Three new species of genus Russula Pers. from Sal dominated forests of tropical India based on morphotaxonomy and multigene phylogenetic analysis Author Ghosh, Aniket Central National Herbarium, Botanical Survey of India, 3 MSO Building, DF block, sector 1, Salt Lake City, Kolkata 700064 (India) ghosh. aniket 87 @ gmail. com Author Buyck, Bart Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d’histoire naturelle, Sorbonne Université, Université des Antilles, CNRS, EPHE, case postale 39, 57 rue Cuvier, F- 75231 Paris Cedex 05 (France) bart. buyck @ mnhn. fr buyck@mnhn.fr Author Chakraborty, Dyutiparna Eastern Regional Centre, Botanical Survey of India, Shillong 793003 (India) dyuti. parna. mail @ gmail. com mail@gmail.com Author Hembrom, Manoj Emanuel Acharya Jagadish Chandra Bose Indian Botanic Garden, Botanical Survey of India, P. O. Botanic Garden, Howrah 711103 (India) manojhembrom @ yahoo. co. in manojhembrom@yahoo.co.in Author Bera, Ishika Author Das, Kanad Central National Herbarium, Botanical Survey of India, P. O. Botanic Garden, Howrah 711103 (India) iamishika 6 @ gmail. com kanaddasbsi @ gmail. com (corresponding author) com@gmail.com text Cryptogamie, Mycologie 2023 2023-03-21 20 3 27 50 journal article 10.5252/cryptogamie-mycologie2023v44a3 1776-100X Russula shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. ( Figs 10-12 ) Russula shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. is separated from North American R. redolens by the absence of a strong celery-like taste and odour and because of its ectomycorrhizal association with Shorea robusta . HOLOTYPE . — India . West Bengal , Jhargram district , Lodhasuli , 22°19’57”N , 87°02’47”E , alt. 80 m a.s.l. , on ground, under Shorea robusta in tropical deciduous forests, 27.VIII.2021 , D. Chakraborty , NPDF917-10 L (holo-, CAL [ CAL 1864 ]!). ADDITIONAL SPECIMENS EXAMINED. — India . West Bengal , West Bengal, Jhargram district, Lodhasuli , 22°19’59”N , 87°02’47”E , alt. 80 m a.s.l., on ground, under Shorea robusta in tropical deciduous forests, 13.VIII.2020 , A. Ghosh , AG 20-027 ( CAL [ CAL 1865 ]); Jhargram district, Jhargram city, 22°25’01.1”N , 87°00’13.5”E , alt. 103 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 12.VIII.2021 , A. Ghosh , AG 21-068 ; Uttar Dinajpur , Kaliyaganj , Dhamja , 25°18’00”N , 88°20’35.9”E , alt. 80 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 07.IX.2020 , D. Chakraborty , RGJ-20-05 ; Uttar Dinajpur , Kaliyaganj , Dhamja , 25°18’00”N , 88°20’35.9”E , alt. 80 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 10.X.2021 , D. Chakraborty , RGJ-21-03 ; Bihar , West Champaran district, Valmiki national Park , Raghia range, Sitalbari enclosure, 27°20’14.4”N , 84°13’05.8”E , alt. 133 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 15.IX.2020 , M.E. Hembrom , MEH-20-114 ; Jharkhand , Rajmahal hills, Sahibganj district, Borio block, Pir-Baba Kairasol forest area, 25°09’41.7”N , 87°40’31.9”E , alt. 126 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 24.VIII.2021 , A. Ghosh , AG 21-02 ( JH ); Ranchi district, Getalsud , 23°28’36.5”N , 85°33’23.8”E , alt. 570 m a.s.l., on ground, under S. robusta in tropical deciduous forests, 09.X.2021 , M.E. Hembrom , MEH-21-32 . GENBANK. —OL461227 (nrITS, holotype ) andOL461230 (nrITS, specimen voucher no. AG 20-027 ); ON365930 (nrLSU, holotype ), ON365931 (nrLSU, specimen voucher no. AG 20-027 ); ON387509 (mtSSU, holotype ), ON387514 (mtSSU, specimen voucher no. AG 20-027 ); ON398069 ( rpb 2, holotype ), ON398070 ( rpb 2, specimen voucher no. AG 20-027 ) ETYMOLOGY. — ‘ shoreae ’ refers to Shorea robusta ( Dipterocarpaceae ), the host tree. MYCOBANK. — MB 844207. FACESOFFUNGI NUMBER. — FoF 11435. DESCRIPTION Pileus small to medium-sized, 12-70 mm in diam., convex when young, becoming plano-convex to