A total of 21 sequences were newly generated and deposited in GenBank (Supplementary table 1). The concatenated sequence alignment of the three loci comprised 100 sequences (38 for ITS, 30 for rpb2 and 32 for mtSSU) from 43 collections (Supplementary table 1). The alignment was 2,004 characters long, including gaps. Multi-locus trees generated from ML and BI analyses showed similar topologies without any supported topological conflict. The multi-locus phylogeny (Figure 1) confirmed placement of all Thai collections within the well-supported R. subsect. Amoeninae (ML=99, BI=1.0). Five collections from northeastern Thailand and two collections from northern Thailand form two strongly supported clades and are described below as the new species R. bellissima sp. nov. and R. luteonana sp. nov. The new species are not resolved as sister. The first species, R. bellissima, is strongly supported as sister to a clade of Australian sequestrate species that includes R. variispora T. Lebel and an undescribed Russula sp. labeled as Macowanites sp. The Indian species R. intervenosa S. Paloi, A.K. Dutta & K. Acharya is placed as sister to them with only ML support of 77%. The second species, R. luteonana, is placed with moderate support as sister to the sequestrate European species R. andaluciana T.F. Elliott & Trappe.
The ITS tree (Figure 2) shows a similar topology and relationships for the studied specimens. In addition, R. intervenosa received good support (ML=84, BI=0.99) as sister to the clade of R. bellissima and R. variispora. Five additional ITS sequences that are grouped with strong support within R. bellissima species clade were recovered, three from Thailand, one from Laos, and one from Singapore. We did not recover any other Amoeninae ITS sequences from Thailand.
Russula bellissima Manz & F. Hampe sp. nov.
Mycobank: MB 840549
Holotype: THAILAND, Theong district, Chaing Rai, 19°36'45''N 100°4'00''E, alt. 500 m, dry dipterocarpus forest in small groups on loamy soil, 12 July 2012, F. Hampe (Holotype: GENT FH 12-127; Isotype: MFLU12-0619).
Etymology: ’bellus’ = latin for beautiful, pretty, lovely; ’bellissima’ = the most beautiful. Resembling the species Russula bella which is also belonging to Russula subsection Amoeninae.
Diagnosis: Pileus small to medium-sized; cuticle dry, smooth, matt and pruinose, red; stipe white or with a red flush; spore ornamentation of moderately distant to dense amyloid spines or warts, frequently fused into short crests or even long wings; suprahilar spot inamyloid; hymenial cystidia and pileocystidia absent.
Pileus: (Figure 3) small to medium sized, 10–50 mm diam., young hemispherical or convex, becoming plane and depressed at the centre; margin first even, when old distinctly tuberculate-striate up to 10 mm from the margin, often radially cracking; cuticle hardly peeling, radially disrupted into small patches, pruinose when young, later dry, smooth, matt and pruinose in the centre, colour near the margin when young varnish red (9C8), later red to coral red (9B6–7); near the centre deep red, blood red, dark red (10C7–8), raspberry red (10D7), strawberry red (10D8) or purple brown (10E-F8). Lamellae: 3–5 mm deep, thin, moderately dense, 6–8 at 1 cm near the pileus margin, adnexed, white, slightly anastomosing at the base; lamellulae absent, occasionally forked near the stipe; edges concolorous, entire but pruinose under lens. Stipe: 10–30 × 3–7 mm, usually narrowed towards the base, sometimes cylindrical, surface smooth, white and mainly with a distinct pastel red to red flush, occasionally completely white or sometimes also almost completely red, interior stuffed. Context: white, fragile, unchanging when damaged, reaction with guaiac after 5 seconds negative on both stipe and lamellae surfaces, reaction to FeSO4 and sulfovanillin negative; taste mild; odour inconspicuous. Spore print: not observed.
