS 298100, HMAS 298101, HMAS 298102, HMAS 298103, and HMAS 298104. Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org (accessed on 10 January 2024)).

2.2. DNA Extraction, PCR Amplification, and Sequencing

DNA was extracted using the Fungal DNA Mini Kit (OMEGA-D3390, Feiyang Biological Engineering Corporation, Guangzhou, China) following the manufacturer’s protocol. Briefly, 100 mg of starting material (fruiting body) was used for DNA extraction. The amplification of the nucleotide sequences of the internal transcribed spacer (ITS), 28S large subunit regions of ribosomal DNA (LSU), the translation elongation factor 1-α (TEF1-α), the ribosomal mitochondrial small subunit (mtSSU), and second largest RNA polymerase II regions (RPB2) was conducted via polymerase chain reaction (PCR) using the primer pairs: ITS4/ITS5 [30], LROR/LR5 [31], TEF1-α [32], mtSSU [30], and RPB2-6F/RPB2-7cR [33], respectively. The PCR reaction volume was 25 μL, comprising 12.5 μL of 2× Rapid Taq Master Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Sangon, Shanghai, China), and 1 μL of template genomic DNA. The reaction mixture was adjusted to a total volume of 25 μL using distilled deionized water. Amplification products were visualized using 1% agarose gel electrophoresis. Sequencing was performed by Fuzhou Tsingke Company (Fuzhou, China) using bidirectional (double-stranded) sequencing.

2.3. Alignment and Phylogenetic Analyses

To construct the phylogenetic tree of Russulaceae, we utilized sequences obtained from six fungal strains and reference sequences for multi-locus phylogenetic analyses which were obtained from Rehner and Buckley [32], Chen et al. [34], Deng et al. [35], Buyck et al. [36], and Roy et al. [37]. The newly generated sequences were screened for similarity through a GenBank BLAST search. The ITS, LSU, mtSSU, RPB2, and TEF1-α sequences were aligned using the MAFFT v. 7.11 online tool (https://mafft.cbrc.jp/alignment/software/ (accessed on 23 December 2023)), followed by manual adjustments in MEGA 7.0. Phylogenetic analyses employed both maximum likelihood (ML) and Bayesian inference (BI) methods. ML analysis was conducted using RaxML-HPC2 on XSEDE v. 8.2.12 via the CIPRES Science Gateway portal, while BI analysis was performed using MrBayes on XSEDE v. 3.2.7a (https://www.phylo.org/ (accessed on 25 December 2023)). The consensus tree was constructed using FigTree v. 1.4.4 and further refined using Adobe Illustrator CS 6.0. Newly generated sequences from this study have been deposited in GenBank. Branches showing ML bootstrap support values (≥70) and Bayesian posterior probability (≥0.90) were considered significantly supported.

3. Results

3.1. Phylogenetic Analyses

The multi-locus sequence matrix spans a length of 4350 bp. Its dataset comprises 700 bp of ITS, 890 bp of LSU, 1100 bp of TEF1-α, 800 bp of RPB2, and 860 bp of mtSSU. For the multi-locus region, the best substitution model for ITS and RPB2 in the BI analysis is SYM + G4, while for LSU and TEF1-α it is SYM + I + G4, and for mtSSU the best substitution model is GTR + F + G4. A total of 148 sequences, including newly generated ones, were deposited in the GenBank database (Table 1 and Table 2). Based on the foundational rank consistency of the phylogenetic topologies obtained from BI and ML analyses, only the ML trees are depicted in Figure 1 and Figure 2. The resulting phylogenetic trees demonstrate strong support for clades of the four new species in multi-locus phylogenetic analyses. These new species exhibit notable distinctions from known species (Figure 1 and Figure 2). Bootstrap and posterior probability values indicate robust support in multi-locus phylogeny for R. junzifengensis (from subsect. Virescentinae), R. zonatus (from subsect. Virescentinae), and L. jianyangensis (L. subsect. Zonarii), forming a distinct clade.

Table 1.

Species and specimens of Lactarius used for the molecular phylogenetic analyses.

