Morphological Characteristics and Phylogeny Reveal Six New Species in Russula Subgenus Russula (Russulaceae, Russulales) from Yanshan Mountains, North China

Species of the genus Russula are key components of ectomycorrhizal ecosystems worldwide, some of which are famous edible fungi. Although many new species have been described in China, their diversity in North China is still poorly known. Based on the morphology observation of specimens and molecular phylogenetic analyses, combined with the current classification frame of Russula, six new species of Russula subgenus Russula are proposed from the Yanshan Mountains in northern Beijing and northern Hebei Province of China in this study: viz. Russula miyunensis (subsection Chamaeleontinae), R. plana (subsection Chamaeleontinae), R. sinoparva (subsection Puellarinae), R. sinorobusta (subsection Puellarinae), R. subversatilis (subsection Roseinae), and R. yanshanensis (subsection Puellarinae). This is the first report of the species of Russula subgenus Russula from the Yanshan Mountains. This study enriches the species diversity of Russula in North China and provides new data support for the systematic study of Russula in subsequent research, including research and development on edibility.


Introduction
Russula Pers. (Russulaceae, Russulales, Agaricomycetes, and Basidiomycota) was established in 1796, which is one of the most abundant genera, including at least 2000 species [1,2]. This genus is mainly characterized by colorless to multi-colored pileus, amyloid warty basidiospores, abundant spherocytes in a heteromerous trama, an absence of latex, and hyphae without clamp connections [3][4][5]. Russula is a large genus of ectomycorrhizal (ECM) fungi that are found in all common ecosystems, such as broad-leaved forest, coniferous forest, mixed coniferous, broad-leaved forest, or scrubland [6][7][8]. Furthermore, some members of Russula not only play an important role in ecology by symbiotic with a variety of plants but also serve as a food source for many animals, including humans. Some species of Russula, e.g., Russula delica Fr., Russula griseocarnosa X.H. Wang, Zhu L. Yang, & Knudsen, Russula nigricans Fr. et al., are famous edible fungi and important commercial trade goods in the world [7,[9][10][11][12]. According to recent statistics on the diversity of Chinese edible macrofungi resources, there are about 70 edible species in China [12]. The previous classification system for Russula was based on morphology, e.g., pileus color, spore print, and spore. Miller and Buyck et al. first used phylogenetic analysis of nrITS loci to compare with the previous classification system of Russula in Europe, resulting in 78 Russula species forming six clades with higher supported values on the phylogenetic tree [13]. Buyck et al. [1,2] demonstrated that Russula was one of four monophyletic groups in non-corticoid Russulaceae and was divided into eight subgenera by multi-locus phylogenetic studies [1,14].
The subgenus Russula Pers. is a species-rich subgenus of Russula, which is morphologically characterized mainly by a great variation of basidiocarp size, pileus thick to extremely thin fleshed; stipe abnormally annulate gills unusually equal or lamellulae; spore print white to yellow; spores with amyloid suprahilar spot. Phylogenetically, this subgenus is divided into two parts: a core and a crown clade [1,2].
The Yanshan Mountains (115 • -119 • 47 E, 39 • 40 -41 • 20 N) are located in North China and have a warm temperate continental monsoon climate. This region is known for its high plant diversity. The main forest types of this region are deciduous broad-leaved forest and mixed coniferous and broad-leaved forest. Dominant ectomycorrhizal trees in this region include Pinus tabuliformis Carr, Betula spp., Quercus spp. and Abies (Mill.) spp. The Yanshan Mountains have an annual precipitation of approximately 350-700 mm, and their altitude ranges from 200 to 2200 m [37,38]. Until the present study, records about the Russula species in this area were very few [39].
In this study, six new species of the subgenus Russula crown clade from the Yanshan Mountains were described based on multi-locus phylogenetic analyses and detailed macroand micromorphological data. The aims of this study are to identify the taxonomic status and phylogenetic position of new species, to establish a comprehensive database on the diversity of macrofungal in North China, especially the status of Russula in the Yanshan Mountains, and to use this as a basis for promoting research on macrofungal diversity and edible Russula species in this region.