Spores: (Figure 4, 6) (6.9–)7.3–7.8–8.3(–8.9) × (6.1–)6.8–7.2–7.6(–8.4) µm, subglobose to broadly ellipsoid, Q=1.01–1.1–1.2(–1.29); ornamentation of moderately distant [(4–)5–6(–7) in a 3 µm diam. circle] amyloid spines or warts, (1.1–)1.2–1.4–1.6(–1.7) µm high, fused or connected by fine line connections into often long crests or wings, [(0–)1–3(–4) fusions and the same number of line connections in a 3 µm diam. circle], crests and wings frequently branched and occasionally form closed loops, isolated elements dispersed, edge of crests and wings irregularly wavy; suprahilar spot moderately large, inamyloid. Basidia: (30.5–)34.5–44.1–53.5(–65.0) × (10.5–)11.5–12.6–14.0(–16.0) µm, broadly clavate or obpyriform, 4-spored; basidiola cylindrical, ellipsoid or broadly clavate, ca. 5–10 µm wide. Hymenial cystidia on lamellae sides: absent. Lamellae edges: covered by densely arranged or fasciculate marginal cells. Marginal cells: (27.0–)38.5–46.4–54.5(–61.0) × (5.0–)5.5–6.7–7.5(–9.0) µm; subulate or narrowly lageniform, apically attenuated and constricted to ca. 1–2 µm, sometimes slightly moniliform or flexuous. Pileipellis: (Figure 5) orthochromatic in Cresyl Blue, gradually passing to the underlying context, 200–300 µm deep; suprapellis 60–130 µm deep, composed of erect or ascending hyphal terminations forming a dry trichoderm, well delimited from 140–210 µm deep subpellis composed of horizontally oriented, strongly gelatinized narrow hyphae. Subpellis not well delimited from the underlying context, elongate hyphae gradually changing to sphaerocytes. Acid- resistant incrustations: absent. Hyphal terminations near the pileus margin: composed of long apically attenuated terminal cell and a chain of 1-4 ovoid to barrel shaped, short unbranched cells with one distinctly longer apical cell; constricted on septa, usually not flexuous, oriented towards the pileus surface, usually thin-walled, sometimes slightly thick-walled (up to 1 µm thick); terminal cells mainly subulate or lageniform, apically attenuated and acute, measuring (19–)27.5–38.3–49.0(–66.5) × (3.3–)4.5–5.8–7.0(–9.0) µm, rarely with a forked apex, mixed with dispersed, cylindrical or ellipsoid, distinctly shorter, obtuse terminal cells measuring (7.5–)11.5–17.8–29.5(–42.5) × (3.0–)4.0–4.5–5.0 µm; subterminal cells measuring (4.5–)5.5–8.3–11.5(–16.0) × 4.5–5.3–6.0(–7.0) µm. Hyphal terminations near the pileus centre: similar in shape and also with a mixture of long acute and short obtuse terminal cells, acute ones measuring (12.0–)22.0–35.2–48.5(–79.0) × (2.5–)3.5–4.9–6.5(–8.0) µm, obtuse ones more frequent, measuring (6.5–)8.5–12.0–15.5(–22.0) x (3.5–)4.0–4.9–6.0(–7.5) µm. Primordial hyphae or pileocystidia: absent. Cystidioid hyphae and oleipherous hyphae not observed.
Additional material studied: THAILAND, Chiang Mai Province, Mae On District, about 3 km from Tharnthong lodges, 18°51'55''N 99°17'23''E, alt. 725 m, Dipterocarpaceae dominated forest with the presence of some Castanopsis trees, in small groups on loamy soil, 17 July 2012, F. Hampe (GENT FH 12-158, duplicate: MFLU12-0648).