Taxon Voucher Location GenBank Accession Number
ITS nrLSU RPB2 TEF1
L. albocarneus - China KX441117 KX441364 KX442105 -
L. aurantiozonatus HCCN10589 Korea MH984993 MH985118 MH936950 -
L. aurantiozonatus SFC20150901-06 Korea MH984976 MH985097 MH936929 -
L. austroscrobiculatus MHHNU 31161 China OL770185 - - -
L. austroscrobiculatus XHW3542 China OL770183 - - -
L. blennius MTB16 Germany MN947353 - - -
L. blennius - Sweden AY606944 - - -
L. brachycystidiatus KUN-HKAS 67933 China MF508951 - - -
L. brachycystidiatus KUN-HKAS 96483 China MF508950 - - -
L. brachycystidiatus KUN-HKAS 96442 China MF508949 - - -
L. brachycystidiatus KUN-HKAS 69233 China MF508948 - - -
L. brachycystidiatus KUN-HKAS 76061 China MF508947 - - -
L. brachycystidiatus KUN-HKAS69519 China MF508952 - - -
L. castaneus KUN-HKAS 47648 China MF508962 - - -
L. castaneus KUN-HKAS 61976 China MF508961 - - -
L. cinereus LM4663F Mexico FJ348708 - - -
L. cinereus CCB9 (TENN) USA MF755272 - - -
L. cinereus 2636 Canada KJ705204 - - -
L. cremicolor KA12-0268 Korea MH985013 - QCH40198 -
L. cremicolor HCCN19933 Korea MH984972 - QCH40153 -
L. dilutisalmoneus HMJAU 42465 China MF152847 - - -
L. dilutisalmoneus KUN-HKAS 61803 China MF152846 - - -
L. fluens IBL83 Poland MZ410712 - - -
L. fluens MC01-525 Denmark AJ889961 - - -
L. gloecorpus LH91 USA GQ268638 - - -
L. gloeocarpus KUN-HKAS 73602 China OL770166 - - -
L. gloeocarpus ZP-2287 (MHHNU) China OL770165 - - -
L. gloeocarpus Pa1-mOTU056 Japan LC315865 - - -
L. haugiae LM4994 Mexico KT583642 - AOF41440 -
L. haugiae LM4988 Mexico KT583641 - AOF41439 -
L. haugiae LM4957 Mexico KT583640 - - -
L. hunanensis MHHNU 31989B China OL770172 - - -
L. hunanensis MHHNU 31989A China OL770171 - - -
L. incrustatus KUN-HKAS 61789 China MK675285 - - -
L. incrustatus KUN-HKAS 61772 China MK675284 - - -
L. jianyangensis HMAS298103T China OR835448 OR826782 OR915862 OR887738
L. jianyangensis HMAS298104 China OR835446 PP033514 OR915863 OR887739
L. lignicola KUN-HKAS 59474 China MF508946 - - -
L. lignicola KUN-HKAS 54122 China MF508945 - - -
L. olivinus KUN-HKAS 116928 China OL770196 - - -
L. olivinus KUN-HKAS 118988 China OL770195 - - -
L. pallidizonatus KUN-HKAS 85389 China MF508932 - - -
L. pallidizonatus KUN-HKAS 62019 China MF508931 - - -
