Species of Russula subgenus Heterophyllidiae (Russulaceae, Basidiomycota) from Dinghushan Biosphere Reserve

. Two new species of subgenus Heterophyllidiae subsection Cyanoxanthinae , Russula fusiformata Y.Song sp. nov. and R. purpureorosea Y.Song sp. nov., collected from the Dinghushan Biosphere Reserve (DHSBR), are described based on both morphology and a phylogenetic analysis of the internal transcribed spacer (ITS), further increasing Heterophyllidiae species diversity in the area. Differences between the two new species and related taxa are analyzed. The other 17 reported species of Russula subgenus Heterophyllidiae that have been collected from DHSBR during mushroom explorations since 2014 are also summarized. The dominant species and the ecological distribution of all 19 species are briefly discussed, and most species are presented in macrofungal plates.


Introduction
Russula Pers. is a hyperdiverse ectomycorrhizal genus with a worldwide distribution from temperate to tropical areas (Buyck 1989;Buyck & Horak 1999;Buyck et al. 2015Buyck et al. , 2018. Due to its edibility, toxicity, ecological functions and diversity, Russula is among the most important lineages of macrofungi (Looney et al. 2016;Wu et al. 2019). Species of Russula are easy to recognize by their brittle context and relatively large and brightly colored surface of the pileus. But the genus displays particular variability in macroand micromorphological phenotype and chemical reactions, which has led to a complex infrageneric classification that is still artificial (Romagnesi 1967;Singer 1986;Bon 1988;Sarnari 1998). According to the newest classification system based on both multi-locus phylogeny, macrofungal morphology and ectomycorrhizal phenotype, Russula is divided into 8 subgenera, namely Archaeae Buyck (Buyck et al. 2018. Species in subgen. Heterophyllidiae mostly share medium to large basidiomata with the pileus coming in almost all possible colors, predominantly equal lamellae, frequently distinct smell, mild to strongly acrid taste, white or various shades of cream to ochre spore print, spores with inamyloid or partly amyloid suprahilar spot and abundant gloeocystidia (except for subsect. Amoeninae (Singer) Buyck).
The Dinghushan Biosphere Reserve (DHSBR, 112°30′39″-112°33′41″ E, 23°9′21″-23°11′30″ N) is located in Zhaoqing City, Guangdong Province, southern China, in the subtropical monsoon climate zone. DHSBR is considered as a region highly diverse in macrofungi (Bi et al. 1994), and possesses three typical forest formations representing the early, middle and late succession stages of the forest: coniferous forest, mixed coniferous and broad-leaf forest, and broad-leaf forest. During the macrofungi explorations carried out since 2014, eighteen new species and one epitype of Russula (Das et al. 2017;Zhang et al. 2017;Song et al. 2018aSong et al. , 2018bSong et al. , 2021Li et al. 2019;Yuan et al. 2019;Zhou et al. 2020), two new species of Lactarius Pers. (Wang et al. 2018) and three new species and one new variety of Lactifluus (Pers.) Roussel (Zhang et al. 2016Song et al. 2017Song et al. , 2018c have been reported from this area. In this study, another two novel species in Russula subgen. Heterophyllidiae subsection Cyanoxanthinae Singer, named as Russula purpureorosea Y.Song sp. nov. and R. fusiformata Y.Song sp. nov., are proposed based on both morphological and phylogenetic evidence. Until now, a total of 19 Russula species in subgen. Heterophyllidiae have been reported, 15 of which were first collected and described from DHSBR. All species of Heterophyllidiae with voucher specimens and ITS sequences collected from DHSBR since 2014 are summarized (Table 1), species distribution in three different vegetation types is briefly analyzed (Table 2), and most species are presented in macrofungal plates (Appendices 1-12).

Sampling and morphological studies
Fruiting bodies of the specimens were all collected from Dinghushan Biosphere Reserve (DHSBR, 112°33′ E, 23°10′ N), Guangdong Province, China. Specimens were dried at about 40°C and deposited in the Herbarium of Guangdong Institute of Microbiology (GDGM). Macroscopic characteristics of the intact fresh fruit bodies were recognized in daylight in the field and were photographed using a Canon IXUS 220 hs digital camera. HTML Color Codes (https://htmlcolorcodes.com/color-names/) were used to describe the color terminology.
Tissue sections were immersed in 5% KOH and then stained with 1% aqueous Congo red solution to observe the microscopic characters. All tissues were also examined in Cresyl blue to verify the presence of ortho-or metachromatic reactions as explained in Buyck (1989). Sulfovanillin (SV) was used to test for staining reactions of cystidia. Micromorphological features were observed and photographed using a Nikon E200 microscope. Basidiospores were also observed in Melzer's reagent and measured in side view, excluding ornamentation and apiculus which were observed by SEM. The notation (x/y/z) indicates that measurements were made on x basidiospores in y fruit bodies from z specimens. In the notation of basidiospore dimensions ʻ(a-) b-m-c (-d)ʼ, b-c is the range including 95% of the measured values for length or width, with ʻaʼ and ʻdʼ corresponding to the extremes of all measurements, and ʻmʼ corresponding to the mean value. Q indicates the length/width ratio of basidiospores.

