Phylogenetic analysis
The final ITS-nrLSU-RPB2-mtSSU sequence dataset was analyzed using ML and BA. Both the ML analysis and BA resulted in similar tree topologies, and only the ML tree is shown in Fig. 1. The posterior probabilities for the BA are also shown along the branches. The phylogenetic analyses showed that subsect. Virescentinae was a monophyletic group; this was strongly supported by BS (100%) and PP (1). Additionally, the remaining 5 subsections of sect. Heterophyllidia were significantly supported as a monophyly.
The samples of the two new species, R. subpunicea and R. albolutea, formed a strongly supported clade (BS 100%, PP 1.00) and were clearly distinct from known and sequenced species of the subsect. Virescentinae. R. subpunicea clustered together with unidentified environmental samples of R. sp. from Japan, which is the sister taxon to a clade comprising R. crustosa Peck and R. mustelina Fr. with 85% bootstrap support and 1.00 posterior probabilities. R. albolutea is the sister taxon, without support, for a clade of all of the abovementioned species.
Taxonomy
Russula subpunicea B. Chen & J. F. Liang, sp. nov., Figs. 2a–g, 3 and 4
MycoBank: MB836124
Diagnosis: Basidiomata small to medium-sized; yellowish white to pinkish to dark pink pileus; adnate to subfree lamellae; spore print white; subglobose to broadly ellipsoid to ellipsoid basidiospores with relatively small amyloid warts or spines; hymenial cystidia mainly clavate; subcylindrical, lageniform or clavate marginal cells; terminal cells of hyphal terminations in pileipellis lageniform or clavate; suprapellis mainly composed of chains of short inflated cells with attenuated terminal cells; and pileocystidia mainly clavate.
Holotype: China, Guangxi Zhuang Autonomous Region, Pingxiang City, Fubo Forest Farm, 22°01′42.11″ N, 106°50′1.07″ E; 641 m asl., in mixed forests of Castanopsis hystrix and Betula alnoides, 19 August 2016, JXM189 (RITF3715).
Etymology: The Latin word “subpunicea” refers to the pinkish pileus.
Basidiomata: Small to medium-sized; pileus 12–87 mm in diameter; initially hemispheric when young, applanate to convex, convex with a depressed center after mature; margin incurved, cracked, and striations up to 1/4 of the radius; surface smooth, glabrous, slightly viscous when humid, peeling to 1/4 of the radius; yellowish white (2A2) with pinkish tinge when young, pinkish or dark pink after maturation, shallower at the margin. Lamellae: Adnate to subfree, 2–4 mm deep, dense, white (1A1) to cream, lamellulae absent, edge entire and concolor. Stipe: 25–40 × 7–15 mm, cylindrical, slightly attenuated at the base, white (1A1) to dirty white, and medulla initially stuffed becoming hollow. Context: 2–3 mm thick in half of the pileus radius, white (1A1), unchanging when bruised, taste slightly bitter or spicy, odor unpleasant. Spore print: White.
Basidiospores: (4.9)5.3–6.3–7.4 × (4.3)4.6–5.3–6.0(7.1) μm, Q = 1.00–1.14–1.35, subglobose to broadly ellipsoid to ellipsoid; ornamentation of relatively small, moderately distant to dense [3–5(–6) in a 3 μm diameter circle] amyloid warts or spines, 0.4–0.8 μm high, occasionally reticulate, fused in short or long branched chains [1–2(–3) in the circle], frequently connected by line connections [0–1(–2) in the circle]; suprahilar spot large, amyloid. Basidia: 30–34.3–39(40) × (5)6.5–8.8–10.7(12.8) μm, mostly 4-spored, some 2- and 3-spored, clavate; basidiola clavate or subcylindrical, ca. 5–9 μm wide. Hymenial cystidia on lamellae sides: Moderately numerous, ca. 1200–1500/mm2, 44–75.5–105(114) × (5.8)6.2–8.4–10 (10.4) μm, clavate or subcylindrical, apically mainly obtuse, often with 2–6 μm long appendage, thin-walled; contents heteromorphous or granulose, mainly in the middle and upper part, turning tawny in SV. Hymenial cystidia on lamellae edges: Sometimes shorter, (51.5)52.4–70.6–93(95) × 6–8–9.6(10.9) μm, clavate or cylindrical, apically mainly obtuse, sometimes with 2–5 μm long appendage thin-walled; contents heteromorphous-crystalline, almost without color change in SV. Marginal cells: (9.5–)12.5–16.8–23.5(25.3) × (3.7)4.4–5.8–7.2(7.5) μm, subcylindrical, lageniform or clavate, sometimes flexuous. Pileipellis: Orthochromatic in cresyl blue, sharply delimited from the underlying context, 120–180 μm deep, two–layered; suprapellis 50–70 μm deep, composed of chains of short inflated cells with