L. pallidizonatus KUN-HKAS 85388 China MF508933 - - -
L. pallidizonatus KUN-HKAS 47641 China MF508930 - - -
L. pallido-ochraceus KUN-HKAS 71574 China MF508942 - - -
L. pallido-ochraceus KUN-HKAS 54492 China MF508941 - - -
L. pallido-ochraceus KUN-HKAS 58675 China MF508940 - - -
L. pallido-ochraceus TC_277 China MW722813 - - -
L. pallido-ochraceus KUN-HKAS 59446 China MF508943 - - -
L. pallido-ochraceus KUN-HKAS 75991 China MF508939 - - -
L. parallelus SFC20150701-55 Korea MH984953 MH985072 MH936904 -
L. parallelus KA12-1557 Korea MH984921 MH985035 MH936867 -
L. pohangensis SFC20130807-68 Korea MH985018 MH985143 MH936975 -
L. pohangensis SN1-3870 China LC622651 - - -
L. psammicola 263825 USA MK607513 - - -
L. psammicola BPL869 USA KY848507 - - -
L. purpureocastaneus KUN-HKAS 61975 China MF508965 - - -
L. purpureocastaneus KUN-HKAS 56384 China MF508964 - - -
L. purpureocastaneus KUN-HKAS 49827 China MF508963 - - -
L. scrobiculatus QHU20107 China OM970920 - - -
L. scrobiculatus HMAS264940 China KX441085 KX441332 KX442073 -
L. scrobiculatus JN01-058 Thailand KF432968 - - -
L. scrobiculatus HMAS267747 China KX441098 MF893430
L. sinozonarius KUN-HKAS 69414 China MF508926 - - -
L. sinozonarius KUN-HKAS 56133 China MF508929 - - -
L. sinozonarius KUN-HKAS 48534 China MF508928 - 0 -
L. sinozonarius KUN-HKAS 96778 China MF508927 - - -
L. sinozonarius KUN-HKAS 54620 China MF508925 - - -
L. spadiceus SFC20121005-03 Korea MH985021 MH985146 MH936978 -
L. spadiceus NIBRFG0000104942 Korea MH984956 MH985076 MH936908 -
L. subbrevipes KUN-HKAS 85379 China MF508938 - - -
L. subbrevipes KUN-HKAS 61409 China MF508937 - - -
L. subbrevipes KUN-HKAS 57562 China MF508936 - - -
L. subbrevipes KUN-HKAS 62032 China MF508935 - - -
L. subbrevipes KUN-HKAS 94947 China MF508934 - - -
L. subzonarius KUN-HKAS 96630 China MF508960 - - -
L. subzonarius KUN-HKAS 73522 China MF508959 - - -
L. uvidus mh0963 Sweden AY606957 AF325293 - -
L. uvidus HMAS269338 China KX441140 KX441387 KX442128 -
L. vulgaris MHHNU 32017 China OL770178 - - -
L. vulgaris HYJ1878 (EFHAAU 476) China OL770175 - - -
L. vulgaris WNW33 (EFHAAU 1602) China OL770173 - - -
Multifurca zonaria FH12-009 Thailand KF432960 - - -
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Superscript “T” denotes the type strain of the new species.

