Morphological Characters and Molecular Phylogeny Reveal Three New Species of Subgenus Russula from China

Three new species are described and illustrated here based on morphological evidence and phylogenetic analysis from China. Russula leucomarginata is recognized by a yellowish red to reddish brown pileus center, a yellowish white to reddish white and sometimes cracked margin, and a reddish white to pastel pink stipe. Russula roseola is characterized by its reddish white to ruby red pileus center, pink to rose margin, adnate to slightly decurrent lamellae with unequal-length lamellulae, reddish white to pink stipe, and occasionally three-celled pileocystidia. Russula subsanguinaria is morphologically characterized by a reddish brown to dark brown pileus center, a reddish orange to brownish red margin with striation, a reddish white to pink stipe with an expanded base, basidiospores with moderately distant to dense amyloid warts, and hymenial cystidia turning to reddish black in SV. In this study, we performed phylogenetic analysis based on ITS sequence and 28S-RPB1-RPB2-mtSSU datasets. Detailed morphological features and phylogenetic analysis indicate that these three new species belong to Russula subg. Russula.


Introduction
Russula Pers. is a cosmopolitan group and occurs across a wide range of habitats, from the arctic tundra to tropical forests, and forms ectomycorrhizal relationships with diverse host plants [1,2]. It is the largest of all genera in Russulaceae, which includes eight subgenera and at least 2000 species within the genus [3][4][5][6]. Some species of Russula are important edible fungi and are also commercially traded around the world [2,[7][8][9]. According to the recent statistics on resource diversity of Chinese macrofungi, there are 78 edible species and 30 medicinal species in China [10]. The precise identification of Russula is all-important for the development and utilization of these fungi.
Recently, some new species of subgenus Russula have been successively reported in Asia [14,15], indicating that many unknown Russula species are waiting to be discovered. To better understand the biological diversity and geographic distribution of Chinese subgenus Russula, extensive surveys have been undertaken in different parts of China. During the investigation and sampling, several interesting specimens were found. Morphological characters and phylogenetic analysis consistently supported their independence from other known Russula species. Here, three taxa are proposed as new to science, based on detailed morphological descriptions coupled with illustrations and phylogenetic relations.

Morphological Study
The macromorphological characteristics of collections were photographed and annotated in the field. Specimens were dried at 45-55 • C and deposited in the herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF). Macromorphological characters were based on detailed notes and photographs of fresh basidiomata. Descriptive terms follow widely used mycological glossaries [16]. Color codes and terms are mostly from Kornerup and Wanscher [17]. Micromorphological features were described using the description templates of Adamčík et al. [5]. All were performed from dried specimens with a ZEISS Imager M2. Basidiospores were observed and measured in Melzer's reagent in side view excluding ornamentation. Other microscopic structures were observed and measured in Congo red after pretreatment in 5% potassium hydroxide (KOH). Estimates of spore ornamentation density followed Adamčík and Marhold [18]. The hymenial cystidia density estimates refer to Buyck [19]. Pileipellis was examined in Cresyl blue to verify the presence of ortho-or metachromatic reactions [20]. Sulfovanillin (SV) was used to observe the color changes of cystidia contents [21]. The ornamentation and structure of basidiospores were illustrated under a scanning electron microscope (SEM-JEOL JSM-6510). Basidiospore measurements are indicated as (Min-)AV-SD-AV-AV+SD(-Max), where Min = the minimum value, Max = the maximum value, AV = average, SD = standard deviation, and Q stands for length/width ratio of the basidiospores.

Molecular Study
Genomic DNA was extracted by an improved CTAB protocol from dry specimens [22]. In the present study, five loci were amplified and sequenced: 600 base pairs of the ITS region of rDNA using primers ITS1 and ITS4 [23]; 900 base pairs of the 28S nuc rDNA (28S) with primers LR0R and LR5 [24]; 1300 base pairs of the largest subunit of the RNA polymerase II (RPB1) using primers RPB1-AF [25] and RPB1-CR [26]; 700 base pairs of the second largest subunit of the RNA polymerase II (RPB2) with primers bRPB2-6f and fRPB2-7cr [26,27]; and 600 base pairs of the ribosomal mitochondrial small subunit (mtSSU) using primers MS1 and MS2 [23]. The amplified PCR products were subsequently sequenced on an ABI 3730 DNA analyzer using an ABI BigDye 3.1 terminator cycle sequencing kit (Shanghai Sangon Biological Engineering Technology and Services CO., Ltd., Shanghai, China). The newly generated sequences were submitted to GenBank (Table 1).

