Taxonomic and phylogenetic evidence reveal two new Russula species (Russulaceae, Russulales) from northern Thailand

Russula is the most diverse genus within the family Russulaceae and has an ectomycorrhizal association with many host trees. During the monsoon season of 2022, five Russula specimens were collected in northern Thailand. After morphological and molecular analyses, the five specimens were determined to belong to Russula subgenus Heterophyllidia. A single specimen belonged to R. bellissima. Four other collected specimens were found distinct from all other known species. We have identified these four specimens as two new species, namely R. pseudomodesta and R. sribuabanensis. Russula pseudomodesta belongs to the subsection Modestinae, while R. sribuabanensis belongs to the subsection Virescentinae. The phylogenetic placement of two new species within Russula has been confirmed based on sequences of the nuclear internal transcribed spacer (nrITS) region and a large subunit (nrLSU) of the nuclear ribosomal RNA (nrRNA). Comprehensive morphological descriptions, field photographs, and comparisons of two new species with other morphologically and phylogenetically closely related species are provided.


Introduction
The genus Russula Pers.was established by Persoon (1796).This is an important ectomycorrhizal mushroom genus that belongs to the family Russulaceae, order Russulales (Sarnari 1998).This genus is distributed worldwide with more than 2000 species that are associated with a wide range of host trees (broadleaf and coniferous plants) in different ecosystems (Paloi et al. 2018;Adamčík et al. 2019;Song et al. 2022).Several species of Russula have exhibited socioeconomic, industrial, ecological, and medicinal importance.In recent days, many Russula species have shown anticancer, antiinflammatory, immunostimulatory, and beneficial antioxidant activities (Taengphan et al. 2019;Li et al. 2020;Khatua et al. 2021;Li et al. 2021;Zhang et al. 2021;Panda et al. 2021).Several taxonomic studies have concluded that Russula is a monophyletic genus (Buyck et al. 2008;2018) . 2018; 2020).Among these eight subgenera, Russula subg.Heterophyllidia is characterized by the presence of medium-to large-sized basidiomata, equal lamellae, context unchanged or sometimes appearing yellowish to rusty brown, generally mild to strongly acrid taste, white or cream spore prints, suprahilar spots inamyloid or partly amyloid, as well as the presence of gloeocystidia, and an absence of primordial hyphae (Buyck et al. 2018).Approximately, 163 species in the Russula subg.Heterophyllidia have been discovered all over the world (Han et al. 2023).Russula subg.Heterophyllidia is divided into four sections, namely Aureotactae Buyck & V. Hofst., Heterophyllae Fr., Ingratae Quél., and Subvelatae Singer (Buyck et al. 2018).
Northern Thailand covers an area of about 22,135 km 2 (18.7883°N, 98.9853° E), and its unique geographical location and vast climatic conditions make it an attractive habitat for the growth of macrofungi.According to Hyde et al. (2018) (Hampe and Manz 2021;Wisitrassameewong et al. 2022).During a survey of macrofungi in Chiang Mai and Lamphun Provinces, Northern Thailand of 2022, five specimens of Russula were collected.Based on morphological characteristics and multigene phylogenetic analyses, five specimens were identified as two distinct new species (introduced as R. pseudomodesta and R. sribuabanensis), as well as one previously known species (R. bellissima) that belongs to Russula subg.Heterophyllida.