Table 2.

Species and specimens of Russula used for the molecular phylogenetic analyses.

Taxon Voucher Location GenBank Accession Number
ITS nrLSU RPB2 mtSSU TEF1
R. adusta RDL 18-039 Belgium OM833079 - - - ON015965.1
R. aeruginea AT2003017 Sweden DQ421999 DQ421999 - - -
R. alboareolata SUT-1 Thailand AF345247 - - - -
R. albolutea RITF4460 China: Chongqing - MW397121 MW411341 MW403834 -
R. albolutea RITF4461 China: Yunnan - MW397122 MW411342 MW403835 -
R. albolutea RITF4462 China: Yunnan - MW397123 MW411343 MW403836 -
R. albolutea RITF2653 China MT672478 MW397120 MW411340 MW403833 -
R. amethystina SJ84 Pakistan KT953615 - - - -
R. amethystina SJ92 Pakistan KT953616 - - - -
R. amoenolens 12838 (MICH) France KF245510 - - - -
R. amoenolens 77763 (PDD) New Zealand GU222264 - - - -
R. ammophila IC09010703 (BCN) Spain MK112566 MK108033 - - -
R. aureoviridis H15060613 China KY767808 - - - -
R. aureoviridis RITF1868 China MW397096 - - MW403842 -
R. aureoviridis H16082612 China KY767809 MK881920 - MK882048 MN617846
R. aureoviridis RITF4709 China MW646980 MW646992 - MW647003 MW650849
R. brunneoaurantiaca GUBH19957 India OP270714 - - - -
R. brunneonigra H5813 (DAR) Australia EU019945 - - - -
R. cerolens HE2720 China KC505578 - - - -
R. cf. amoenolens 12794 (MICH) USA KF245512 - - - -
R. cf. vesca BB 06.525 Mexico - KU237465 KU237751 KU237309 -
R. crassotunicata UBCOGTR0439s Canada EU597082 - - - -
R. crustosa BPL265 USA: Tennessee - KT933826 KT933898 - -
R. cyanoxantha UE29.09.2002-2 France - DQ422033 DQ421970 - -
R. cyanoxantha FH 12-201 Germany KR364093 KR364225 - - -
R. farinipes UE28.09.2002-4 Sweden DQ421983 - - - -
R. fuliginosa R74 CZECH REPUBLIC HG798529 - - - -
R. grisea BB 07.184 Slovakia - KU237509 KU237795 KU237355 -
R. grisea UE2005.08.16-01 Sweden DQ422030 DQ422030 - - -
R. grisea FH12234 Germany KT934006 KT933867 - - -
R. ilicis MF 00.300 Italy - KU237595 KU237880 KU237443 -
R. illota UE26.07.2002-3 (UPS) Sweden DQ422024 DQ422024 DQ421967 - -
R. indoalba AG 15-628 India KX234820 - - - -
R. junzifengensis HMAS298101T China OR826832 OR826833 OR915864 OR941507 OR887742
R. junzifengensis HMAS298102 China OR880061 OR880054 OR915865 OR941508 OR887743
R. langei BB 07.792 France - KU237510 KU237796 KU237356 -
R. livescens F0177 China GU371295 - - - -
R. maguanensis XHW4765 China MH724918 MH714537 MH939990 - MH939983
R. mariae BB 07.038 USA - KU237538 KU237824 KU237384 -
R. medullata BB 07.252 Slovakia - KU237546 KU237832 KU237392 -
R. mustelina FH12226 Germany - KT933866 KT933937 - -
R. mustelina SA 09.88 Slovakia - KU237596 KU237881 KU237444 -
R. nigricans - Germany AF418607 - - - -
R. pallescens UBC:F23913 Canada KJ146729 - - - -
R. pallidula RITF2613 China - MH027960 MH091698 MW403845 -
R. pallidula RITF3331 China - MH027961 MH091699 MW403846 -
R. parvovirescens SDRM 6280 USA MK532789 - - - -
R. prasina HMAS 281232 China MH454351 - - - -
R. pseudocrustosa HBAU15015 China MT337520 - - - -
R. pulverulenta RF566 (pers. herb.) USA AY061736 - - - -
R. rufobasalis H17052204 China MH168570 MK881947.1 - MK882075.1 MT085585.1
R. subpunicea RITF1435 China: Hunan - MW397126 MW411346 MW403839 -
R. subpunicea RITF2615 China: Hunan - MW397127 MW411347 MW403840 -
R. subpunicea RITF3715 China MN833635 MW397124 MW411344 MW403837 -
R. subpunicea RITF2648 China MN833638 MW397125 MW411345 MW403838 -
R. substriata XHW4766 China MH724921 MH714540 MH939993 - MH939986
R. variata BPL241 USA - KT933818 KT933889 - -
R. vesca BPL284 USA KT933978 KT933839 - - -
R. vesca AT2002091 Sweden DQ422018 DQ422018 DQ421959 - -
R. violeipes BB 07.273 Slovakia KU237534 KU237820 KU237380 -
R. virescens HJB9989 Belgium DQ422014 DQ421955 - -
R. xanthovirens GDGM 71145 China MG786056 - - - -
R. zonatus HMAS298099 China OR826839 OR826846 OR915866 OR941505 OR887740
R. zonatus HMAS298100T China OR880062 OR880056 OR915867 OR941506 OR887741
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Superscript “T” denotes the type strain of the new species.

Figure 1.

Figure 1

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Phylogeny inferred from Lactarius multigene sequences (nrLSU, ITS, mtSSU, rpb2, and tef1-α) using Bayesian analysis. Support values in normal type are bootstrap support (BS, significant when ≥70%). Values in bold are Bayesian Posterior Probabilities (PP, significant when ≥0.95). The scale bar indicates the number of nucleotide substitutions per site. New species are highlighted in red. Arrows show the support values at the branching points. Superscript “T” denotes the type strain of the new species.

Figure 2.