Sampling and Morphological Observations
Specimens were collected from 2017 to 2021. Fresh specimens were photographed in the field, and characteristics such as color, odor, and viscosity were noted. Specimens were dried with a Dorrex dryer at 45 • C and deposited in the Herbarium of the College of Life Science, Capital Normal University, Beijing, China (BJTC). Macroscopic characteristics were recorded from fresh specimens. Microscopic characteristics were observed from thin sections of dried material mounted in 3% KOH or sterilized water. Congo Red (1%) was used to make the structures more visible. Melzer's reagent was used to test the amyloid reaction of the spores [40]. All tissues were also examined in cresyl blue to verify the presence of ortho-or metachromatic reactions, as explained in Buyck [1]. Cystidia contents were examined in sulfovanillin (SV) solution [40]. Microscopic structures (e.g., basidiospores, basidia, cystidia) were observed and measured using a light microscope (Olympus DP71, Tokyo, Japan) and Image Pro Plus 6.0. The basidiospore structures were further observed under a field emission scanning electron microscope (SEM, Hitachi S-4800, Tokyo, Japan), digital cameras (Olympus U-TV0.5XC-3, Tokyo, Japan), and measuring software (Image Pro Plus 6.0). Basidiospore measurements were presented as (Min-) AV-SD-AV-AV + SD (-Max), where Min is the minimum value, Max is the maximum value, AV is the average value, SD is the standard deviation, and Q represents the length/width ratio of the basidiospores [8]. Statistics for the microscopic characteristics (e.g., basidiospores and basidia) were based on 30 measurements per specimen. The descriptive terms follow Adamčík et al. [2]. In this study, color codes were used from the reference website colorhexa (https://www.colorhexa.com (accessed on 8 September 2022)).

DNA Extraction and Sequencing
DNA extraction was achieved via the M5 Plant Genomic DNA Kit (Mei5 Biotechnology, Co., Ltd., Beijing, China). The DNA obtained was dissolved in 1 × TE buffer/sterile water and stored at −20 • C for later use. The PCR amplifications were performed in a Bio-Rad S1000 TM Thermal Cycler (Bio-Rad Laboratories, Inc, Hercules, CA, USA). The primer set nrITS 1f/nrITS 4 was used to amplify for rDNA ITS region [41], T LR0R/LR5 for the large subunit nuclear ribosomal DNA (nuLSU rDNA) region [42], MS1/MS2 for the ribosomal mitochondrial small subunit (mtSSU) region [41], RBP2-6f/RBP2-7r for the second largest subunit of RNA polymerase II (rpb2) region [43] and tef1F/tef1R for the second largest subunit of transcription elongation factor 1-alpha (tef-1α) region [44], respectively. The PCR volume was 25 µL, and the detailed composition was described by Zhou et al. [39]. PCR amplification conditions for nrITS and nrLSU refer to Li et al. [25]. PCR amplification conditions for mtSSU and rpb2 refer to Song et al. [8]. PCR amplification conditions for tef-1α refer to Morehouse et al. [44]. DNA sequences were sequenced by Zhongkexilin Biotechnology, Co., Ltd., Beijing, China. Newly obtained sequences in this study were submitted to the NCBI GenBank database (https://submit.ncbi.nlm.nih.gov/ (accessed on 20 July 2022)). Accession numbers of sequences used for phylogenetic analyses are provided in Figure 1 and Table 1.

Molecular Phylogenetic Analyses
Raw reads of the generated DNA sequences were used to obtain consensus sequences using SeqMan v.7.1.0 (DNASTAR Inc., Madison, WI, USA). All sequences (nrITS, nrLSU, rpb2, tef-1α and mtSSU) were analyzed using MAFFT v.6 and manually trimmed using MEGA 6 [45]. All reference sequences of subgenus Russula of dataset were chosen for phylogenetic analyses based on previous studies and GenBank database in NCBI.
The nrLSU-rpb2-tef-1α-mtSSU multi-locus phylogenetic analysis included 83 ingroup samples, which were used to analyze the phylogenetic position of our specimens in the genus Russula. Moreover, the nrITS phylogenetic analysis included 129 ingroup samples, which were used to analyze the relationships among our collections and other species in the subgenus Russula. All reference sequences of subgenus Russula of the dataset were chosen for phylogenetic analyses based on previous studies and the GenBank database in NCBI and UNITE (accession number in Table 1 and Figure 2 [38,[46][47][48][49]. ML analysis used a GTR locus substitution model by running 1000 bootstrap replicates with all default settings parameters [50]. Bayesian inference (BI) phylogenetic analysis was performed using MrBayes v.3.1.2 [51]. Branch supports were calculated using a bootstrapping (BS) method with 1000 replicates [52]. Bayesian inference (BI) analysis was performed by the Markov chain Monte Carlo (MCMC) algorithm [53]. MrModeltest v. 2.3 was used to estimate the best model for Bayesian inference (GTR + I + G for nrITS, nrLSU, rpb2, and mtSSU; SYM + I + G for tef-1α) [51]. Two MCMC chains were run from the random trees for 10,000,000 generations and stopped when the mean standard deviation of split frequencies fell below 0.01. Trees were saved once every 1000 generations by the default settings. The first 25% of trees were discarded as the burn-in phase of each analysis. In the remaining trees, branches with significant Bayesian posterior probabilities were estimated, it has relatively stable topologies, and clades with high Bayesian posterior probability (pp) values can also illustrate relative relationships between species [54]. ML bootstrap support (BS) ≥ 50% and Bayesian posterior probability (PP) ≥ 0.95 were shown on the nodes in Figures 1 and 2.    Table 1. Asterisks (*) denote branches with pp = 1.00, MLb = 100%.

