Four New Species of Russula Subsection Sardoninae from China

Four new species of Russula subsection Sardoninae from northern and southwestern China under coniferous and deciduous trees are proposed as R. begonia, R. photinia, R. rhodochroa, and R. rufa. Illustrations and descriptions of R. gracillima, R. leucomarginata, R. roseola, and the above four new species are provided based on evidence of morphological characters and phylogenetic analyses of the internal transcribed spacer (ITS), as well as the multi-locus of mtSSU, nLSU, rpb1, rpb2 and tef1-α. The relationships between these new species and allied taxa are discussed.


Introduction
Russula Pers. is an ectomycorrhizal genus of high species diversity which is commonly distributed throughout boreal, temperate and tropical ecosystems worldwide [1][2][3]. It often appears as one of the main ectomycorrhizal macrofungal groups in forest and shrubs [4][5][6]. This group, which has been estimated at at least 2000 species worldwide, plays a significant role in preserving the biodiversity of forest ecosystems. The symbiotic association of ectomycorrhiza established by Russulas and their host plants is able to improve the absorption of nutrients including nitrogen, water and minerals, which are helpful in overcoming barriers caused by adverse conditions [7,8]. Anatomical features of ectomycorrhiza are the conserved, morphological, infrageneric taxonomic bases for Russula [2]. The developed ectomycorrhizal network of Russula also breeds quantities of fruiting bodies, some of which are regarded as edible mushrooms with considerable regional economic value [9][10][11][12][13]. Taxonomy of Russula are based on macro-morphological characters in the early stage, such as pileus surface tinge, context taste, and spore print colour. Then, micro-morphological characters are introduced as taxonomic evidence of this genus, e.g., the shape and size of hymenial and pileipellis elements. The phylogenetic topology of certain DNA regions have been regarded as an important taxonomic basis in recent decades. The genes commonly analysed in Russula taxonomy are ITS, mtSSU, nLSU, rpb1, rpb2 and tef1-α [14,15].
The fairly large basidiomata, brightly coloured pileus, brittle context, and absence of annuluses or volva make Russula species distinguishable in the field. The Russula subsection Sardoninae Singer, a member of Russula section Russula Pers., is a representative group of the reddish-capped species of the genus [14]. This subsection is characterized by pink-to-lilac tinged stipes, adnate or subdecurrent lamellae, stiff, acrid tasted context, cream-to-ocher basidiospore print, and non-crusted pileocystidia in pileipellis [14,15]. 6f/frpb2-7cR [32][33][34]. Partial translation elongation factor 1-α (tef1-α) gene was amplified with primer pair EF-983F/EF-1567R [35]. PCR conditions in aforementioned references were applied. Amplification products were detected by 1.5% agarose gel electrophoresis. Then PCR product depurations were performed using a Biomed Nucleic Purification Kit. Sanger sequencing was performed with the same primers as for PCRs in a ThermoFisher ABI 3730XL DNA Analyzer by Biomed Gene Technology Company (Beijing, China). An ABI BigDye 3.1 Terminator Cycle Sequencing Kit was also applied in sequencing procedure. The newly acquired sequences in this analysis were deposited in GenBank (bold text in Supplementary Tables S1 and S2).