Figure 2

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Phylogeny inferred from Russula multigene sequences (nrLSU, ITS, mtSSU, rpb2, and tef1-α) using Bayesian analysis. Support values in normal type are bootstrap support (BS, significant when ≥70%). Values in bold are Bayesian Posterior Probabilities (PP, significant when ≥0.95). The scale bar indicates the number of nucleotide substitutions per site. New species are highlighted in red. Arrows show the support values at the branching points. Superscript “T” denotes the type strain of the new species.

Lactarius jianyangensis showed the greatest similarity to Lactarius pallido-ochraceus, with an additional 84 sequences from Lactarius collected to construct the tree (Table 1, Figure 1). L. jianyangensis exhibited the highest genetic similarity to L. pallido-ochraceus and clustered with two other species, L. vulgaris and L. pallidizonatus (Figure 1). However, despite clustering with these three species, the substantial phylogenetic distance between L. jianyangensis and the other members of this clade supports its classification as an independent species. Two putative new species within the Russula genus, Russula junzifengensis, formed a strongly supported cluster (BS 100%) and were notably distinct from other known species within the Virescentinae group. Russula junzifengensis clustered together with an unidentified sequence from China (voucher: HMAS250919), which served as the sister clade to R. indoalba, supported by 98% bootstrap support and a posterior probability of 1. R. zonatus clustered alongside R. brunneoaurantiaca and formed a clade sister to R. brunneoaurantiaca with a posterior probability of 1.

3.2. Taxonomy

Russula zonatus S. Liu & Jun Z. Qiu, sp. nov. (Figure 3a,b and Figure 4).

Figure 3.

Figure 3

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SEM photos of basidiospores. (a,b) R. zonatus, (c,d) R. junzifengensis, and (e,f) L. jianyangensis. Scale bars: (a–f) = 10 μm.

Figure 4.

Figure 4

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Morphological characteristics of Russula zonatus (HMAS298100). (a,b) Basidiomata; (c) pileipellis in 5% KOH; (d) lamellae in 5% KOH. (e,f) Basidium in Congo Red reagent; (g) cystidium in Congo Red reagent; (h) basidiospores in Congo Red reagent; bars: (a,b) = 1 cm; (c–h) = 10 µm.

MycoBank: MB 851147.

Etymology: The epithet “zonatus” refers to the morphological feature of ring patterns on the surface.

Holotype: CHINA. Fujian Province, Mingxi County, Xiayang Town, Ziyun Village, in mixed forests, alt. 379 m, 26.34323138° N, 117.45805533° E, 30 August 2021, S. Liu and Jun Z. Qiu (holotype HMAS298100; paratype HMAS298099).

Description: Basidiocarps medium-sized to big. Pileus 7.5–11.9 cm in diam., convex-expanded to infundibuliform with a central depression and slight incurved margin, shallowly infundibuliform when mature, surface glabrous and dry with unclear or golden brown zone lines, no dark brown ring patterns on the surface, brown to grayish brown. Context 3 mm thick, satin white. Lamellae adnate, crowded, marble white, no forking, concolorous with the pileus when fully mature. Stipe 2.5 × 6–3.5 × 10 cm, central, equal, sometimes with fibrils, whitish or sub-concolorous with the pileus.

Basidiospores (6.4) 6.5–7.8 (8) × (5.2) 5.5–6.8 (7) µm (Q = (1.05) 1.06–1.19 (1.23), Q = 1.13 ± 0.05), broadly ellipsoid, with almost isolated warts, plage not amyloid. Basidia 32.5–49 × 10–13 µm, clavate, four-spored. Cystidium common, 30–50 × 5–7.5 µm, fusiform to lanceolate with campulitropal head. Lamella edge sterile, lamellae 8.74 µm thick.

Pileipellis duplex: gelatinous epicutis, 100–230 µm thick, hyphae 2–7 µm wide, hyaline to light yellow in 5% KOH, smooth. Hypodermium well developed, yellowish to lightly yellow intracellular pigment in 5% KOH.

Ecology and distribution: Gregarious in subtropical mixed forests (fagaceous forests or mixed forests with fagaceous trees). Known to inhabit Fujian Province, China.