Phylogenetic Analyses
The nrITS phylogenetic analysis included 129 ingroup samples, M. zonaria (DED7442) and M. ochricompacta (BB02.107) were used as the outgroups. The dataset of nrITS loci comprised 611 characters including alignment gaps. The best Bayesian tree is shown in Figure 1. The nrLSU-rpb2-tef-1α-mtSSU multi-locus phylogenetic analysis included 83 ingroup samples, M. ochricompacta (580/BB 07.010) and M. aurantiophylla (644/BB 09.119) were used as the outgroups. The dataset of multi-locus comprised 2511 characters including alignment gaps. The nrLSU-rpb2-tef-1α-mtSSU dataset was analyzed by ML analysis and BI analysis. Phylogenetic analysis generated topologies from ML analysis and BI analysis were almost identical, and the Bayesian tree are shown in Figure 2.
The nrLSU-rpb2-tef-1α-mtSSU and nrITS phylogenetic analyses revealed that the subgenera proposed by Buyck et al. [1] were well-supported with significant Bayesian posterior probability (PP) values and maximum likelihood bootstrap (MLB). Sequences of our collections all fell into the Russula subgenus Russula crown clade and formed six new lineages (marked in red and bolded in Figures 1 and 2) with significant support. Thus, they were considered as six distinct clades and described as new species in this paper, i.e., Russula miyunensis, R. plana, R. sinoparva, R. sinorobusta, R. subversatilis, and R. yanshanensis.
The nrLSU-rpb2-tef-1α-mtSSU multi-locus phylogenetic analysis showed that two sequences of the new species Russula sinoparva (BJTC C540, BJTC Z441) were supported as one clade (pp = 1.00, MLB = 100%) and clustered with Russula odorata Romagn. The nrITS phylogenetic analysis showed similar topologies to that of multi-locus phylogenetic tree, and sequences of our collections also formed six strong support end branches. Notebly, R. subversatilis and R. sinoparva were clustered together with R. khinganensis G.J. Li & R.L. Zhao. Russula miyunensis and R. plana formed a well-supported clade. Russula sinorobusta formed a clade clustered with Russula lepidicolor Romagn. and Russula intermedia P. Karst. but without support values.

Taxonomy
Based on phylogenetic analyses and morphology, six new species of Russula subgenus Russula from the Yanshan Mountains were recognized and described in this study. MycoBank: MB 845047 Diagnosis: Russula miyunensis is diagnosed by small to big-sized basidiomata, light pink or grayish-yellow, central deep red to deep brown pileus, basidiospores ornamented with amyloid warts, and more or less chain-like, suprahilar spot obvious, longer basidia, shorter terminal cells near the pileus margin, pileocystidia without color change in sulfovanillin. Morphologically, R. miyunensis is similar to Russula olivascens Fr. and Russula clavatohyphata R.P. Bhatt, A. Ghosh, Buyck, & K. Das, but pileus of R. miyunensis has severely cracked when mature.    Etymology: The epithet "miyunensis" referred to the locality "Miyun District" where the type specimen was collected.

MycoBank: MB 845048
Diagnosis: Russula sinoparva is diagnosed by small basidiomata, light pink to pink pileus, subglobose to broadly ellipsoid basidiospores ornamented with small amyloid warts, the absence of hymenial cystidia on lamellae edges, bigger hymenialcystida on lamellae sides. Russula sinoparva and Russula cessans A. Pearson have similar morphological characteristics, but R. sinoparva has light pink to pink pileus and smaller basidiospore, and bigger hymenial cystidia on lamellae sides. Etymology: The epithet "sinoparva" refers to this Chinese species that has smaller basidiomata resembling the Russula parva Carteret & Reumaux.

MycoBank: MB 845050
Diagnosis: Russula sinorobusta is diagnosed by small to medium-sized basidiomata, gray-red to rose red, central deep red pileus, basidiospores ornamented with small amyloid warts, suprahilar spot small, longer basidia, shorter hymenial cystidia on lamellae sides, pileocystidia absent. Morphologically, R. sinorobusta is similar to R. intermedia and Russula vinosa Lindblad., but R. sinorobusta has gray-red to rose red, central deep red pileus, and smaller basidiospore. Etymology: The epithet "sinorobusta" refers to the stipe of this Chinese species that is relatively sturdy, resembling the Russula robusta R. Heim.