Phylogenetic Analyses
The ITS sequences of representative and closely related species of R. subsection Sardoninae were selected from GenBank (https://www.ncbi.nlm.nih.gov, accessed on 18 November 2022) and UNITE databases (https://unite.ut.ee, accessed on 18 November 2022) referred to in previous analyses [1,16,17,[19][20][21][23][24][25]28,[36][37][38][39][40][41]. Referential sequences for multi-locus phylogenetic analyses were from Looney et al. [1], Buyck et al. [2], and Chen et al. [25]. Newly generated sequences were screened for identity through a BLAST in GenBank. Voucher specimens from this analysis are listed in Supplementary Tables S1 and S2. Sequences were assembled and aligned in MAFFT 7.487 with E-INS-I strategy [42]. The matrix of dataset was manually adjusted with BioEdit 7.2.6 [43]. Multi-locus datasets were concatenated using SequenceMatrix v1.9 [44]. Species of Russula subsection Atropurpurinae Romagn, R. atropurpurea (Krombh.) Britzelm., R. krombholzii Shaffer, R. ochracea Fr. and R. vinacea Burl. were used as outgroups in ITS and multi-locus of phylogenetic analyses. Maximum likelihood (ML), maximum parsimony (MP), and Bayesian analyses were performed to clarify the ITS phylogenetic relationships between the new species and closely related known ones. Maximum likelihood and Bayesian analyses were performed for multi-locus phylogenetic analyses of mtSSU, nLSU, rpb1, rpb2 and tef1-α regions. Maximum likelihood analyses were carried out in raxmlGUI 2.0.3 [45]. The number of bootstrap (BS) replications was set to 1000. Substitution models of GTR + I, GTR + G, and GTR + G + I were used in ML analyses. The Bayesian analysis (BI) was conducted in MrBayes 3.2.7 using Metropolis-coupled Markov-chain Monte Carlo (MCMCMC) methods [46,47]. MrModeltest 2.3 was used for the estimation of best-fit model with Akaike information criterion (AIC) [48] for Bayesian analyses. The four Markov-chain runs were set to 1.5 × 10 6 generations. Trees were saved at frequency of every 100th generation. The standard deviation of split frequencies was stably kept below the 0.01 threshold when the running was over. The discard for burn-in phase of each analysis was the first 25% of sampling trees. The remaining 75% of trees were used for computation of Bayesian posterior probabilities (PPs), following a 50% majority-consensus rule. The convergences of runs were assessed using Tracer 1.7 [49]. MP analysis was performed in PAUP * 4.01 [50]. Gaps in alignment were regarded as missing data. All sites in the matrix were treated as unordered and unweighted. The algorithm in heuristic search of MP analysis followed the tree bisection-reconstruction (TBR). The stabilities of clades were assessed through bootstrap analysis of 1000 replicates [51]. A Kishino-Hasegawa test (KH test) was carried out to determine the significances of tree differences [52]. The values of consistency index (CI), retention index (RI), rescaled consistency index (RC), homoplasy index (HI), and tree length (TL) were also calculated. Trees were depicted and polished in Figtree 1.4.4 (http://tree.bio.ed.ac.uk/, accessed on 18 November 2022).
The ITS sequence matrix of this analysis was 576 bp in length, representing 41 species. The dataset contains 182 bp of ITS-1, 169 bp of 5.8S and 212 bp of ITS-2. Of the 576 characters in the ITS phylogenetic analyses, 368 characters were constant, 35 variable characters were parsimony-uninformative, and 173 characters were parsimony-informative. The tree has a CI of 0.442, an RI of 0.865, an RC of 0.382, an HI of 0.558, and a TL of 638. For BI analysis of the ITS region, the best substitution model is GTR + I + G. The total 142 sequences, including 60 newly generated ones, corresponded to 24 species and six complexes of R. subsection Sardoninae, as well as four species of an out group from Russula section Atropurpurinae.
The multi-locus sequence matrix is 4067 bp in length, representing 15 species. The dataset contains 564 bp of mtSSU, 879 bp of nLSU, 1253 bp of rpb1, 776 bp of rpb2, and 595 bp of tef -1α. For BI analysis of the multi-locus region, the best substitution model is GTR + I + G for mtSSU, nLSU, rpb1 and rpb2, and SYM + I + G for tef -1α. The total 211 sequences, including 136 newly generated ones, corresponded to 16 species of R. subsection Sardoninae and two species of an out-group species from Russula section Atropurpurinae.
