Morphological and phylogenetic evidence for two new species of Russula subg. Heterophyllidia from Guangdong Province of China

Abstract Two new species of Russulasubg.Heterophyllidia from Guangdong Province of China were described and illustrated based on morphological characters, and their identity supported by molecular phylogeny. R.luofuensis is morphologically characterized by a grayish yellow to brownish orange pileus center with a purplish gray to grayish magenta margin, a surface that is cracked and broken into small golden-brown patches, subglobose to broadly ellipsoid basidiospores with warts fused in short or long chains and a suprapellis composed of hyphal extremities with inflated, ellipsoid or globose cells and attenuated terminal cell. R.subbubalina is distinguished by the blanched almond to dark salmon pileus that is cracked with age, subglobose to broadly ellipsoid basidiospores with wart fused in short or long chains and frequently connected by line connections, a suprapellis with hyphal ends composed of inflated or ellipsoid cells and attenuated terminal cell, and pileocystidia that are mainly clavate and sometimes with round or ellipsoid appendage. The phylogenetic analyses based on ITS-nrLSU-mtSSU-TEF1 dataset were performed using maximum likelihood and Bayesian analysis. In terms of morphological features and molecular data, the former species belongs to subsect. Virescentinae, whereas the latter comes under subsect. Heterophyllinae.


Introduction
Russula Pers. is the largest genus of Russulaceae, estimated at least to contain 2000 species, which has resulted in many complex and multilevel classifications Adamčík et al. 2019;Wijayawardene et al. 2020 Hofst., and R. subg. Russula . The infrageneric classification system of Russula based on a multi-locus phylogenetic analysis was followed in this study. This genus is globally distributed and occurs across a wide range of habitats from Arctic tundra to tropical forests and forms ectomycorrhizal relationships with diverse plants (Knudsen and Borgen 1982;Buyck et al. 1996;Looney et al. 2018). Some species of Russula are famous edible fungi and are also commercially traded worldwide (Looney et al. 2018;Wang 2020). According to recent statistics on the resource diversity of Chinese macrofungi, there are 78 edible Russula species in China (Wu et al. 2019).
Guangdong Province is located in the southern coastal area of China, which is one of the Chinese provinces with tropical and subtropical climates. The climate can be divided into the middle subtropical, the southern subtropical, and the tropical climate zones, from north to south. The annual average temperature in Guangdong Province is 19-24 °C and the annual average rainfall is 1500-2000 mm. Abundant moisture, moderate to high temperatures, and variegated physiography support luxuriant and highly diversified plant growth. Broad-leaved evergreen forests, intermixed with coniferous and deciduous trees, cover much of the land. During the rainy season, the forest ecosystem can facilitate the fruiting of most ectomycorrhizal fungi, among which the members of Russula are very common. Recently, 16 new species and one epitype of Russula from Guangdong Province have been reported (Das et al. 2017;Zhang et al. 2017;Song et al. 2018a, b;Li et al. 2019;Yuan et al. 2019;Zhou et al. 2020). Obviously, Guangdong Province has become a hotspot in research on biodiversity of Chinese Russula, which makes it more vital for us to continue to explore it.
Northern hemisphere species within subg. Heterophyllidia are mainly characterized by the mostly medium to large basidiomata, equal lamellae, mild to strongly acrid taste, white or cream and rarely ochre spore print, basidiospores with inamyloid or partly amyloid suprahilar spot, mostly abundant gloeocystidia that are typically mucronate to obtuse-rounded, and absence of primordial hyphae. During a survey of the habitat diversity and geographic distribution of Russula in Guangdong Province, some interesting specimens of subg. Heterophyllidia were found that were different from known species. In this study, two new species from Guangdong Province are presented based on the morphological characters and molecular data.

Morphological study
Fresh specimens were collected and photographed in Luofu Mountain Provincial Nature Reserve, Guangdong Province, South China. Collections were dried at 45-55 °C and deposited in the herbarium of the Research Institute of Tropical Forestry, Chinese Academy of Forestry (RITF). The macromorphological characters were described based on detailed notes and photographs. The color codes mostly refer to Kornerup and Wanscher (1981). The description templates and terminology of the micromorphological characters were taken from Adamčík et al. (2019). Estimates of spore ornamentation density from scanning electron microscopy pictures follow Adamčík and Marhold (2000). The hymenial cystidia density estimates refer to Buyck (1991). Experiments were performed on dried specimens using a ZEISS Imager M2 (Carl Zeiss AG; Germany). The basidiospores were observed and measured in Melzer's reagent from a lateral view excluding ornamentation. After pretreatment of dried specimens in 5% potassium hydroxide (KOH), other micromorphological characters were identified and measured in Congo red. The coloring of the cystidia contents was observed in a sulfovanillin (SV) solution (Caboň et al. 2017). The pileipellis were examined in cresyl blue to verify the presence of ortho-or metachromatic reactions (Buyck 1989). The structure and ornamentation of the basidiospores were illustrated using a scanning electron microscopy (SEM-JEOL JSM-6510). Basidiospore measurements are 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.

