Two novel species of subgenus Russula crown clade (Russulales, Basidiomycota) from China

Two novel species of Russula (Russulaceae, Russulales), R. coronaspora Y.Song sp. nov. and R. minor Y.Song sp. nov. belonging to subgenus Russula crown clade, are described based on both morphological and phylogenetic evidence. In morphology, R. coronaspora sp. nov. is mainly characterized by its distinct spores ornamented with sparse, cylindrical and isolated spines, which resemble coronavirus, and gelatinized pileipellis with pileocystidia mostly septate and sometimes branched; R. minor sp. nov. possesses a very small basidiocarp with pileus less than 2.5 cm in diameter., small basidia, easily peeling and gelatinized pileipellis with slender terminal cells and abundant SV+ pileocystidia. Positions of the two new species in both phylogenetic trees based on ITS and 5-locus sequences (nLSU, mtSSU, rpb1 , rpb2 and tef1 ) confi rm their distinct taxonomic status.


Introduction
Russula Pers. is the type genus of family Russulaceae and the largest genus in the order Russulales. Russula can be considered as the second most taxonomically diverse genus of ectomycorrhiza-forming fungi after the genus Cortinarius (Pers.) Gray, playing important ecological roles in the maintenance of different ecosystems (Looney et al. 2016). At least 78 species of Russula are consumed and 30 species are used in traditional medicine for at least 440 years in China (Wu et al . 2019). . Subgenus Russula crown clade has a very high species richness worldwide, at least 43 novel species have been described in this crown clade based on both morphological characters and molecular data since 2006, of which 34 were reported from Asia and 15 from China (Das et al . 2006Wang et al . 2009Wang et al . , 2019Adamčík et al . 2010Adamčík et al . , 2015Adamčík et al . , 2016Adamčík et al . , 2019Li et al . 2012Li et al . , 2013aLi et al . , 2013bLi et al . , 2015Li et al . , 2016Li et al . , 2018aLi et al . , 2018bMiller et al . 2012;Ariyawansa et al . 2015;Paloi et al . 2015Paloi et al . , 2016Paloi et al . , 2018Ghosh et al . 2016Ghosh et al . , 2021Jabeen et al . 2017;Jiang et al . 2017;Khatua et al . 2017;Tibpromma et al . 2017;Li & Deng 2018;Caboň et al . 2019;Phookamsak et al . 2019;Hampe & Manz 2021).
The Dinghushan Biosphere Reserve (DHSBR) is located in southern China (112°30′39″-112°33′41″ E, 23°09′21″-23°11′30″ N) and subjected to subtropical monsoon climate. The region possesses three typical forest formations: coniferous forest, coniferous and broad-leaf mixed forest, and broad-leaf forest. Recently, during the survey for macrofungi carried out in DHSBR, a well-known reserve for its high biological diversity, another two novel species of subgen. Russula crown clade were discovered in a deciduous forest composed mainly of trees of the Fagaceae family. Both new species are here described based on morphological and phylogenetic evidence.

Morphology
Specimens were collected from Dinghushan Biosphere Reserve (DHSBR, 112°30′39″-112°33′41″ E, 23°09′21″-23°11′30″ N) from June to October during the years 2016-2019. Specimens were dehydrated at 40-50 °C and deposited in the Herbarium of Microbiology Institute of Guangdong (GDGM). Fresh basidiocarp were photographed using Canon IXUS 220 hs digital camera or Canon EOS M50 under daylight in the fi eld, and macroscopic characteristics were recorded. Macromorphological descriptions are based on the fi eld notes and photographs taken from fresh basidiocarps. Basidiocarps are considered as "very small" when pileus diameter less than 3 cm.
Microscopic characters were observed under a Nikon Eclipse Ni-U microscope from hand-sectioned dried materials. Scanning electron images of basidiospores were captured with a Field Emission Scanning Electron Microscope (JSM-6330F). Basidiospores were examined in Melzer's reagent and measured in side view, excluding ornamentations. Other micromorphological structures (basidia, hymenial cystidia, gloeocystidia, elements of pileipellis and stipitipellis) were observed from handsectioned dried tissues rehydrated in 5% or 10% KOH solution and then mounted with 1% aqueous Congo red reagent. All tissues were examined in cresyl blue (Buyck 1989) to verify the presence of ortho-or metachromatic reactions. Sulphovanillin (SV) was used to test color reactions of cystidia (Sarnari 1998). Descriptive terminology followed Vellinga (1988). Color designation refers to HTML Color Codes ( https://htmlcolorcodes.com ). The abbreviation [x/y/z] indicates that measurements were made on x basidiospores in y basidiocarp from z collections. Basidiospore size and length / width ratio (Q) are given in the form of (a-) b-m-c (-d). The range b-c contains 95% of the measured values, a and d refer to the extremes of all measurements, and m means average value.

DNA extraction, PCR and sequencing
Genomic DNA was extracted from fresh fruiting bodies using a modifi ed CTAB procedure of Doyle & Doyle (1987). ITS was amplifi ed using primer pair ITS1 and ITS4 (White et al. 1990). The amplifi cation protocol consists of a 5 min pre-degeneration at 94°C, followed by 32 cycles of 30s at 94°C, 30s at 52°C and 1 min at 72°C, and a fi nal 12 min extension at 72°C. nLSU, mtSSU, rpb1 , rpb2 and tef1 were amplifi ed using the primers and protocols described in Buyck et al. (2018). The products obtained were purifi ed using E.Z.N.A Gel Extraction Kit (OMEGA) and sequenced on an ABI3730xl DNA Analyzer (IGE, Guangzhou, China). The newly generated sequences were deposited in GenBank ( www.ncbi.nlm.nih.gov ).