attenuated terminal cells; subpellis 80–120 μm deep, composed of horizontally oriented, relatively dense, intricate, 2–6 μm wide hyphae. Hyphal terminations near the pileus margin typically branched, occasionally flexuous, thin-walled; terminal cells (10)11–19.5–34(42) × (2.3)2.6–4.6–7.2(8) μm, mainly attenuated or narrowly lageniform, occasionally clavate or cylindrical, apically constricted or obtuse; subterminal cells frequently shorter, ca. 3–8 μm wide, typically unbranched. Hyphal terminations near the pileus center similar to those near the pileus margin; terminal cells (9.2)9.5–16.6–20.5(23.8) × 2.8–4.6–6 μm, cylindrical, clavate, lageniform, apically obtuse or occasionally constricted; subterminal cells often shorter, rarely branched, ca. 4–9 μm wide. Pileocystidia near the pileus margin always one-celled, (30)35–45–55 × 4.2–5.4–6.5(–7) μm, mainly clavate, occasionally fusiform, apically typically obtuse, sometimes with ellipsoid 2–5 μm long appendage, thin-walled; contents granulose or crystalline, turning reddish in SV. Pileocystidia near the pileus center similar in size, always one-celled, (26)26.7–36.7–56.7(61.5) × (4)4.5–6–7.3(9) μm, thin-walled, mainly clavate, occasionally fusiform, apically often obtuse or occasionally mucronate, occasionally with 2–5 μm long appendage, contents granulose, turning reddish in SV. Cystidioid hyphae: In subpellis and context with granulose contents, oleiferous hyphae in context with granulose or crystalline contents.
Additional specimens examined: China, Zhejiang Province, Lishui City, Baiyunshan Ecology Forest Farm, 28°28′50.14″ N, 119°55′0.46″ E, 141 m asl., 20 August 2015, JXM33 (RITF2648); Hunan Province, Yongzhou City, Dongan County, Shunhuang Mountain National Forest Park, 26°23′54.40″ N, 111°02′54.97″ E, 457 m asl., 5 July 2012, yy37 (RITF1435); ibid., 13 July 2015, LYK49 (RITF2615).
Notes: The combination of morphological features and phylogenetic analysis place R. subpunicea in subsect. Virescentinae. Phylogenetically, our new species R. subpunicea is clustered with an unidentified environmental sample of “R. sp.” from Japan, which share 97% query coverage and 99.82% identity with the new species. Unfortunately, there are no details available except the sequence information in GenBank. Russula subpunicea is sister to a clade comprising the North American species R. crustosa and the European species R. mustelina with good support. However, R. crustosa differs from R. subpunicea in having a brownish yellow, greenish or subolivaceous pileus with a cuticle that is cracked and forms small spot-like areolae or pseudoverrucous papules and often has short hymenial cystidia on the lamellae sides [(59)63–69–75(80) × (7)8–9.5–11(13) μm] (Peck 1886, Adamcik et al. 2018); R. mustelina is distinct in having a honey brown or tawny brown to dark brown pileus and often larger basidiospores (7–9.7 × 5.8–7.8 μm) and basidia (50–70 × 6–10 μm, Sarnari 1998).
Morphologically, R. subpunicea may be confused in the field with two recently reported new species: R. maguanensis J. Wang, X.H. Wang, Buyck & T. Bau and R. substriata J. Wang, X.H. Wang, Buyck & T. Bau from China. However, R. maguanensis has a sticky tuberculate-striate pileus, a finely to coarsely cracked pileus cuticle, larger basidiospores [(7.5) 8.0–8.5–9.5 × (6.5) 7.0–7.4–8.0(8.5) μm] with isolated warts, shorter hymenial cystidia on the lamellae edges (32–52 × 7–12) and a thicker pileipellis (500–800 μm, Wang et al. 2019), whereas R. substriata possesses a sticky tuberculate-striate pileus, a finely to coarsely cracked pileus cuticle, larger basidiospores [8.0–9.0–10.0 (11.0) × (6.5) 7.0–7.5–8.0 (9.0) μm] with isolated warts, shorter hymenial cystidia on the lamellae edges (23–45 × 6–11 μm), and typically one-layered and locally two-layered pileipellis (220–400 μm, Wang et al. 2019).
Russula albolutea B. Chen & J. F. Liang, sp. nov., Figs. 2 h–k, 5 and 6
MycoBank: MB836126
Diagnosis: Basidiomata small to medium-sized, yellowish white pileus, white stipe with yellowish tinge, subglobose to broadly ellipsoid to ellipsoid basidiospores with relatively small ornamentation, hymenial cystidia mainly clavate, apically often obtuse, contents granulose or crystalline, turning mauve in SV, suprapellis mainly composed of chains of short inflated cells with attenuated terminal cells, pileocystidia clavate, apically typically obtuse.