Morphological study
Fresh basidiomata were collected from Chiang Mai and Lamphun Provinces, Northern Thailand, during the rainy seasons of 2022.Macro-morphological characteristics, ecological data, and certain chemical reactions to different chemicals (10% KOH and guaiacol) were noted in the field.Color codes and terms were used according to Kornerup and Wanscher (1981).Specimens were dried at 45 °C for 48 h.Microscopic characteristics were obtained from dried specimens using free hand sections by mounting them in 5% KOH, Congo red, and Melzer's reagent.They were then viewed through an Olympus CH30 microscope.The size of the basidiospores was documented based on 30 measurements recorded from each specimen.The Q value denotes the length/width ratio of the basidiospores.Basidiospore statistics have been presented as mean values (underlined).
Scanned electron micrographs (SEM) of basidiospores were obtained from spore prints coated with gold and viewed under a high vacuum mode to observe patterns of spore ornamentation.This work was carried out with the use of a JEOL JSM-IT800 scanning electron microscope (SEM) at Chiang Mai University.The holotype and other examined specimens were deposited at the Chiang Mai University Herbarium of the Department of Biology (CMUB) and the Sustainable Development of Biological Resources Laboratory (SDBR), Faculty of Science, Chiang Mai University, Thailand.

DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from fresh specimens using a DNA Extraction Mini Kit (FAVORGEN, Taiwan) according to the manufacturer's protocol.The nrITS, nrLSU, and RPB2 regions were amplified by polymerase chain reaction (PCR) with ITS4/ITS5 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), and RPB2-6F/RPB2-7cR (Matheny et al. 2007) primer pairs, respectively.The amplification program for these three domains was performed in separate PCR reactions.PCR programs of nrITS and nrLSU were established by following the methods employed by Paloi et al. (2023).The amplification program for RPB2 consisted of an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, an annealing step at 54 °C for 40 s, and an extension step at 72 °C for 1 min on a peqSTAR thermal cycler (PEQLAB Ltd., UK).PCR products were checked on 1% agarose gels stained with ethidium bromide under UV light.PCR products were purified using a PCR clean-up Gel Extraction NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Germany) following the manufacturer's protocol.The purified PCR products were directly sequenced.Sequencing reactions were performed, and the sequences were automatically determined by genetic analyzer at the 1st Base Company (Kembangan, Malaysia) with the PCR primers mentioned above.

Phylogenetic analyses
The sequences associated with a high degree of similarity to the newly generated sequences were retrieved from GenBank and from recent publications (Chen et al. 2019;Ullah et al. 2020;Chen et al. 2021a, b;Khatua et al. 2021;Wisitrassameewong et al. 2022;Zhou et al. 2023), as is shown in Table 1.Three Russula species, R. nigricans, R. dissimulans, and R. camarophylla, were used for rooting purposes.Multiple-sequence alignment was performed with MUSCLE (Edgar 2004) using default settings and improved where necessary using BioEdit v. 6.0.7 (Hall 2013).The final alignment was submitted to TreeBASE (30517).Phylogenetic analyses were carried out based on only the nrITS and  (Hillis and Bull 1993;Alfaro et al. 2003).Tree topologies were then visualized in FigTree version 1.4.0 (Rambaut 2009).

Phylogenetic analysis
The newly generated nrITS and nrLSU sequences were deposited in the NCBI database and their accession numbers are indicated in Table 1.The combined nrITS and nrLSU sequence dataset consisted of a total number of 62 taxa, and the aligned dataset was comprised of 1751 characters including gaps (nrITS: 1-818 and nrLSU: 819-1751).The best scoring RAxML tree was established with a final ML optimization likelihood value of −11,706.494.The final average standard deviation value of the split frequencies at the end of the total MCMC generations was calculated as 0.00874 through BI analysis.Notably, the phylograms of the ML and BI analyses were similar in topology.Therefore, the phylogenetic tree obtained from ML analysis was selected and is presented in this study (Fig. 1).Diagnosis: Differs from R. modesta by the presence of a larger pileus, no interspace venose between lamellae, globose to subglobose basidiospores, and ornamentation up to 1.1 µm high.
Etymology: The name "pseudomodesta" refers to the similarity of the new species to R. modesta.

Russula sribuabanensis
Diagnosis: Differs from R. pallidula by the presence of larger pileus, longer pileus terminal cells (up to 63 µm) and growing in association with Shorea sp.dominating mixed forest; from R. shanglaensis by the presence of smaller caulocystidia and white stipe.
Etymology: The Latin name "sribuabanensis" refers to Sribuaban Subdistrict, Lamphun Province where type species was collected.
Ecology and distribution: Solitary, growing in a forest dominated by trees of Shorea spp.(Dipterocarpaceae).Known only from the type locality in Northern Thailand.

