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

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).


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].
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.

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)).

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.

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 (Tables 1 and 2).Based on the foundational rank consistency of the phylogenetic topologies obtained from BI and ML analyses, only the ML trees are depicted in Figures 1 and 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 (Figures 1 and 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    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.
Ecology and distribution: Gregarious in subtropical mixed forests (fagaceous forests or mixed forests with fagaceous trees).Known to inhabit Fujian Province, China.
Basidiospores (6.4) 6.5-7.8 (8) × (5.2) 5.5-6.8Description: Basidiomata with a small size.Pileus 25-45 mm broad, initially h spheric, becoming plano-convex and planate when mature, convex with inrolled ma shallowly infundibuliform when mature, surface greasy when wet, aniline yellow, b sweet pink, Titian red to agate, sometimes center salmon-orange or Mars yellow w raw sienna margin, margin glabrous, sub-transparently striate.Context 3-4 mm whitish to brown.The lamella 1-2 mm broad, Mikado orange to cadmium orange young, xanthine orange, amber brown when mature, concolorous with the pileus fully mature, sub-crowded to crowded, unequal length and extended.Additionally Description: Basidiomata medium-sized, with a diameter of 40-60 mm.Initially hemispherical, later broadly convex to flat with a shallow depression, featuring a subtransparently 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.
Ecology and distribution: Gregarious in subtropical mixed forests (solitary or gregarious in Fagaceae forest).Known to inhabit Fujian Province, China.

Discussion
All Russula and Lactarius species characterized thus far form ectomycorrhizal sy oses with higher plants and trees, and both genera contain cosmopolitan as well as m host-specific members, with both edible and toxic species having been identified [20 Lactarius is characterized by the production of latex, although the genus has now separated into two (Lactarius and Lactifluus), with an additional separation of se 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 Ecology and distribution: Gregarious in subtropical fagaceous forests.Known to inhabit Fujian Province, China.

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.

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.

Figure 1 .
Figure 1.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 .
Figure 2. Phylogeny inferred from Russula multigene sequences (nrLSU, ITS, mtSSU, rpb2, and α) using Bayesian analysis.Support values in normal type are bootstrap support (BS, signifi when ≥70%).Values in bold are Bayesian Posterior Probabilities (PP, significant when ≥0.95) Figure 2. 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.

J
. Fungi 2024, 10, x FOR PEER REVIEW 10 of 18 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 5 .
Figure 5. 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 subtransparently 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 Lactarius jianyangensis S. Liu & Jun Z. Qiu, sp.nov.(Figures 3e,f and 6).

Table 2 .
Species and specimens of Russula used for the molecular phylogenetic analyses.