applanate, uplifted with age, centrally depressed to umbilicate at maturity; margin decurved to plane with age, entire; cuticle viscid and shiny when moist, dull upon drying, peeling to 1/2 of the radius, when young dark green (27F5-6) to dull green (26C-D3-4) with paler margin (26D4), at maturity with dark green (27F5-6) centre with alternating dark green and greyish green concentric rings (26D3-4, 25-26F7-8). Pileus context up to 6 mm thick at the disc, thinning towards the margin, compact, brittle, chalky white (1-2A1), unchanging after bruising or cutting; turning salmon pink (6A4) with FeSO 4 and deep to dark turquoise (24E-F7-8) in guaiacol. Lamellae up to 4 mm high, narrowly adnate to adnexed, subdistant to close (9-13/cm at pileus margin), chalky white (1-2A1), forked near the stipe apex, midway to the margin, or near the margin; lamellulae present in different lengths; edges entire and concolorous. Stipe 10-67× 5-22 mm , cylindrical to clavate, central, firm and brittle; surface dry, smooth, chalky white (1-2A1) with dull green (26D4) tinges. Stipe context solid when young, becoming stuffed to hollow with maturity, surface chalky white (1-2A1), unchanging after bruising or cutting, becoming salmon pink (6A4) with FeSO 4 and deep to dark turquoise (24E-F7-8) in guaiacol. Odour not distinctive. Taste mild. Spore print not obtained. Basidiospores subglobose, broadly ellipsoid to ellipsoid, rarely globose, (5.5-)6.3-7.0-7.7(-8.4)×(4.8-)5.4-6.0-6.6(-7.8) µm, Q=(1.02-)1.09-1.17-1.25(-1.41); ornamentation composed of amyloid isolated warts; warts up to 0.5 µm high, pustu - lose or rounded, sometimes fused with each other; suprahilar spot distinct, large but inamyloid, apiculi up to 1.5 µm long. Basidia (46-)51-57-62(-65)×(9-)10-11-12(-13) µm, 4-spored, subclavate, tapering towards base, sterigmata up to 6 µm long. Hymenial cystidia rare on the lamellae sides, (50-)53.5-61-68 (-76)×(7-)10-11.5-13.5(-15) µm, cylindrical,subclavate, clavate to fusiform with rostrate to moniliform apex, emergent up to 22 µm above the other elements of the hymenium; contents finely crystalline, near the lamellae edges usually smaller and narrower, measuring 46-51-55× 9-11-12 µm; all hymenial cystidia not reacting in sulfovanillin. Lamellae edges fertile with basidia and cystidia. Subhymenium layer 35-40 µm thick, pseudoparenchymatous. Hymenophoral trama mainly composed of large nests of sphaerocytes and intermixed with hyphal elements. Pileipellis orthochromatic in Cresyl blue, sharply delimited from the underlying sphaerocytes of the context, 276-307 µm deep, two-layered and vaguely divided in a relatively dense suprapellis, 96-91 µm deep, composed of erect or ascending hyphal terminations forming a trichoderm, and a subpellis 180-216 µm deep, composed of more densely and more horizontally oriented hyphae.Acidoresistant incrustations absent.Hyphal terminations near the pileus margin often slightly flexuous, thin-walled, composed of chains of 1-3 cells, branched at the subterminal cells or the cells just below; terminal cells measuring (11-)13-21-29(-44.5)×(3-)4-4.5-5.5(-7) µm, mainly subulate or cylindrical, apically acute and distinctly attenuated or obtuse-rounded; subterminal cells mainly cylindrical, but sometimes wider. Hyphal terminations near the pileus centre of similar structure, terminal cells slightly less wide, measuring (9-)12.5-19.5-26.5(-30.5)×(2.5-)3-4-4.5(-5) µm, mainly subulate or cylindrical, apically acute and distinctly attenuated or obtuse-rounded; subterminal cells mainly cylindrical, but sometimes wider or with lateral appendages. Pileocystidia near the pileus margin typically one-celled,flexuous, thin-walled, (14-)17.5-40-62(-96)×(2.5-)3.5-4.5-5.5(-6) µm, mainly subulate, apically mostly mucronate or with short appendages; those near the pileus centre