Russula junzifengensis S. Liu & Jun Z. Qiu, sp. nov. (Figure 3c,d and Figure 5).

Figure 5.

Figure 5

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Morphological characteristics of Russula junzifengensis (HMAS298101). (a,b) Basidiomata; (c) pileipellis in 5% KOH; (d) lamellae in Congo Red reagent. (e) Basidium in Congo Red reagent; (f) cystidium in Congo Red reagent; (g,h) basidiospores in Congo Red reagent; bars: (a,b) = 1 cm; (c–h) = 10 µm.

MycoBank: MB 851146.

Etymology: Named after the Junzifeng Nature Reserve where the fungus was collected.

Holotype: CHINA. Fujian Province, Mingxi County, Junzifeng Nature Reserve, Xiafang Town, Zhushe Village, in mixed forests, alt. 410 m, 26.56490316° N, 117.03322856° E, 7 August 2021, S. Liu and Jun Z. Qiu (holotype HMAS298101; paratype HMAS298102).

Description: Basidiomata medium-sized, with a diameter of 40–60 mm. Initially hemispherical, later broadly convex to flat with a shallow depression, featuring a sub-transparently striate margin. The lamellae are densely crowded and sometimes slightly decurrent in mature and dry conditions, with a sharp, incurved, and even margin. The surface is glabrous, ranging from dry to slightly glutinous, presenting a satin white appearance, marble white at the center. In the mature stage, the central color turns to a shade of light yellow, pinard yellow, occasionally displaying maize yellow or light orange–yellow to capucine buff. The lamellae are adnate, densely packed, and of yellowish white color, without forking, becoming fragile and matching the pileus color when fully mature. The stipe measures 5.5 × 1.2 cm, central, cylindrical to slightly tapered upwards, rarely becoming subcylindrical to clavate, slightly narrowing towards the base, without an annulus. The stipe is white, appearing yellowish white, smooth in youth, later exhibiting fibrils on the surface. While young, it is full-bodied, eventually becoming hollow. The odor is indistinct.

Basidiospores (5.4) 6–7.5 (7.5) × (5.2) 5.5–6.5 (6.6) µm, [Q = (1.04) 1.05–1.25 (1.25), Q = 1.15 ± 0.06], ellipsoid, composed of small amyloid conical warts, mostly isolated, sometimes fused; indextrinoid, ornamentation small to medium-sized. Basidia 38.5–46.5 × 9–12 µm (smaller), clavate, four-spored, clavate, hyaline to light yellow in 5% KOH. Cystidia, 42 ×13.5 µm (smaller), thin-walled, hyaline in 5% KOH. Pileipellis well developed, separated from the surrounding spherocytes of the context, yellowish to lightly yellow intracellular pigment in 5% KOH.

Ecology and distribution: Gregarious in subtropical mixed forests (solitary or gregarious in Fagaceae forest). Known to inhabit Fujian Province, China.

Lactarius jianyangensis S. Liu & Jun Z. Qiu, sp. nov. (Figure 3e,f and Figure 6).

Figure 6.

Figure 6

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Morphological characteristics of Lactarius jianyangensis (HMAS298103). (a) Basidiomata; (b) pileipellis in Congo Red reagent (c) lamellae in Congo Red reagent. (d) Basidium in Congo red reagent; (e) cystidium in Congo red reagent; (f,g) basidiospores in Congo red reagent; bars: (a) = 1 cm; (b–g) = 10 µm.

MycoBank: MB 851149.

Etymology: Named after Jianyang District, where the fungus was collected.

Holotype: CHINA. Fujian Province, Nanping City, Jianyang District, in mixed forests, alt 841 m, 27.34220872° N, 118.16609715° E, 19 August 2021, S. Liu and Jun Z. Qiu (holotype HMAS298103; paratype HMAS298104).