Note: Russula bellissima is a small species with a bright red pileus and pink colour on the stipe. This colour is distinctive and resembles North American R. mariae, Indian R. intervenosa and Asian R. bella. It is very unlikely that the distribution of any European or North American species is overlapping with the Thai species. However, little is known about the distribution ranges and the ecological niches of other Asian Russula species, therefore discussing the morphological distinguishing characters between them and R. bellissima is more relevant. Russula bellissima is not closely related to R. bella and it differs from this species by larger spores with a more prominent spore ornamentation, absence of hymenial cystidia on lamellae sides and subterminal short, ellipsoid cells in the suprapellis arranged in unbranched chains of up to four7. The Thai species resembles and is closely related to the Indian R. intervenosa, but it has a more prominent spore ornamentation, hymenial cystidia (on lamellae sides) are absent, and hyphal terminations in the pileipellis are wider39.
Russula luteonana M. Pobkwamsuk & K. Wisitrassameewong sp. nov.
Mycobank: MB 840550
Holotype: THAILAND, Amnat Charoen province, Hua Taphan district, Junction near Watbochaneng , dry dipterocarp forest, alt. 145 m, 15°41'28''N 104°31'41''E, 13 July 2016, Thitiya Boonpratuang, Rattaket Choeyklin, Prapapan Sawhasan, Maneerat Pobkwamsuk, Pattrachai Juthamas, Nattawut Wiriyathanawudhiwong, Patcharee Patangwesa (BBH41120).
Etymology: ‘Luteolus’= yellow colour, ‘Nanus’ = small. Refer to pileus color and size of the species.
Diagnosis: Pileus medium-sized, dry, usually yellow, spores with subreticulate amyloid ornamentation and inamyloid suprahilar spot, hymenial cystidia on lamellae sides large, lamellae edges with combination of subulate, clavate and pyriform marginal cells.
Pileus: (Figure 7) medium-sized, 28‒53 mm diam., plano-convex with depressed centre, infundibuliform when mature; margin striated and radially cracking in dry condition; cuticle dry, peeling to almost ½ of radius, smooth to minutely wrinkled, dull in dry condition, color very variable, some collections pale cream and with darker pale brownish-yellow centre, other yellow brownish and with darker orange-brown centre, sometimes also bright red-brown and with discolored centre, always with rusty-brown spots especially when near the centre. Lamellae: 3‒5 mm deep, moderately distant, intervenose, forking near the stipe, white to cream, edges even, concolorous. Stipe: 26‒40 × 6‒9 mm, cylindrical or narrowed at the base, surface dry, longitudinally wrinkled, white, turning brown when bruised. Context: 2‒4 mm in at the half pileus radius, soft, solid, becoming partially hollow when mature, white, unchanging when cut. Taste mild; odour rather strong, fishy. Spore print: not observed.
Spores: (Figure 8, 10) (7.4‒)8.1‒8.6‒9(‒10.1) × (6.1‒)7.4‒7.5‒7.9(‒9.1) μm, subglobose to broadly ellipsoid, Q = (1.03‒)1.09‒1.15‒1.20(‒1.30), ornamentation of moderately distant, obtuse, (0.7‒)1.1‒1.3‒1.5(‒1.9) μm high spines, connected by abundant line connections [(0‒)3‒6(‒8) in in a 3 µm diam. circle], branched, forming an incomplete reticulum, crest irregularly wavy and occasionally fused [(0‒)1‒2(‒5) fusions in the circle], isolated elements rare; suprahilar spot inamyloid. Basidia: (29‒)34.5‒39.1‒44(‒51.5) × (10‒)12‒13.2‒14.5(‒16.5) μm, clavate, 4-spored, rarely 2-spored, basidiola subcylindrical to subclavate, (25.5‒)30‒35.4‒41(‒47) × (9‒)11‒12.2‒14 (‒16) μm. Hymenial cystidia on lamellae sides: usually protruding over other elements