Phylogenetic Analysis
Species in the subg. Russula core clade with high similarity to our new species and representative species were selected for phylogenetic analyses. Accession numbers for ITS sequences are shown in Figure 1. Accession numbers and references of the sequences used in the multigene phylogenetic tree are given in Table 1. Initial sequence alignment was performed by MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/ (accessed on 1 March 2022)). To obtain reliable and reasonable results, the online program Gblocks 0.91b (Gblocks_server.html) was used in default parameters; approximately 77.8% of sites retained after Gblock analysis. The final sequence alignment was deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S28485 (accessed on 1 March 2022)). Maximum Likelihood (ML) and Bayesian Analysis (BA) were implemented for phylogenetic analyses. ML analysis was performed using RAxML-HPC2 on XSEDE (8.2.12) through the Cipres Science Gateway (www.phylo.org (accessed on 1 March 2022)). The analysis was executed by applying the rapid bootstrap algorithm with 1000 replicates to affirm the consistency of the results under the GAMMA model. Only the maximum-likelihood best tree from all searches was kept. For BA analyses, the GTR model was selected as the best substitution model by MrModeltest [29]. BA was carried out with MrBayes on XSEDE (3.2.7a) through the Cipres Science Gateway (www.phylo.org (accessed on 1 March 2022)) under the GTR model. Four Markov chains were run for a total of 50,000,000 generations and trees were sampled every 100 generations, the first 25% of each sampled topology being discarded as part of the burn-in procedure. Bootstrap Support (BS) ≥ 70% was considered significantly supported. Bayesian Posterior Probability (PP) values were calculated from the 50% majority rule consensus trees, and PP ≥ 95% were regarded as significant.

Phylogeny
The ITS and 28S-RPB1-RPB2-mtSSU datasets were used in Maximum Likelihood and Bayesian Analysis. Both the ML and BA analyses resulted in essentially the same tree topologies, and only the ML tree is shown in Figures 1 and 2. BA posterior probabilities are also shown along the branches.
The ITS phylogenetic analysis showed that subg. Russula obtained a high support (BS 100%, PP 1). The samples of the three new species R. leucomarginata, Russula roseola, and R. subsanguinaria formed strongly supported clades (BS 100%, PP 1.00) and were clearly distinct from known species of subg. Russula. Russula leucomarginata clusters together with R. rhodocephala Bazzicalupo, D. Miller and Buyck. by 83% bootstrap support and forms a

Results Phylogeny
The ITS and 28S-RPB1-RPB2-mtSSU datasets were used in Maximum Likelihood and Bayesian Analysis. Both the ML and BA analyses resulted in essentially the same tree topologies, and only the ML tree is shown in Figures 1 and 2. BA posterior probabilities are also shown along the branches.
The ITS phylogenetic analysis showed that subg. Russula obtained a high support (BS 100%, PP 1). The samples of the three new species R. leucomarginata, Russula roseola, and R. subsanguinaria formed strongly supported clades (BS 100%, PP 1.00) and were clearly distinct from known species of subg. Russula. Russula leucomarginata clusters together with R. rhodocephala Bazzicalupo, D. Miller and Buyck. by 83% bootstrap support and forms a sister clade to R. subsanguinaria without support. Russula sanguinaria (Schumach.) Rauschert For multigene phylogenetic analysis, the results were similar to those of the ITS phylogenetic analysis. Subg. Russula formed a well-supported monophyletic group. The three new species were clearly separated from the known species.  For multigene phylogenetic analysis, the results were similar to those of the ITS phylogenetic analysis. Subg. Russula formed a well-supported monophyletic group. The three new species were clearly separated from the known species. R. subsanguinaria without support. However, R. rhodocephala is different from R. leucomarginata with a very firm basidiomata, a reddish orange pileus margin without striations, a strongly amyloid patch suprahilar spot, often longer basidia ((36.5-)41-45-49(-55) × (9-)10-11-12(-14) μm), and the association with Pinus [30]. Russula subsanguinaria differs from R. leucomarginata by its association with Pinus, a reddish orange to brownish red pileus margin, shorter hymenial cystidia on lamellae sides and marginal cells, and often longer pileocystidia. Similarly, European species R. sanguinaria is different from R. leucomarginata. Russula sanguinaria has a habitat mostly under Pinus and variable pileus diameter (27-120 mm), always smooth pileus margin, not peelable cuticle, fruity odor, often subacute to acute hymenial cystidia on lamellae sides, and usually shorter hymenial cystidia on lamellae edges (33-70 × 5-7.5 μm) [31,32].  MycoBank: MB838150 Diagnosis: Recognized by its cracked pileus, yellowish red to reddish brown pileus center, yellowish white to reddish margin, and reddish white to pastel pink stipe. It is mainly separated from similar-looking species by the internal transcribed spacers (ITS) sequence data. The similarity is less than 97% with all these species.
Etymology: Subsanguinaria (Latin), named so because it is similar to R. sanguinaria. MycoBank: MB838149 Diagnosis: Mainly characterized by its reddish white to ruby red (12D8) in the pileus center, pink (11A4) to rose margin, adnate to slightly decurrent lamellae, often present lamellulae, reddish white to pink stipe, clavate or ellipsoid basidiola, and occasionally three-celled pileocystidia. The similarity of ITS sequence data of R. roseola is less than 97.5% with all similar-looking species.