slightly shorter, (22-)24-31.5-39(-46) ×(3.5-) 4-4.5-5 µm; all with contents finely crystalline and without reaction in sulfovanillin.Clamp connections absent in all parts. FIG. 10. — Phylogram generated by Maximum Likelihood analysis based on nrITS sequence data of Russula shorae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. and their allied species. Maximum Likelihood bootstrap support values (MLbs) ≥70% are shown on the left of “/” and Bayesian Posterior Probabilities (BPP) ≥ 0.95 are shown on the right above or below the branches at nodes. Russula shorae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. is placed in red font to highlight their phylogenetic positions in the tree. FIG. 11. — Russula shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. (from holotype): A -C , fresh and dissected basidiomata in the field and basecamp; D , E , transverse section through pileipellis showing elements; F , transverse section through lamellae showing basidia; G , H , transverse section through lamellae showing hymenial cystidia near the lamellae edges; I -M , transverse section through lamellae showing hymenial cystidia near the lamellae sides. Scale bars: A, B, 20 mm; D, 20 μm; E-M, 10 μm. FIG. 12. — Russula shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. (from holotype): A , basidiospore; B , elements of the pileipellis near the pileus margin: hyphal terminations and pileocystidia; C , hymenial cystidia near the lamellae sides; D , basidia; E , hymenial cystidia near the lamellae edges; F , elements of the pileipellis near the pileus centre: hyphal terminations and pileocystidia. Scale bars: 10 µm. NOTES The nBLAST of the obtained ITS sequence places R. shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. in subg. Heterophyllidiae , which was also clearly suggested by its morphological characters, including the inamyloid suprahilar spot, the typical ramifying hyphal extremities at the pileus surface composed of chains of more or less inflated, short cells that become gradually narrower toward the terminal cell and onecelled, narrow and mucronate pileocystidia. In our ITS phylogenetic analysis, the here newly described R. shoreae D.Chakr., A.Ghosh, K.Das & Buyck , sp. nov. is placed sister to the Chinese R. verrucospora , a subtropical species that has smaller spores and a more variable pileus colour with grey and vinaceous tints. Both are again placed sister to the North American and equally green R. redolens which has a unique and strong smell of parsley. These three species form a strongly supported clade, which is placed sister, again with strong statistical support, to the annulate R. brunneoannulata Buyck of the African subsect. Aureotactinae Heim ex Buyck ( Buyck 1994 ). All these species have very similar microscopic features of pileipellis and share the same type of spore ornamentation consisting of isolated blunt warts. All of these species differ from subsect. Cyanoxanthinae Sing. in the absence of strong metachromatic reactions in Cresyl blue. Some of the above-mentioned species were also grouped with strong support in recent multilocus phylogenies. Indeed, a representative sampling of species belonging to subg. Heterophyllidiae was distributed over four significantly supported clades in the combined multilocus phylogeny, based on 28S, rpb 2 and tef -1 loci, that was published by Wang et al . (2019) . In that phylogeny, subsect. Substriatinae was introduced as a new subsection that grouped with Aureotactinae as one of the four strongly supported clades in the subgenus. This topology was never recovered in ITS-based phylogenies, nor in the combined multilocus (based on the same loci) published by Vera et al . (2021) where Aureotactinae , impacted by the introduction of R. redolens , no longer grouped with Substriatinae .