Taxon
Voucher specimen ITS accession no.
A BLAST query of the ITS sequences in GenBank indicated that the two novel species should be assigned to Russula subgenus Heterophyllidiae. A maximum likelihood (ML) analysis was conducted using RAxML ver. 7.2.6 (Stamatakis 2006) for Russula subgen. Heterophyllidiae based on the rDNA ITS matrix. The ITS dataset was aligned using MAFFT ver. 7 (https://mafft.cbrc.jp/alignment/server/), then manually adjusted and trimmed with BioEdit ver. 7.0.9 (Hall 1999). The final ITS alignment for R. subgen. Heterophyllidiae comprised 135 sequences and 875 characters including gaps. A rapid bootstrapping (BS) algorithm of 1000 replicates was executed in RAxML, followed by a heuristic ML search for the best tree using the GTRGAMMA model.

Phylogeny
From the phylogram of Russula subgen. Heterophyllidiae we can see that most of the 19 species collected from DHSBR are significantly supported ( Diagnosis Russula fusiformata sp. nov. is distinguished by its ITS sequence with a similarity less than 95% compared to other known species. In morphology, it is mainly characterized by the lavender blush to rosy brown pileus with crenate margin, adnate lamellae, metachromatic pileipellis with slender furcated and septate terminal elements, and fusiform cheilocystidia with sharp apices.

Etymology
The name refers to the fusiform cheilocystidia with sharp apices.

Remarks
Our phylogenetic analysis based on ITS shows that Russula fusiformata sp. nov. is well nested in subsect. H.Qiu. The three species have a common habitat and distribution, namely all are gregarious in monsoon evergreen broad-leaf forest and mixed pine-broad-leaf forest in DHSBR. Russula dinghuensis (Appendix 1) is characterized by the olive green pileus with acute and incurved margin, white and rarely forked lamellae, thick metachromatic pileipellis. Russula subpallidirosea (Appendix 3) can be recognized by the pale pink to pale grayish-pink pileus, white and forked lamellae, metachromatic pileipellis with short terminal elements. Russula fusiformata can be differentiated from related species by its lavender blush to rosy brown pileus and fusiform cheilocystidia with sharp apices. In addition, the margin of R. dinghuensis and R. subpallidirosea is even and incurved while in R. fusiformata it is crenate. The lamellae of R. dinghuensis and R. fusiformata are rarely forked, but those of R. subpallidirosea are often forking. Moreover, the chemical reactions of cystidia and pileipellis of the three species are totally different. Pleurocystidia in R. fusiformata are SV negative, but positive in R. dinghuensis and R. subpallidirosea.  Diagnosis Russula purpureorosea sp. nov. can be separated from other known species by its ITS sequence with a similarity less than 95.5% when aligned. The species is morphologically characterized by the pale pinkish purple pileus with even or incurved margin, adnate and crowded lamellae with lamellula, thick metachromatic pileipellis, terminal elements of pileipellis with some inflated terminal and subterminal cells.

Etymology
The name refers to pale pinkish purple pileus.

Remarks
Russula purpureorosea sp. nov. has equal gills, lamellulae not frequent, context unchanging, spore print whitish, spores with inamyloid suprahilar spot, primordial hyphae absent, gloeocystidia abundant, terminal elements inflated; all these characters indicate that R. purpureorosea belongs to subgen. Heterophyllidiae subsect. Cyanoxanthinae in morphology. Our phylogenetic analysis based on ITS shows that R. purpureorosea is closely related to R. pallidirosea Kropp within subsect. Cyanoxanthinae. Russula purpureorosea can be distinguished from related species by its pinkish purple to rosy brown pileus with lamellulae. Compared with R. purpureorosea, R. pallidirosea has smaller pinkish pileus (cap 1.5-2.5 cm in diam.) with margins inrolled at first, becoming uplifted with age, lamellae occasionally forked, lamellula present and stipe tapering toward the base. Basidiospores of R. pallidirosea have low unconnected amyloid ornamentations which are generally shorter than in R. purpureorosea.

Discussion
The phylogenetic analyses based on ITS showed that R. purpureorosea sp. nov. clusters with its sister species R. pallidirosea (Kropp 2016) forming a distinct clade, while R. fusiformata sp. nov. forms a distinct clade parallel to the clade comprising R. dinghuensis and R. subpallidirosea. A detailed morphological comparison of the two novel species and their related taxa is given in Table 3. Both morphological and phylogenetic analyses demonstrate that R. purpureorosea and R. pallidirosea are two novel species in R. subgen. Heterophyllidiae subsect. Cyanoxanthinae.
The Dinghushan Biosphere Reserve is located in southern China. It is one of three reserves in China selected in 1980 to join the Biosphere Reserve Network of UNESCO for their rich biological diversity and ecological importance. The region is subjected to a subtropical monsoon climate and possesses three vegetation types: coniferous forest, mixed coniferous and broad-leaf forest, and broad-leaf forest. The distinct climate and ecological environment allow for a diversity of species of Russula. Until now, up to 141 specimens representing 19 species (including the two novel species proposed in this study) of Russula subgen. Heterophyllidiae have been collected, of which 15 species were first described from DHSBR. All the collected species of Heterophyllidiae with voucher specimens and distribution are listed in Table 1 and Table 2, respectively. Of all the 141 specimens, 106 samples representing 18 species were collected from broad-leaf forest, 34 samples representing 10 species from mixed coniferous and broad-leaf forest, and only one sample from coniferous forest. The species richness and diversity of Russula in broad-leaf forest is higher compared to the other two vegetation types. Russula aureoviridis J.W.Li & L.H.Qiu, distributed in all three types of forest and collected every year with the most specimens, seems to be the dominant species among the native Russulas in DHSBR.