Discussion
The topological structure of the two trees of the nrITS phylogenetic analysis (Figure 1) and the nrLSU-rpb2-tef-1α-mtSSU phylogenetic analysis ( Figure 2) are basically similar, but the Bayesian posterior probability values and maximum likelihood bootstrap were higher in the nrLSU-rpb2-tef-1α-mtSSU analysis. In recent studies, many new species of Russula have been described only by nrITS loci phylogenetic analysis [14,25,62], which also results in the lack of other gene sequences and may cause some difficulties when performing multi-gene phylogenetic analysis. Therefore, it is crucial to discover better-differentiated DNA barcodes within the subgenus Russula in subsequent studies.
Through the current investigation, most species of the Russula were found in broadleaved forests and associated with Carpinus turczaninowii, Castanea mollissima, J. mandshurica, B. costata, and P. davidiana trees, few species of the Russula occurred in coniferous forests, such as P. tabuliformis. The distributing characteristics may be related to the large area of broad-leaved forest in the Yanshan Mountains.
Russula Subgenus Russula has a very high species richness worldwide. At least 50 novel species have been described based on both morphological characters and molecular data since 2006, of which at least 30 species were reported from Asia [35]. In previous research, 16 species of Russula subgenus Russula are reported from southern China. [9,18,23,[32][33][34][35]63,64]. Moreover, six species are from Northeast China [18,24,65,66]. Regarding the provincial distribution in China, more species of subgenus Russula are found in Guangdong Province and Heilongjiang Province. The reason for this phenomenon may be that these two provinces are actively investigated by mycologists.
In this study, six new species belonged to three subsections under the subgenus Russula, namely subsection Chamaeleontinae (R. miyunensis and R. plana), subsection Puellarinae (R. sinoparva, R. sinorobusta, and R. yanshanensis) and subsection Roseinae (R. subversatilis). Subsection Chamaeleontinae belongs to species of normally small size. The cap is not much fleshy, the cuticle is detachable, and the margin is smooth or just grooved, especially when ripe; the stem is white, frail, and meaty, then hollow. Two new species, R. miyunensis and R. plana, described in this study, also fit these characteristics. Referring to Sarnari's classification system, subsection Chamaeleontinae belongs to section Amethystinae Romagn. [5], and the position of this group in the systematic tree is also relatively stable. No Chinese Russula species belonging to subsection Chamaeleontinae have been identified in previous studies. Subsection Puellarinae was established by Singer in 1932 [67]. Singer had studied American and tropical Russulas prior to proposing his classification. Subsections Chamaeleontina Singer, Subcompactinae Singer, and Puellarinae were separated out within section Constantes Singer [68]. Russula khinganensis, described by Li et al., is also a member of subsection Puellarinae [24]. Subsection Roseinae was recognized by Sarnari in 1998 [5]. Morphologically, members of the subsection Roseinae provide a persistent bright red-colored reaction in dried fruit bodies with sulfovanilin. No species of subsection Roseinae were reported before among Russula species in China.
Russula contains a number of wild edible fungi in the world, and there are also a certain number of poisonous fungi. The classification of Russula is a difficult point in the classification of macrofungi [11]. Therefore, a classification study of the Russula is also essential to promote the study of edible species within the genus. According to incomplete statistics, 128 species of Russula are used as edible mushrooms in 28 countries worldwide [69]. Moreover, 78 edible species of Russula in China were recorded by Wu et al. [12], e.g., Russula delica Fr., Russula densifolia Secr. ex Gillet, Russula griseocarnosa X.H. Wang, Zhu L. Yang, & Knudsen.
Through literature review, all three subsections in which the six new species of this study are located have edible species distributed in China [70]. In subsection Chamaeleontinae, Russula turci Bres. and Russula roseipes Secr. ex Bres. are representative species. In subsection Puellarinae, Russula puellaris Fr. is a representative species and is more widely distributed in China. In subsection Roseinae, there are more edible mushrooms known in this subsection, including Russula pseudointegra Arnould & Goris, Russula rosea Pers., Russula lepidicolor Romagn., and all the above species are also distributed in China. Therefore, the six new species discovered this time should also contain edible species that worth to be studied.
This study is the first report of the species of Russula subgenus Russula from the Yanshan Mountains in northern Beijing and northern Hebei Province. Considering the large area of China and its diverse forest types, it is reasonable to infer that many more species of the genus Russula are expected to be found in the following studies. On the premise of determining the number of Russula species, relevant studies on edible species can be further deepened.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.