Description: Basidiomata with a small size. Pileus 25–45 mm broad, initially hemispheric, becoming plano-convex and planate when mature, convex with inrolled margin, shallowly infundibuliform when mature, surface greasy when wet, aniline yellow, bittersweet pink, Titian red to agate, sometimes center salmon-orange or Mars yellow with a raw sienna margin, margin glabrous, sub-transparently striate. Context 3–4 mm thick, whitish to brown. The lamella 1–2 mm broad, Mikado orange to cadmium orange when young, xanthine orange, amber brown when mature, concolorous with the pileus when fully mature, sub-crowded to crowded, unequal length and extended. Additionally, the flesh of Lactarius has an aroma. Stipe 35–40 × 7–10 mm, central or tapering downwards, sometimes with longitudinal grooves, surface smooth, greasy, with scattered pits, whitish or sub-concolorous with the pileus, the end of the stipe was slightly enlarged, succulent and hollow, latex white or watery–milky.

Basidiospores (5.99) 6–7.7 (7.8) × (4.8) 4.9–6.3 (6.44) μm, [Q = (1.07) 1.09–1.34 (1.36), Q = 1.20 ± 0.09], broadly ellipsoid, surface has protuberance ridge to reticulate pattern, colorless to hyaline in KOH. Basidia 30.55–39.03 × 8.21–11.32 μm, four-spored, narrowly clavate, colorless to hyaline in KOH, sterigmata 2.84–3.87 μm. Clamp connections abundant in all tissues. The head of pleuromacrocystidia is warping, trama 4.53–4.81 μm.

Ecology and distribution: Gregarious in subtropical fagaceous forests. Known to inhabit Fujian Province, China.

4. Discussion

All Russula and Lactarius species characterized thus far form ectomycorrhizal symbioses with higher plants and trees, and both genera contain cosmopolitan as well as more host-specific members, with both edible and toxic species having been identified [20,38]. Lactarius is characterized by the production of latex, although the genus has now been separated into two (Lactarius and Lactifluus), with an additional separation of several species from Lactarius as well as Russula into Multifurca [39]. Most species of Lactarius form symbioses with broadleaf or coniferous hosts, consistent with their discovery in the Fujian forests of pine.

Russula are distinguished by their bright-colored caps, but do not produce latex and are often characterized by their brittle caps [40]. Due to the difficulty in separating species by their morphological characteristics alone, the modern identification of species within the Russula and Lactarius genera has relied on utilizing the ITS sequence in phylogenetic analysis as the primary molecular method for distinguishing and interpreting these closely related species [41]. However, an overreliance on ITS-based phylogenetic structures can lead to inaccurate subgenus classifications and may overlook the presence of known species, such as the R. queletii complex and the rhodochroa-subsanguinaria complex, often manifested within ITS-based phylogenetics [24]. Additionally, earlier studies focusing on Lactarius species found minimal consistency between Asian Lactarius species and those from other continents. Relying solely on ITS-based phylogenetic analysis and morphological characteristics for Lactarius species’ identification appears insufficient. Hence, the utilization of multi-locus phylogenetic analysis has become the preferred method for revealing the genetic relationships within the Russula and Lactarius genera.

Here, we employed a combined ITS-nrLSU-RBP2-mtSSU-TEF-1α multi-locus phylogenetic analysis method to support the identification of three species that have been named R. junzifengensis, R. zonatus, and L. jianyangensis. These assignments are based on combined morphological characterizations and molecular multilocus phylogenetic analyses. Russula zonatus appears to be a very common red mushroom in the subtropical-tropical Quercus forests of Fujian and is a member of the subgenus Crassotunicata. Key features for its identification include medium basidiocarps, a convex expanded to infundibuliform pileus with a central depression and slight incurved margin, glabrous and dry surface with indistinct or golden brown zone lines, brown to grayish brown color, very crowded lamellae, moderately ornamented basidiospores with isolated warts, and a subtropical habitat. R. zonatus forms a clade with R. brunneoaurantiaca (with a highest ITS identity of ~99%), R. adusta, and R. nigricans. All these species have a mucilaginous pileus and comparatively large spores and basidia. R. zonatus has a high similarity to R.brunneoaurantiaca’s ITS sequence (0.99%), but they have significant morphological differences. R. zonatus belongs to the subgenus Crassotunicata of the Russula genus and has medium to large basidiocarps, with the convex expanded to the infundibuliform pileus with a central depression and slight incurved margin. It has a glabrous and dry surface with indistinct or golden brown zone lines, a brown to grayish brown color, very crowded lamellae, and moderately ornamented basidiospores with isolated warts. R. zonatus has smaller basidia and cystidia compared to R. brunneoaurantiaca, and, at the macroscopic scale, additional differences are quite obvious, with R. brunneoaurantiaca having a surface that is mucilaginous, brownish orange turning yellowish brown to light brown, and a smooth stipe surface [37].