Phylogenetic analysis
Phylogenetic analyses based on both ITS and combined sequences of nLSU-mtSSU-tef1 -rpb2 -rpb1 were performed with Maximum Likelihood method. The datasets were automatically aligned by MAFFT ver. 7.427 (Katoh & Standley 2013), then manually adjusted and trimmed with BioEdit ver. 7.0.9 (Hall 1999). The fi nal ITS and 5-locus alignments comprised 804 bp and 4137 bp (nLSU 892 bp, mtSSU 548 bp, tef1 933 bp, rpb2 766 bp, rpb1 998 bp), respectively. Incongruence length difference test (ILD test) was performed with PAUP* ver. 4.0b10 to check the homogeneity of different gene locus, the resulting P value is 0.05 (equal to the threshold for concatenated analysis), indicating that the 5-locus alignment can be used for further phylogenetic analysis. Phylogenetic analysis based on ITS consisted of 70 sequences (Table 1), R. emetica (Schaeff.) Pers. belonging to core clade was chosen as outgroup; the concatenated 5-locus tree consisted of 39 sequences (  (Stamatakis 2006), followed by a rapid bootstrapping (BS) algorithm of 1000 replicates and heuristic ML search for the best tree using the GTRGAMMA model. All parameters in RAxML analysis were kept at default. Bootstrap value (BS) exceeding 70% was considered to be signifi cantly supported.

Phylogeny
The proposed two novel species, Russula coronaspora Y.Song sp. nov. and R. minor Y.Song sp. nov., are both nested in subgen. Russula crown clade in both ITS and 5-locus tree ( Fig. 1 and Fig. 2 , respectively). R. coronaspora formed a signifi cantly supported clade (BS = 93%) sister to R. brunneoviolacea Crawshay in the maximum likelihood tree based on ITS, while clustered with R. cf. brunneoviolacea and R. melliolens Quél. in the 5-locus tree with clade highly supported (BS = 95%). Russula minor sp. nov. clustered with 5 undescribed sequences collected from North America in ITS tree, but showed evident genetic distance with the latter ones, with sequence similarity less than 95.2%; while formed a distinct clade in the 5-locus tree, showing uniqueness just as its morphological characters refl ected. Russula minor sp. nov. gained moderate support in the ITS tree with 96% BS value.

, 7 A Diagnosis
Russula coronaspora sp. nov. is mainly characterized by its small basidiocarps with pinkish brown pileus, interveined lamellae with few lamellulae, small spores ornamented with sparse, cylindrical and isolated spines, hymenial and dermatocystidia weakly SV+ or SV-, pileipellis gelatinized and orthochromatic in cresyl blue, pileocystidia mostly septate and sometimes branched.   Table 1. Newly generated sequences used in phylogenetic analysis based on ITS. Newly generated sequences are shown in bold.

Diagnosis
Russula minor sp. nov. is mainly characterized by its very small basidiocarp with thin context and pink to rosy pileus less than 2.5 cm in diam., pileipellis very easy to peel off, white to cream lamellae with few lamellulae, often curved and hollow to multi-chambered stipe, spores with conical to cylindrical warts never connected, very small basidia and gelatinized pileipellis with slender terminal cells and abundant pileocystidia, and pleurocystidia weakly SV+ (slightly becoming brownish) or SV-, while pileocystidia becoming brown in SV.

Etymology
Referring to its very small basidiocarp.

Discussion
Our multigene phylogeny ( Fig. 2 ) places Russula coronaspora sp. nov. and R. minor sp. nov. in a signifi cantly supported subclade (BS = 97%) within the crown calde of subgen. Russula . This subclade corresponds largely to subgen. Tenellula Romagn. as it is principally composed of species of the subsections Puellarinae Singer and Laricinae Romagn., both harboring typically small species with abundant dermatocystidia and short basidia and various colors of spore print. Russula coronaspora sp. nov. is here placed outside of Puellarinae and Laricinae but in a strongly supported subclade (95%) together with R. melliolens (subsect. Melliolentinae Singer ) and a R. 'cf brunneoviolacea' (the European species is part of Romagnesi's subsect. Sphagnophilinae Romagn., equally placed in subgen. Tenellula , but this specimen was collected in Canada and is likely a still undescribed American taxon). In the phylogenetic tree based on ITS, R. coronaspora is closely related to R. brunneoviolacea , while R. minor sp. nov. clustered with several undescribed taxa from North America.
The basidiospores of Russula coronaspora sp. nov. which are exceptionally small, yet ornamented with large, very sparse, cylindrical and completely isolated warts or spines, resembling coronavirus, make R. coronaspora very special compared with its phylogenetically related species. Russula brunneoviolacea Crawshay and R. melliolens Quél. are phylogenetically related to R. coronaspora sp. nov., but the former two species all have a much darker pileus and spores with ornamentations more or less connected (Schaeffer et al . 1949;Kaur et al. 2011).
Russula minutula var. minor Z.S.Bi was previously described from Dinghushan Biosphere Reserve (Bi & Li 1986), and it resembles R. minor sp. nov. in the fi eld because of small size and similar color, but their size of spores, basidia and hymenial cystidia (also form of cystidia on gill sides) proved to be different.