Habitat and distribution: Single or scattered in coniferous and broad-leaved mixed forests. Note: The pileus surface of Asian collections contains mainly reddish tinges for this, one of the most conspicuous macroscopic characters. The pileus colours of the European collections are more complicated. Pale green, dirty violet, and, rarely, citrine tinges are also present on pileus. The European specimens differ in several characteristics, such as the following: basidiospore ornamentations composed of mostly isolated warts with few connections, hymenial cystidia prolonged into a voluminous attenuated or lanceolate apex, and a habitat of the Betula forest [14,15]. The morphological and phylogenetic analyses of this study reveal a vast distribution of R. gracillima in China. The geographic locali-ties of these samplings are from northern and northeastern parts of China. The flora of these known distribution areas are mainly Holarctic. It should be noted that the Asian distribution of this species was once reported as R. gracilis subsp. altaica Singer based on specimens of the Russian Altai Mountains. This taxon now has been regarded as a synonym of R. gracillima in subsequent studies. It has lower basidiospore ornamentations 0.2-0.5 µm in height [14,15]. As the Altai Mountains lie at the midpoint between Europe and East Asia, molecular phylogenetic analyses of R. gracillima in this region are needed to reveal its actual distribution in further studies. There are four collections from southwestern China (HMAS274621, HMAS274614, HMAS156221, and HMAS156227) nested with R. gracillima specimens in ITS phylogenetic analyses (Figure 1). The flora of ectomycorrhizal symbiotic plants for these four collections are different from the Holarctic flora for that of R. gracillima. HMAS274621 and HMAS274614 are collected from Hengduan Mountain region of Sino-Himalayan floral subkingdom. HMAS156221 and HMAS156227 are sampled from Yunnan-Myanmar-Thailand region of the Malay floral subkingdom. These specimens may represent some cryptic species; thus, they are not identified as R. gracillima in this study. Intensive morphological and multi-locus phylogenetic analyses are necessary for these samplings in future. Russula subsanguinaria can be distinguished from R. gracillima by the presence of brightly reddish tinged pileus, wider basidia of (25. Description: Basidiomata medium sized. Pileus 33-65 mm in diam., flat-hemispheric, umbonate to convex when young, turning planate at last, sometimes centrally concave, brightly reddish tinged, Carmine (I1i), Nopal Red (I3i), and Brazil Red (I5i) at the centre, occasionally faded to English Red (II7i) to Orange-Rufous (II11i), turning Sanford's Brown (II11k), Pecan Brown (XXVIII11 i) to Cacao Brown (XXVIII9 i) when mature, glossy, often glutinous when wet; margin acute, slightly incurved when young, the flat, occasionally wavy or uplifted, often cracked, non-striate, rarely indistinctly striate 1/6-1/5 from the edge inwards, peeling 1/6-1/4 of the radius, often with a reddish tinge of Testaceous (XXVIII9 ), Terra Cotta (XXVIII7 ), and Scarlet (I5). Lamellae adnate to adnexed, 2-4 mm in height at midway of the disc radius, occasionally forked near the stipe attachment, slightly interveined, White (LIII), unchanging when bruised, slowly becoming the pale cream tinge of Cartridge Buff (XXX19 f), Cream Color (XVI19 f) to Maize Yellow (III19f) when old; edge even, tapering towards the margin, crowded, 8-15 blades at 1 cm from the pileus margin, lamellulae rare. Stipe central, 41-79 × 9-17 mm, cylindrical, rarely clavate to subclavate, sometimes ventricose towards the base; annulus absent, smooth first, longitudinally rugulose when mature, surface mostly reddish fringed of Scarlet Red (I3), Nopal Red (I3i), and Spectrum Red (I1), often faded with pinkish tinges of Geranium Pink (I3d), Eosine Pink (I1d), and Strawberry Pink (I5d) towards the upper parts, initially stuffed, turning fistulous to hollow when mature. Context 