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 1,751 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 − 11706.494. 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). The final phylogenetic tree is divided into eight clades (clades a to h) that represent the subsect. Virescentinae, Amoeninae, Griseinae, Heterophyllinae, Modestinae, Ilicinae, and Cyanoxanthinae, and sect. Ingratae, respectively. Our specimens CMUB40006 and SDBR-CMUNK1760, introduced as R. sribuabanensis were placed in the subsect. Virescentinae (clade a). Both specimens formed a separate subclade from other members of the subsect. Virescentinae with higher support values (BS = 100% and PP = 1.00). Another collection of CMUNK1785 clumped together with R. bellissima (FH12-185 and FH12-127) was previously reported from Thailand (clade c). Additionally, two specimens, CMUB40005 and SDBR-CMUSOU33, introduced as R. pseudomodesta were monophyletic and formed a distinct subclade under the subsect. Modestinae (clade e) with high support values (BS = 100% and PP = 1.00).
Taxonomy
Russula pseudomodesta Paloi, Suwannarach N. & Kumla J., sp. nov. (Figs. 2a, 2c and 3)
MycoBank: MB 848802
Holotype: THAILAND. Chiang Mai Province, Chiang Mai University, 18°48'0.8496'' N; 98°57'21.2256'' E, elevation 335 m, 20 August 2022, S. Paloi (Holotype, CMUB40005). GenBank numbers OQ957419 (nrITS), OQ968300 (nrLSU), and OQ973265 (RPB2).
Diagnosis: Differs from R. modesta, by the presence of a larger pileus, no interspace venose between lamellae, globose to subglobose basidiospores, and ornamentation upto 1.1 µm high.
Etymology: The name “pseudomodesta” refers to the similarity of the new species to R. modesta.
Basidiomata large to medium. Pileus 60−125 mm diam., convex or ovoid at early stage, become broadly convex to plano-convex with slightly depressed center; surface smooth to pruinose, moist to semi moist, greenish grey (1A2), yellowish grey (2B2−3B2), grayish yellow (3C3), orange grey (5B2−6B2), greyish orange (5B3−6B3), greyish green (25B2) to sometimes reddish grey (7B2) at center, becoming fed to ward margin greyish white (1A2), yellowish white (2A2), gray (3B1) to sometime white (1A1), no change after bruising, turned orange with KOH, light pinkish with guaiacol; margin wavy, cracked at maturity, striate; context up to 5 mm at the middle, white to off-white, no change after exposed with air. Lamellae adnate, up to 8 mm wide, regular, sometimes bi-forked near stipe, white (1A1), no change after bruising; edge smooth, concolorous; lamellulae zero to one in a row. Stipe 35−52 × 11−18 mm, central, more or less equal, cylindrical, sometimes slightly bulbous base; surface smooth to pruinose, semi-moist, white (1A1), no change after bruising, turned yellow with KOH, light pinkish with guaiacol; context firm, white (1A1), becoming light yellowish after exposed with air. Odor not recognized, not hard. Taste mild. Spores print white to off white.
Basidiospores 5.5−6.4−7.2 × 5.1−5.6−6.5 µm, Q = 1.0−1.1−1.1, globose to sub-globose, ornamentation amyloid, composed with 0.3−0.5 µm low to 0.7−1.1 µm high warts, few connected (1−3) together with fine line, 0.1 µm high, in between two wart very small wart present, never reticulate, suprahilar spot not amyloid. Basidia 35.5−53.6 × 9−11.3 µm, clavate to sub-clavate, sometimes cylindrical, thin-walled, hyaline, oil granules present when viewed with KOH, 4-spored; sterigmata 2.9−5.5 µm long, cylindrical. Lamellae edge composed of cystidia and basidia. Subhymenium pseudoparenchymatous. Hymenial cystidia 66−87 × 10.2−13 µm, cylindrical to sub-cylindrical or fusiform with short appendiculate to moniliform or sometimes obtuse apex, thin-walled, hyaline, heteromorphous contents present near gill edge; same as gill side. Pileipellis orthochromatic in cresyl blue, context composed with sphaerocytes; subpellis composed with densely arranged hyphae, 125−150 µm deep, gelatinized, hyphae 3.98−2.76 µm broad, thin-walled, hyaline, branched; suprapellis 95−150 µm deep, less gelatinized, composed with erect to sub-erect hyphal end, 30−65 × 2.2−7.1 µm, thin-walled, sometimes thick walled, septet, hyaline, hyphal end obtuse to sometimes pointed, subterminal cells 5.5−16.5 × 5−10.5 µm, globose, sub-globose to ellipsoid, thin-walled, hyaline, 3−5 cells in a row. Pileocystidia rare at margin, comparatively frequent toward center, different types, clavate to sub-clavate or sub cylindrical with obtuse to less pointed or moniliform to mucronate, 25.0−50.5 × 5.0−6.5 µm, sometimes very long 75−90 × 5.5−7.5 µm, cylindrical with obtuse to mucronate apex, one celled, thin-walled, hyaline, oil granule present with KOH. Stipitipellis composed with tightly arranged, interwoven hyphae, 3.0−4.5 µm broad, thin-walled, hyaline, branched, septate, hyphal end obtuse to clavate; context composed with sphaerocytes. Caulocystidia abundant, different shaped, 17.0−54.0 × 3.5−6 µm, few are very long, 4.0−6.0 µm wide, clavate to sub-clavate or sub-cylindrical with obtuse to round apex to moniliform or mucronate thin-walled, hyaline, oil granules present with KOH. Clamp connection absent in all tissue.