Russula junzifengensis is characterized by a white or slightly stained white–yellow or yellow pileus, which is broadly convex to flat with a shallow depression, slightly crowded lamellae, medium basidiospores with isolated warts, and a subtropical habitat. This species is similar to R. pseudocrustosa, R. indoalba, and R. xanthovirens, and phylogenetic analysis showed that R. junzifengensis formed a highly supported sister group with R. indoalba, but their ITS sequence similarity is less than 90%. Macromorphologically this species seems to be indistinguishable from R. indoalba; both species have whitish gray basidiomata, a clavate stipe, and ellipsoid basidiospores. But the lamellae of R. junzifengensis are not attached to the stipe and appear with fibrils on the stipe.

The phylogenetic results indicate that L. jianyangensis is closest to L. pallido-ochraceus and L. pallidizonatus. In comparison to L. pallido-ochraceus, both species have few pits on their stipe, a surface that is greasy when wet and basidiospores with reticulate ornamentation. In comparison to L. pallido-ochraceus, the basidiomata of the new species described here has a deeper color, smaller basidia, and narrower pleuromacrocystidia. The ITS similarity between the two species was 94%. Southern China’s Lactarius pallidizonatus X.H. Wang seems to be another closely related species [39]; L. pallidizonatus can be distinguished by its light orange–yellow margin, cream–whitish context, pits near the base on stipes, and basidiospores (80/4/3) (7.0) 7.5–9.0 (9.5) × (5.5) 6.0–7.5 µm [Q = (1.10) 1.12–1.29 (1.35), Q = 1.21 ± 0.05], however, L. pallidizonatus has a lighter color and bigger basidiospores than L. jianyangensis.

5. Conclusions

In this study, we employed a multi-locus phylogenetic analysis method, combined with morphological characteristics, to identify three new fungal species in the Quercus forests of Fujian province, namely R. junzifengensis, R. zonatus, and L. jianyangensis. These new species belong to the Russula and Lactarius genera, which form ectomycorrhizal symbioses with higher plants and trees. We found that relying solely on ITS sequences and morphological characteristics for species identification is insufficient, as it may lead to inaccurate subgenus classifications and the omission of known species. Therefore, we suggest using multi-locus phylogenetic analysis methods to reveal the genetic relationships within the Russula and Lactarius genera, as well as their phylogenetic affinities with species from other geographical regions. Our study provides new data on the fungal diversity and distribution in Fujian province, and also contributes new insights to fungal taxonomy and phylogeny.

Acknowledgments

We would like to thank Ling Wang, Longbin Lin, Huili Pu, Tianyu Tan, Taichang Mu, and Jie Zhao for their help with sample collection.

Author Contributions

Acquisition of funding, Y.C., H.S. and J.Q.; Collection of data, S.L., M.Z., N.O.K., Z.H., J.C., R.C., Y.D., Y.H., Z.W., X.G., C.Y., P.L., W.Z. and H.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 10 January 2024)). All new taxa were linked with MycoBank (https://www.mycobank.org/ (accessed on 10 January 2024)).

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the National Natural Science Foundation of China (No. 32270029, U1803232, 31670026), the National Key R & D Program of China (No. 2017YFE0122000), a Social Service Team Support Program Project (No. 11899170165), Science and Technology Innovation Special Fund (No. KFB23084) of Fujian Agriculture and Forestry University, a Fujian Provincial Major Science and Technology Project (No. 2022NZ029017), a Key Project from Fujian Provincial Department of Science and Technology (No. 2020N5005), and the Young and Middle-aged Teacher Education Research Project of Fujian Province (No. JAT210075).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 10 January 2024)). All new taxa were linked with MycoBank (https://www.mycobank.org/ (accessed on 10 January 2024)).

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