2-4 mm thick at centre of disc, White (LIII) first, unchanging, slowly becoming pale cream fringe of Maize Yellow (III19f) to Cream Color (XVI19 f) when old, brittle, taste more or less acrid, even mild when old, odour indistinct. Spore print pale cream (Romagnesi IIa-IIb). ; suprahilar area disctinct, verruculose and amyloid. Basidia 30-41 × 9-13 µm, clavate to subclavate, rarely subcylindrical, four-spored, sometimes two-spored, hyaline; sterigmata 5-7 µm in length, often straight, at times slightly tortuous. Cheilocystidia 54-83 × 9-13 µm, clavate to fusiform, rarely subclavate, apex acuminate, often bluntly acuminate, moniliform, mucronate to lanceolate, contents sparsely distributed, granular, greyish in SV. Pleurocystidia dispersed to moderately numerous, ca. 700-1300/mm 2 , 50-85 × 8-13 µm, subfusiform to subclavate, rarely fushiform and subcylindrical, apex bluntly acuminate, sometimes moniliform or lanceolate, contents scattered, granular, greyish in SV. Subhymenium 30-40 µm thick, pseudoparenchymatous, not well-developed, composed of ellipsoid to subglobose cells 8-11 µm in width. Pileipellis composed of two layers, sharply distinguished from the sphaerocytes beneath. Suprapellis 80-110 µm thick, a trichoderm at pileus centre, composed of slantly oriented, nongelatinized hyphae, primordial hyphae absent, terminal cells cylindrical, 14-25 × 3-7 µm, apex obtuse, subapical cells cylindrical, rarely branched or inflated, a trichoderm at pileus margin, mostly horizontal to ascending, rarely vertical, terminal cells 15-35 × 3-7 µm, cylindrical, obtuse at apex. Pileocystidia abundant in pileus margin, often embedded from subpellis in pileus centre, long, cylindrical, multi-septate, 5-13 µm in width, apex obtuse, rarely constricted, contents granulate, partly sparse, greyish in SV. Subpellis a cutis, 150-230 µm thick, composed of mostly repent, cylindrical to inflated hyphae 4-11 µm in width. Clamp connections absent in all tissues.
Habitat and distribution: Single or scattered in broad-leaved forests. Note: This species is similar to R. exalbicans in its reddish-to-pinkish pileus usually distinctly fading and a habitat of the Betula forest, the latter differs in its greenish tinges intermixed on pileus surfaces, context turning greyish when wet, ochreous spore print (IIIa-IIIb), and larger basidiospores of 7-9.6 × 5.4-7 µm. Another species in R. subsect. Sardoninae whose pileus sometimes completely fade into citrine is R. gracillima; it can be distinguished from R. photinia by its greenish and dirty-violet tinges on pileus surfaces, longer hymenial cystidia of up to 100 µm in length, and higher ornamentations of up to 1.1 µm in height [14,15]. The red form of this species can be mistaken for R. begonia in the forest of northern China due to its reddish-to-pinkish tinged pileus and stipes, cream basidiospore print, and habitat of Fagaceae trees. Russula begonia differs in its having shorter basidia of 29-42 × 8-12 µm, longer hymenial cystidia of up to 75 µm in length, and an ixotrichoderm at the pileus centre. For those pure reddish-capped species in R. subsect. Sardoninae, R. helodes is different from R. photinia in its distinctly greyish context, ochreous spore print (Romagnesi IIIa-IIIb), and larger basidiospores of 8-10.5 × 6.6-8.2 µm with ornamentations forming a complete network; R. luteotacta differs in its conspicuous discolouring, sometimes even all white, very acrid tasting context, white spore print (Ia-Ib), and larger basidiospores 7-9 × 5.7-7.5 µm; R. sanguinea can be distinguished by its larger basidiospores of 7.2-9.6 × 6.3-7.4 µm with mostly isolated ornamentations, longer and wider hymenial cystidia of 60-150 (and more) × 8.5-18.5 µm, and a habitat of the Pinus forest; R. rhodopus is different from R. photinia in its having ochreous spore print (IIc-IIIb), basidiospores ornamentations subreticulate to reticulate, longer hymenial cystidia of up to 100 µm, and a habitat of coniferous forest [14,15].