Ecology and distribution: Solitary to caespitose, growing under Shorea spp. (Dipterocarpaceae) tree. Known only from the type locality in northern Thailand.
Material examined: THAILAND. Chiang Mai Province, Chiang Mai University premises, 18°48'0.8496'' N; 98°57'21.2256'' E, elevation 335 m, 28 August 2022, S. Paloi and W. Phonrob, (SDBR-CMUSOU33). GenBank numbers OQ957482 (nrITS) and OQ968301 (nrLSU).
Russula sribuabanensis Paloi, Suwannarach N. & Kumla J., sp. nov. (Figs. 2b, 2d & 4)
MycoBank: MB 848803
Holotype: THAILAND. Lamphun Province, Mueang Lamphun District, Sribuaban Subdistrict, 18°32'11'' N; 99°07'29'' E, elevation 414 m, 19 August 2022, S. Paloi, N. Suwannarach and J. Kumla, (Holotype, CMUB40006). GenBank number OQ957188 (nrITS), OQ968132 (nrLSU), and OQ973264 (RPB2).
Diagnosis: Differs from R. pallidula, by the presence of larger pileus, long 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 coloured stipe.
Etymology: The Latin name “sribuabanensis” refers to Sribuaban Subdistrict, Lamphun Province where type species was collected.
Basidiomata medium to large. Pileus 25−105 mm diam., hemispherical to convex at early stage, becoming broadly convex, applanate to plano-concave with a central depression when mature, surface pruinose, moist, viscid when humid, greyish green (25C3−26C3), greenish grey (25B2‒26B2), greenish white (25A2) at center, becoming fed toward margin grey (26B1), greenish white (25A2) to greenish grey (25B2), no change after bruising, turned light yellowish to yellowish brown with KOH; margin incurved, regular when young, becoming striate, cracked, sometime wavy at maturity. Lamellae adnate to narrowly adnate, up to 6 mm wide, regular, white (1A1) to cream; edge even, concolorous; lamellulae none, sometimes if present, one in a row. Stipe central, 45−70 × 7−19 mm, cylindrical, tapered towards the base, sometimes curved, smooth, white (1A1), turned light yellowish after bruising, light yellowish or yellowish-brown with KOH; context firm, white (1A1), becoming very light yellowish after exposed with air. Odor unknown. Taste mild. Spores print cream.
Basidiospores 6.2−7.4−8.2 × 5.6−6.2−7.2 µm, Q = 1.0−1.1−1.2, sub-globose to sub-ellipsoid, ornamentation amyloid, composed with 0.15–0.25 low to 0.3−0.4 µm high lines connected together, produced a complete reticulate ornamentation, suprahilar spot not amyloid to weak amyloid. Basidia 35.5−51.8 × 6.6−8.9 µm, sub-clavate to sub-cylindrical, thin-walled, hyaline, oil granules present with KOH, 4-spored, sterigmata up to 4 µm long. Lamellae edge composed with numerous cystidia, sterile. Subhymenium pseudoparenchymatous. Hymenial cystidia near gill edge, 61.2−108.8 × 7.9−13.7 µm, subclavate to sub-cylindrical with appendiculate to moniliform or pointed apex, thin-walled, hyaline, oil granules present with KOH, same as gill side, up to 53 µm long projection. Pileipellis orthochromatic in cresyl blue, context composed with sphaerocytes; subpellis 250−350 µm deep, composed with more or less loosely arranged hyphae, 2.5−4 µm broad, thin-walled, hyaline, sometimes oil granules present when viewed with KOH; suprapellis 49−90 µm deep, gelatinized, composed tightly arranged suberect to repent hyphal end, 21.5−63 × 3.5−5.6 µm, thin-walled, hyaline, sometimes fills with cytoplasmic contains, obtuse to pointed apex, subterminal cell 8.5−19.7 × 5.5−9.7 µm, globose, sub-globose to ellipsoid, thin-walled, hyaline. Pileocystidia absent or very rarely present in margin, frequently found in centre, 30.0−42.5 × 3.8−5.5 µm, clavate to cylindrical with mucranate, moniliform to obtuse apex, thin-walled, hyaline, fill with cytoplasmic contains, sometimes long cystidia observed 4.5−6.5 µm wide. Stipitipellis composed with tightly arranged, terminal hyphal, 2.5−4 µm broad, thin-walled, hyaline, pointed or obtuse apex; subterminal cell sub-globose to ellipsoid. Caulocystidia 20.5−41 × 3−5.5 µm, sub-clavate to obtuse apex, very rare appendiculate or mucronate apex, thin-walled, hyaline, fill with cytoplasmic contains, sometimes very long, 3.5−6.0 µm in diam. Clamp connection absent in all tissue.
Ecology and distribution: Solitary, growing in a forest dominated by Shorea spp. (Dipterocarpaceae). Known only from the type locality in northern Thailand.
Material examined: THAILAND. Lamphun Province, Mueang Lamphun District, Sribuaban Subdistrict, 18°32'11'' N; 99°07'29'' E, elevation 414 m, 25 August 2022, S.N. Suwannarach and J. Kumla (SDBR-CMUNK1760). GenBank: nrITS: OQ957228; nrLSU: OQ968132.