Morphological Characteristics and Molecular Evidence Reveal four New Species of Russula subg. Brevipedum from China

In this study, four new species of Russula subg. Brevipedum collected from China are described based on morphological characteristics and molecular evidence. Russula brevispora has a white body whose whole parts often stain brownish-orange or grayish-orange, extremely crowded lamellae with the presence of lamellulae, basidiospores with low warts and an inamyloid suprahilar spot, and clavate or lageniform hymenial cystidia often with a papillary or submoniliform appendage. Russula flavescens is characterized by a white pileus often turning yellowish brown when touched, white lamellae turning brown or light orange, basidiospores with an inamyloid suprahilar spot, and fusiform hymenial cystidia often with an appendage. Russula longicollis is morphologically characterized by a white pileus, turning grayish orange when bruised, white lamellae turning pale yellow when bruised, basidiospores with isolated warts and an amyloid suprahilar spot, and fusiform hymenial cystidia usually with a long appendage. Russula pseudojaponica has a yellowish-brown pileus center, yellowish lamellae unchanging when bruised, basidiospores with low warts and an inamyloid suprahilar spot, clavate hymenial cystidia often with a papillary appendage, and clavate pileocystidia with granulose contents. Phylogenetic analyses showed that R. flavescens, R. brevispora, and R. pseudojaponica are members of the subsect. Pallidosporinae, whereas R. longicollis belongs to subsect. Lactarioideae, and is somewhat related to R. leucocarpa.


Introduction
Russula Pers. is a genus with a great diversity of mushroom-forming fungi species, predictably containing at least 2000 species within the genus [1,2]. The members of this genus are important plant root symbionts in forest ecosystems, occurring across a wide range of habitats, from the arctic tundra to tropical forests [3]. Recently Hofst.), and R. subg. Russula [4,5].
The Russula subg. Brevipedum, typified by R. brevipes Peck, was originally described in 2015 as R. subg. Brevipes; however, this was an invalid name and was changed to Brevipedum in 2020 [5]. The members of this subgenus mostly have a medium to very large basidiomata, which often stains yellowish-brown to reddish-brown, regularly unequal lamellae, a distinct smell or acrid to strongly acrid taste, a whitish to yellow spore print, and mucronate to obtuse-rounded cystidia in all parts of the fruiting body [4]. The species of the R. subg. Brevipedum was initially divided into two subsections: R. subsect. Lactarioideae Maire

Morphological Study
Fresh basidiomata from five provinces of China were collected and photographed. The samples were dried at 50 • C and deposited in the herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF). Macromorphological descriptions were based on detailed notes and photographs. Color codes and terms follow the Methuen Handbook of Colour [36]. Micromorphological characteristics were observed using a ZEISS Imager M2 (Carl Zeiss AG; Oberkohen, Germany) with oil-immersion lenses at a magnification of 1000×. Macroscopic characters were observed in 5% KOH, 1% phloxin, and 1% ammoniacal Congo red. Tissues were mounted in cresyl blue [37], sulfovanillin [1], and treated with carbolfuchsin [38] to observe the presence and color changes of the incrustations and cystidium contents. Basidiospores were observed in Melzer's reagent and measured in lateral view excluding the height of ornamentations. Basidiospores measurements are represented 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 [2]. Basidium length excludes the length of the sterigmata. Hyphal terminations and pileocystidia were observed both near the pileus margin and in the pileus center. Scanning electron microscopy (SEMJEOL JSM-6510) was used to photograph the shape and ornamentation of the basidiospores.

DNA Extraction, PCR, and Sequencing
Total genomic DNA was extracted from the dry specimens following an improved CTAB protocol [39]. The ITS region of rDNA was amplified with the primers ITS1 and ITS4 [40]. The amplification protocol consists of a 5 min initial denaturation at 95 • C, followed by 35 cycles of 30 s at 95 • C, 30 s at 53 • C, and 2 min at 72 • C, with a final extension of 10 min at 72 • C. The remaining three loci (nrLSU, RPB2, and mtSSU) were amplified using the primers and protocols complying with Buyck [4]. The PCR products were purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit according to the operation manual. The amplified PCR products were subsequently sequenced on an ABI 3730 DNA analyzer using an ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Shanghai Songon Biological Engineering Technology and Services Co., Ltd., Shanghai, China). The newly generated sequences were submitted to the GenBank database (https: //www.ncbi.nlm.nih.gov/genbank, URL (29/9/23) Table 1). Type specimens are marked with an asterisk (*) symbol, and the newly generated sequences are in bold.