Habitat and distribution: Single or scattered in coniferous and broad-leaved mixed forests. Note: The morphological character of the Xizang collections of this study are similar to that of Sichuan [25], except for the latter has narrower, mostly one-celled pileocystidia (3.8-)4.7-6.2-7.6(-9.5) µm in width. This species is reminiscent of R. torulosa and R. thindii in R. subsection Sardoninae due to its rosy red pileus and stipes, as well as coniferous-tree habitat. Russula thindii can be distinguished from its slightly bitter-tasting context, longer pleurocystidia of 55-123 × 12-15 µm with appendiculate apices, and shorter and narrower pileocystidia of 27-50 × 5-7 µm [23]; R. torulosa differs in its larger basidiomata up to 90 µm in pileus diameter, basidiospores with lower ornamentations up to 0.6 µm in height, long hymenial cystidia up to 150 µm in length [14,15]. For the other species close to the ITS phylogeny in Figure 1, R. americana is different from R. roseola in its larger basidiospores of 9-11.5 in diam. and habit of Abies forest [18]; R. fuscorubroides can be distinguished by its dark-purple-or brownish-violet-tinged pileus surface, lamellae later turning coarsely ochraceous and rarely greenish, and appendiculate hymenial cystidia apex [56]; R. queletii can be distinguished from the new species through its gooseberry-purple, wine-purple, and dark purple with brown tinges on its pilus surface, the fruity smell of its context, longer basidia of up to 60 µm in length, and hymenial cystidia attenuated to an acute, obtuse or finely capitate apexes [14,15]; R. salishensis differs in its occasional yellow-tobrown splotches on its pileus surface; fruity context odour; or, reminiscent of Pelargonium, subglobose to broadly ellipsoid basidiospores and narrower pileocystidia of up to 7.5 µm in width [20].

Discussion
Although ITS phylogeny analyses have been regarded as one of the standardized methods in the delimitation and clarification of closely related Russula species [57], accurate macro-and microscopic morphology based on statistics and symbiotic plant information in detail are essentials in taxonomy [2,58]. The preference of ITS phylogenetic topology may lead to inaccurate infrageneric classifications and the oversight of existing species. Russula queletii complex 1 and the rhodochroa-subsanguinaria complex in ITS phylogeny are representative examples (Figure 1). Multi-locus phylogenetic analyses have become the preferred technique for revealing ties of consanguinity and cryptic species from Russula genus in recent years [57,59]. The lowest amplification success rate in this study are rpb1 and tef-1α regions (ca. 57%). All of these four species, including R. begonia, R. photinia, R. rhodochroa and R. rufa, are supported by the morphological and multi-locus phylogenetic evidence of this study. This study also demonstrates wider distributions of R. gracillima, R. leucomarginata, and R. roseola. The seven species detailed in this study can be distinguished from the others in morphology.
The Russula subsection Sardoninae members described and illustrated in this study have mostly narrow, erect to synclinal, intricate, rarely branched terminal cells in pileipellis [14]. There are more inflated terminal elements in the pileipellis of R. begonia. Pileocystidia in suprapellis are always cylindrical, and often contain 1-3 septa. Russula rufa has wider pileocystidia compared with other species. The basidiospore ornamentation comparisons show that R. roseola has more interconnected warts and ridges, while R. rhodochroa and R. rufa have more isolated warts.
Some new Asian species of the R. subsection Sardoninae of this study may have been identified as European members such as R. exalbicans, R. gracillima, R. luteotacta, R. persicina, R. queletii, R. sardonia and R. sanguinea in morphology [60,61]. Intensive observations and ITS phylogenetic analyses indicated that some Chinese specimens have both similarities and differences [17,27,[62][63][64]. Combined support of morphology, ITS and multi-locus phylogenies and broad-leaved forest habitats suggest R. gracillima have certain possible distributions in both Europe and North China (Figures 1 and 2). This is because of the continuously distributed symbiotic host plants Betulaceae and Fagaceae members in the Holarctic region [65]. Recent phylogenetic analyses have not yet shown any support for the existence of R. luteotacta and R. sardonia in China. Certain degrees of ITS sequence differences (ca. 1-2%) have been detected in R. exalbicans, R. queletii and R. sanguinea collections from different continents [17,27,[62][63][64]. Multi-locus phylogenetic analyses of Chen et al. [25] and this study further revealed more Asian discoveries in R. subsection Sardoninae: specimens of "R. sanguinea" are R. leucomarginata, R. rhodochroa, R. roseola, and R. subsanguinaria; "R. queletii" samplings are R. rufa; specimens of "R. persicina" are R. begonia; a portion of "R. gracillima" specimens are R. photinia. Whether these Chinese collections of "R. exalbicans" are cryptic species or intraspecific geographical populations still needs further analyses. Several misidentifications in GenBank were detected in this work, such as North American samplings identified as R. fragilis of R. subsection Russula.