Discussion
A combination of certain morphological characteristics, such as gray to grayish green pileus, a mild taste, cream spore prints, erect to suberect terminal hyphae attached with globose to suglobose or ellipsoid subterminal cells, and the presence of caulocystidia, confirmed that R. sribuabanensis belongs to the subsect Virescentinae.During the last few years, several members of this subsection were reported from different Asian countries.These are namely R. pallidula Bin Chen & J.F. Liang (Chen et (Dutta et al. 2015) from India, and R. shanglaensis S. Ullah, Khalid & Fiaz (Ullah et al. 2020) from Pakistan.However, our Thai collections of R. sribuabanensis have been found to be closely related to R. pallidula and R. shanglaensis morphologically and phylogenetically.However, R. pallidula has a smaller pileus (40-55 mm diam.), safranopink color lamellae, shorter pileipellis terminal hyphae (22-37 µm long), and is frequently associated with Pinus sp., Picea sp., and Castanopsis sp.(Chen et al. 2019).Russula shanglaensis has been reported from Pakistan in association with mixed coniferous forests at high altitudes (2300-3000 m).It has larger caulocystidia (40-130 µm long) and yellowish tints at the stipe base (Ullah et al. 2020).The other three Chinese species, viz.R. albolutea (Chen et al. 2021b), R. subpunicea (Chen et al. 2021b), and R. pseudopunicea (Zhou et al. 2023), were determined to belong to the same subsection, but R. pseudopunicea, frequently in association with broadleaf forests dominated by Betula costata, has a reddish brown, light brown to brownish orange pileus, thicker suprapellis (60-190 µm), and longer pileocystidia (Zhou et al. 2023).Russula albolutea is found in mixed hardwood forests dominated by Fagus sp. and has white spore prints, smaller basidia (28-43 µm long), and thicker suprapellis (120-160 μm), while R. subpunicea has a yellowish white, pinkish, or grayish orange pileus, white spore prints and grows in association with Castanopsis hystrix and Betula alnoides (Chen et al. 2021b).
Russula bellissima was previously reported from Thailand.Our collection is similar to the type material in most cases.However, our samples were mostly smaller in size (12−25 mm diam.) with a basidiospore size a little smaller than the type material.The suprapellis and subpellis are also thinner than the type material.However, the species can easily be recognized based on small-to medium-sized pileus with red color, a smooth to purinose pileus surface, inamyloid suprahilar spots, basidiospores ornamented with spines or warts connected by fine lines, the presence of hymenial cystidia, and the absence of pileocystidia (Wisitrassameewong et al. 2022).Russula bellissima is morphologically and phylogenetically closely related to R. intervenosa from India which frequently grows in association with Shorea robusta dominating forests.However, our collection differs from R. intervenosa by the presence of hymenial cystidia on the gill side, very thin suprapellis (47-64 µm deep), and a stipe surface that turned red to brownish red with guaiacol (Crous et al. 2016).

Fig. 1
Fig. 1 A combined (nrITS+nrLSU) phylogram derived from maximum likelihood analysis of 62 taxa.Three Russula species, R. nigricans, R. dissimulans, and R. camarophylla were used for rooting purposes.The numbers above branches represent maximum likelihood bootstrap percentages (right) and Bayesian posterior probabilities (left).Bootstrap

Table 1
Names, voucher numbers, countries, references and corresponding GenBank accession numbers of the taxa used in the phylogenetic analyses of this study (Ronquist et al. 2012).The best substitution models for BI analyses were estimated by Akaike Information Criterion (AIC) in jModeltest 2.1.10(Darribaet al. 2012).The best

Table 1
"NA" = Not available in GenBank database.Specimens obtained from this study are in bold