Sequence Alignment and Molecular Phylogenetic Analyses
All the available sequences of species in the subg. Brevipedum from GenBank were included for phylogenetic analyses. The original bidirectional sequences were assembled with the help of Contigexpress software [41]. The sequences were aligned using the MAFFT 7.0 online version [42] and manually adjusted in BioEdit 7.0.9 [43]. The four datasets were concatenated using Phyutility v2.2 for further analysis [44]. The final aligned result was submitted to TreeBase (S29992). Maximum likelihood (ML) analysis was carried out in RAxML 7.0.3 [45]. All parameters were kept at the default settings, except the models set as GTRGAMMA, and statistical support was obtained using nonparametric bootstrapping with 1000 replicates. Bootstrap support (BS) greater than 75% was considered significant. Bayesian inference (BI) analysis was conducted in MrBayes 3.2.6 [46]. The best-fit model was estimated with MrModelTest v. 2.3 [47] using the Akaike information criterion (AIC). Four chains were run for 5 million generations sampling from the posterior distribution every 100 generations. Other parameters were kept at the default settings. The analysis was terminated when the average standard deviation of split frequencies was stable below 0.01. The parameters and tree samples were then summarized, and the Bayesian posterior probabilities (BPPs) were calculated after discarding the first 25% of the samples as the burn-in. BPPs over 0.95 were considered significant.
The combined dataset included sequences from 73 specimens representing 35 taxa. The dataset had an aligned length of 3018 characters including gaps, of which ITS contains 772 characters, nrLSU contains 896 characters, mtSSU contains 564 characters, and RPB2 contains 786 characters. The best model for the Bayesian analysis of the nrLSU and mtSSU sequence datasets was GTR+I+G; for ITS, the best model was HKY+I+G, and for RPB2 it was SYM+I. Six partitions were implemented for further phylogenetic analyses (ITS, nrLSU, mtSSU, RPB2 1st, RPB2 2nd, and RPB2 3rd).

Phylogeny
The phylogenetic trees generated from the RAxML and Bayesian analyses were similar in topology; thus, only the ML tree is shown in Figure 1. The phylogenetic analyses confirmed that R. subg. Brevipedum formed an independent clade with strong support (BS/BPP = 100/1), and can be divided into two lineages, subsect. Pallidosporinae and subsect. Lactarioideae.

Discussion
Russula subg. Brevipedum were formerly placed in R. subg. Compactae (Fr.) Bon as section Brevipes and elevated to subgenus in 2015 [5]. In China, 13 species of subg. Brevipedum were reported in the past, with six species-R. byssina (G.J. Li and C.Y. Deng), R. callainomarginis (J.F. Liang and J. Song), R. cremicolor, R. leucocarpa, R. luteolamellata, and R. subbrevipes-described as new in recent years [9,25,26]. The other species (R. chloroides, R. delica, R. pallidospora, R. pseudodelica, R. flavispora, R. japonica, and R. brevipes) were described in detail in various publications [24,27,30]. The species described in China were similar to those of Europe and North America in terms of macroscopical morphology, but there were obvious differences in microscopic characteristics. Moreover, most of the species reported in China lack support from molecular evidence.
Russula japonica was first described by Hongo [28], and was reported to be poisonous, causing gastroenteritis [33][34][35]. Over the past decades, most of the specimens with similar characteristics were recorded as R. japonica in China. Our morphological study results indicate that the Chinese species differ from R. japonica in their hymenial cystidia. Moreover, we selected a large ribosomal submit sequence from the location of type provided by Shimono [50] for molecular phylogenetical analyses. The molecular phylogeny results are consistent with the morphology.
In some other species, the differences between Chinese and North American and European specimens mainly appear in terms of the size of the basidiospores, hymenial cystidia, and basidia, the presence or absence of pileocystidia, and so on. For example, R. chloroides has longer basidiospores (7.5-8.9 × 5-6.7 µm in Li [9] and 8.3-11.4 × 7.7-10.3 µm in Liu [24] and shorter hymenial cystidia (34-67 µm in Li [9] and 55.3-91.1 µm in Liu [24]), but smaller basidiospores (6.5-8 × 6-6.7 µm) and longer hymenial cystidia (65-115 µm) are present in Romagnesi [30]. Russula brevipes has shorter basidia (17-46 × 5-12 µm in Li [9] and 37.8-48.2 × 11.8-13.6 µm in Liu [24]), shorter hymenial cystidia (29-64 × 5-12 µm in Li [9] and 56.3-81.5 × 6.9-12.6 µm in Liu [24]), and the absence of pileocystidia; however, there are longer basidia (55.5-68 × 9.5-14 µm) and hymenial cystidia (62-93 × 7.5-11 µm), along with the presence of pileocystidia, weakly greying in SV, in Buyck [8]. This indicates that these species reported from China might be misidentified. The main reason for this is that most descriptions of Russula species in the past have been incomplete. Besides being incomplete, the description style has not been consistent, with regional or author-specific patterns [2]. The combination of these factors makes comparing descriptions difficult, or even impossible. Therefore, we still need more reliable sequences as well as detailed macroscopic and microscopic descriptions to confirm the existence of these species in China. In this study, we propose four new species, including three species that are closely related to R. japonica and one species that is close to R. leucocarpa from China based on molecular phylogeny and morphology data. It is worth noting that R. flavescens, R. pseudojaponica, and R. brevispora were recently reported to have gastrointestinal toxicity [34,35], whereas the toxicity of R. longicollis is still unknown.
Key to four new species and other closely related taxa within subg. Brevipedum