The phylogenetic topology ITS region of this study (Figure 1) generally corresponds with that of Chen et al. [25]. Clade F corresponds to the not highly supported branch composed of R. americana, R. fuscorubroides, R. leucomarginata, R. pseudopelargonia, R. rhodocephala, R. roseola, R. sanguinea (as "R. sanguinaria"), R. sardonia, R. subsanguinaria, and R. thindii in Figure 1 of Chen et al. [25]. Clade G matches the strongly supported branch of R. exalbicans, R. gracillima and R. persicina in Figure 1 of Chen et al. [25]. Similar correspondences are also present in multi-locus phylogenetic analyses of these two studies. Still, there are some topological differences between ITS and multi-locus phylogenies, e.g., R. luteotacta clusters with members of clade G in Figure 1 (Figure 2). There are a number of species in Figure 1 that still need further multi-locus analyses for a lack of reference sequences other than ITS. The representatives of these species are R. choptae, R. renidens, R. rhodopus, R. suecica, and R. vinosoflavescens. Habitat features can be detected from our analyses, which show that members of Clade F grow in coniferous forest, and those of Clade G live in broad-leaved forest. The taxonomic ideology of Sarnari [14] on R. subsection Sardoninae has been partially supported by molecular phylogeny. Thus, concrete symbiotic tree species are important in field work. The identification of ectomycorrhiza is necessary in future Russula taxonomy, especially for those samplings from mixed forest. Four series, Exalbicans, Sardonia, Persicina and Sanguinea, were proposed according to the criteria of pileus colour and forest types in Sarnari [14]. Chen et al. [25] and our analyses show a polyphyly of the R. subsection Sardoninae members with similar pileus colours in molecular phylogenetic topologies, even for those species with the same habitat. This work also provides further evidence of intraspecific pileus colour variations in R. subsection Sardoninae (Figures 3 and 6). Therefore, it is error-prone to use pileus as the taxonomic standard in this subsection. Our work indicates the plant flora of a habitat can be used as an interspecific delimitation standard for those species with high ITS similarities. Russula rhodochroa and R. subsanguinaria can be distinguished by their habitats. As there is an obvious floristic difference between Northeast China and the Yunnan-Guizhou-Guangxi regions in the Sino-Japanese forest subkingdom [65]. Intensive analyses are needed for the Chinese record of R. thindii (RITF2712) collected from the Hubei Province of the Sino-Japanese forest subkingdom, because the type specimen of this taxon is reported in forest of the Sino-Himalayan floral subkingdom [23].
Although the context and lamellae of R. subsection Sardoninae members are more or less acrid in taste, R. sanguinea and R. sardonia of this group have been recorded as edible fungi in China [66,67]. This can be explained by the fact that maturation and dehydration often turn the basidiome context from acrid to insipid in taste. As the Chinese distribution of these two species has not been supported by molecular phylogeny, the edibility of R. subsection Sardoninae still needs further sampling and food safety analyses. Basidiomata of R. rhodochroa are occasionally collected and sold with other mild-tasting Russulas in southeastern region of Guizhou Province, according to our recent surveys. Red-capped Russula members in northeastern China are often regarded as inedible species with the local name "coffin lid". As raw and incomplete cooked R. subsection Sardoninae members may cause gastrointestinal symptoms [68], it is highly risky to label these species as edible fungi.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof9020199/s1, Table S1: The species, vouchers, locations, databank accession numbers and references of ITS phylogenetic analyses; Table S2: The species, vouchers, locations, GenBank accession numbers and references of multi-locus phylogenetic analyses. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence data are available in UNITE and NCBI GenBank following the accession numbers in the manuscript.