of hymenium, widely dispersed (< 350/mm2), (65‒)78‒92.1‒106.5(‒132) × (10.5‒)14‒17.4‒21(‒24) μm, fusiform to lageniform, apically obtuse to subacute, thin- or slightly thick-walled; contents homogenous, optically empty, negative in sulfovanillin. Lamellar edges: with dispersed basidia; Marginal cells: very abundant, mainly long and apically acute, resembling terminal cells in the pileipellis, (28.5‒)41.5‒55‒69(‒93) × (5.5‒)7.5‒9.0‒10.5(‒13) μm, fusiform, rarely lanceolate or lageniform, often fasciculate; mixed with less frequent, distinctly shorter, broadly clavate or obpyriform elements μm, (12.4‒)20.1‒25.7‒31.2(‒44.0) × (5.2‒)‒8.9‒10.9‒12.8(‒14.8) μm. Pileipellis: (Figure 9) orthochromatic in Cresyl blue, sharply delimited from the underlying context, 110‒350 um deep; suprapellis a trichoderm of ascending or erect hyphal terminations, non-gelatinized, subpellis composed of dense, strongly gelatinized, horizontally oriented, narrow hyphae. Acid-resistant incrustations: absent. Hyphal terminations near the pileus margin: mainly unbranched, apically often flexuous, usually composed of distinctly longer terminal cells and a single subterminal short cells, thin-walled; terminal cells of two distinct types, either long and apically attenuated or short, subcylindrical and obtuse, constricted on septa, the long type (27.5‒)44.5‒60.7‒76.5(‒100.5) × (4.0‒)5‒6.6‒8(‒11.3) μm, subulate, narrowly fusiform or narrowly lageniform, apically acute ; the short type (16.4‒)24.7‒36.2‒47.8(‒71.9) × (3.7‒)4.7‒5.7‒6.8(‒7.7) μm, cylindrical, rarely narrowly clavate or somewhat apically narrowed, occasionally moniliform; subterminal cells shorter, mainly unbranched, 5‒8 μm wide. Hyphal terminations near the pileus centre: similarly with terminal elements of two types, the longer type (20‒)37.5‒53‒69(‒90) × (3.5‒)5‒6.1‒7.5(‒9) μm, subulate and acute or subcylindrical and obtuse, the shorter type (12‒)18.5‒31.4‒44.5(‒65.5) × (3‒)4‒5.0‒6(‒7) μm, subcylindrical, cylindrical, rarely narrowly clavate. Primordial hyphae or pileocystidia: absent. Cystidioid hyphae and oleipherous hyphae: in subpellis absent.
Additional material studied: THAILAND, Amnat Charoen province, Hua Taphan district, Junction near Watbochaneng, dry dipterocarp forest, 13 July 2016, 3 collections from different mycelia at this site, Thitiya Boonpratuang, Rattaket Choeyklin, Prapapan Sawhasan, Maneerat Pobkwamsuk, Pattrachai Juthamas, Nattawut Wiriyathanawudhiwong, Patcharee Patangwesa, (BBH41121, BBH41122, BBH41125); ibid., 29 May 2017, Thitiya Boonpratuang, Rattaket Choeyklin, Maneerat Pobkwamsuk, Nattawut Wiriyathanawudhiwong, Tuksaporn Thummarukcharoen (BBH42510).
Note: Russula luteonana is very variable but based on the pileus colour we can eliminate some species with purple or green tints. If we combine this with the white or nearly so stipe, it can only be confused with either R. bella (if it has redder colouration) or R. orientipurpurea. The unique character of R. luteonana is the large cystidia which range 14–21 μm in width and are often also obtuse. While R. bella has many microscopic characters that distinguish it from this proposed species (e.g. smaller spores, narrower hymenial cystidia), R. orientipurpurea resembles, in many aspects, the Thai species (i.e. relatively large spores, obtuse and relatively wide hymenial cystidia on the lamellae sides, and usually only one unbranched short cell below the terminal cell of hyphae in pileipellis). Distinction between these two species can be made by the more prominent spore ornamentation and the often acute hymenial cystidia of R. luteonana .