Molecular study
The total genomic DNA was extracted from dry specimens following an improved CTAB protocol (Zhou and Liang 2011). We amplified and sequenced the following four loci with standard primer sets: 600 base pairs of the ITS region of rDNA using the primers ITS1 and ITS4 (White et al. 1990); 900 base pairs of the nuclear ribosome large subunit (nrLSU) using the primers LROR and LR5 (Vilgalys and Hester 1990); 600 base pairs of the ribosomal mitochondrial small subunit (mtSSU) with primers MS1 and MS2 (White et al. 1990); 900 base pairs of the translation elongation factor 1-alpha (TEF1) using primers EF1-F and EF1-R (Morehouse et al. 2003). Successful PCR products were subjected to automated DNA sequencing on an ABI 3730 DNA analyser 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 database (Table 1).

Phylogenetic analysis
Species in the subg. Heterophyllidia with high similarity to our new species and partially representative species that are closely related to subsect. Heterophyllinae (Fr.) Jul. Schäff. and subsect. Virescentinae Singer were selected for phylogenetic analyses. Russula maguanensis J. Wang, X.H. Wang, Buyck & T. Bau and R. substriata J. Wang, X.H. Wang, Buyck & T. Bau were used as outgroup. NCBI accession numbers and references of sequences used in the phylogenetic tree are listed in Table 1. Initial sequence alignment was performed using the online version MAFFT 7.0 (http://mafft.cbrc.jp/alignment/server/) with manual evaluations and adjustments in BioEdit when necessary to obtain reliable and reasonable results (Hall 1999). The final aligned result was submitted to TreeBASE (S27792). Maximum likelihood (ML) and Bayesian analysis (BA) were implemented for the phylogenetic analyses. The maximum likelihood was carried out by using RAxML-HPC2 on XSEDE (8.2.12) through the CIPRES Science Gateway (www.phylo. org). The ML analysis was executed by applying the rapid bootstrap algorithm with 1000 replicates to affirm the consistency of the results under the GAMMA model. Bootstrap support (BS) ≥70% on the final tree was regarded as significant. The BA was performed on XSEDE (MrBayes 3.2.7a) through the CIPRES Science Gateway (www.phylo.org) under the GTR model. Four independent Markov chains were run for a total of 50000000 generations, trees were sampled every 100 generations, and the first 25% of the trees were discarded as the burn-in phase of each analysis. The Bayesian posterior probability (PP) values were obtained from the 50% majority-rule consensus trees, and nodes with PP ≥0.95 were considered to be significantly supported.

Phylogeny
Both the ML analysis and BA of combined ITS-nrLSU-mtSSU-TEF1 sequences dataset resulted in similar tree topologies, and only the ML tree is shown in Fig. 1. The posterior probabilities for the BA are also shown along the branches. The phylogenetic analyses confirmed that both subsect. Virescentinae and subsect. Heterophyllinae were a monophyletic group; each strongly supported by BS (100%) and PP (1). Additionally, the monophyly of the remaining 4 subsections of subg. Heterophyllidia was also significantly supported.
The samples of the two new species, R. luofuensis and R. subbubalina, formed each a strongly supported clade (BS 100%, PP 1.00) and were clearly distinct from known and sequenced species of the subg. Heterophyllidia. R. luofuensis clustered together with Chinese species R. albidogrisea J. W. Li   Diagnosis. Basidiomata medium-sized to large; grayish yellow to brownish orange pileus center, purplish gray to grayish magenta towards the margin, surface cracking and broken into small golden-brown patches, peeling to 1/2 of the radius; subglobose to broadly ellipsoid basidiospores with warts fused in short or long chains; hymenial gloeocystidia mainly clavate; suprapellis composed of hyphal extremities with inflated, ellipsoid or globose cells and attenuated terminal cell; pileocystidia always one-celled, apically typically obtuse. Holotype. China. Guangdong Province, Huizhou City, Boluo County, Luofu Mountain Provincial Nature Reserve, 23°15'47.13"N, 114°3'45.42"E, 90 m asl., in mixed Fagaceae forests of Cyclobalanopsis and Castanopsis, 22 August 2020, leg. CB446 (RITF4708).
Etymology. The species name refers to the type locality, Luofu Mountain Provincial Nature Reserve.
Etymology. Referred to its morphological resemblance to R. bubalina. Description. Basidiomata medium-sized to large; pileus 50-100 mm in diameter; initially hemispheric when young, applanate to convex, convex with a slightly depressed center after mature; margin incurved, cracked with age, striation short and inconspicuous; surface dry, glabrous, peeling to 1/4 of the radius, pruinose in some part; dark salmon with rusty spots when young, blanched almond after maturation, shallower at the margin. Lamellae adnate to slightly adnexed, 3-5 mm deep, 11-13 at 1 cm near the pileus margin, white (1A1) to cream; lamellulae sometimes present and irregular in length; furcations present especially near the stipe; edge entire and concolor. Stipe 30-55 × 5-15 mm, cylindrical, slightly inflated towards the base, white (1A1) to blanched almond, with rusty tinge towards the base, and medulla initially stuffed becoming hollow. Context 3-4 mm thick in half of the pileus radius, white (1A1), unchanging when bruised, taste mild, odor inconspicuous. Spore print white (1A1) to cream.