Holotype: China, Hubei Province, Shennongjia Forestry District, Hongping Town, 31°40′17.86″ N, 110°26′40.53″ E; 2560 m asl., in mixed hardwood forests dominated by Fagus; 10 August 2016, LYK89 (RITF2653).
Etymology: The Latin word “albolutea” refers to the yellowish white pileus.
Basidiomata: Small to medium-sized, pileus 50–75 mm in diameter, hemispheric when young, convex with a depressed center when mature; margin incurved, not cracked, striations up to 1/3 of the radius; surface smooth, glabrous, slightly viscous when humid, peeling to 1/3 of the radius; yellowish white (2A2) at the center; margin white (1A1). Lamellae: Adnate, 3–4 mm deep, moderately distant, white (1A1), lamellulae absent or rare, edge entire and concolor. Stipe: 30–40 × 8–15 mm, cylindrical or clavate, curved and slightly inflated at the base, rugulose longitudinally, white with yellowish tinge (2A2), medulla hollow. Context: 2–3 mm thick in half of the pileus radius, white (1A1), without color change when bruised, taste mild to slightly bitter, odor indistinct. Spore print: White.
Basidiospores: 5.8–6.6–8.2 × 5–6–6.8(–7.2) μm, Q = 1.00–1.11–1.32(1.38), subglobose to broadly ellipsoid to ellipsoid; ornamentation of relatively small, moderately distant [4–6(–8) in a 3 μm diameter circle] amyloid warts, 0.3–0.5 μm high, subreticulate, rarely isolated, fused in long branched chains [1–2(–3) in the circle], occasionally connected by line connections [0–1(–2) in the circle]; suprahilar spot large, imamyloid. Basidia: 28(28.5)–32.8–39(43) × (7)8.3–10–11.6(14) μm, mostly 4-spored, some 2- and 3-spored, clavate or fusiform; basidiola clavate or subcylindrical, ca. 5–11 μm wide. Hymenial cystidia on lamellae sides: Dispersed to moderately numerous, ca. 600–800/mm2, (38)41–50.6–63(68) × 5–8.1–12 μm, clavate, fusiform or subcylindrical, apically always obtuse, occasionally mucronate, thin-walled; contents abundant granulose or crystalline, turning mauve in SV. Hymenial cystidia on lamellae edges: Dispersed, often smaller in size, 27(27.5)–40–63(66) × (6)6.5–8.1–9.8 μm, clavate or cylindrical, apically often obtuse or mucronate, sometimes with 2–4 μm long appendage, thin-walled; contents granulose or crystalline, mainly in the middle and upper part, turning mauve in SV. Marginal cells: (14)14.7–20–26.2(26.7) × (4.4)5.3–7.2–9.8 μm, clavate or subcylindrical, sometimes flexuous. Pileipellis: Orthochromatic in cresyl blue, sharply delimited from the underlying context, 160–220 μm deep, two–layered; suprapellis 120–160 μm deep, mainly composed of chains of short inflated cells with attenuated terminal cells; subpellis 50–70 μm deep, composed of horizontally oriented, relatively dense, intricate, 2–6 μm wide hyphae. Hyphal terminations near the pileus margin occasionally branched, sometimes flexuous, thin-walled; terminal cells (10.4)10.8–18.8–34(42) × (3.6)4–5.2–6.8(7.1) μm, mainly subcylindrical or clavate, apically obtuse; subterminal cells often wider, ca. 5–8 μm wide, always unbranched. Hyphal terminations near the pileus center similar to those near the pileus margin; terminal cells (11.4)12.5–21.7–28(40) × 3.7–4.8–6.4(7.3) μm, mainly subcylindrical, occasionally ellipsoid, apically obtuse; subterminal cells often wider, ca. 5–7 μm wide, always unbranched. Pileocystidia near the pileus margin always one-celled, (35)39–49–60(85) × (3.6)4–5.4–8.1(9.6) μm, cylindrical, clavate or fusiform, occasionally curved at the middle part, apically usually obtuse, sometimes constricted; contents abundant, granulose or crystalline, turning reddish in SV. Pileocystidia near the pileus center often smaller, always one-celled, (28)32–43–54(58) × 4.2–5.1–6.1(6.3) μm, thin-walled, clavate or cylindrical, apically typically obtuse or occasionally mucronate, contents granulose or occasionally crystalline, turning reddish in SV. Cystidioid hyphae: In subpellis and context with heteromorphous-granulose contents, oleiferous hyphae in context with granulose or crystalline contents.
Additional specimens examined China, Chungking City, Shizhu County, Huangshui Town, Dafengbao Nature Reserve Communities, 30°14′33.12″ N, 108°23′31.10″ E, 1557 m asl., 28 July 2016, LHJ160728-04 (RITF4460); Yunnan Province, Yuxi City, Huaning County, 24°12′48.36″ N, 102°56′44.72″ E, 1600 m asl., August 2016, LHJ-3735 (RITF4461); Yunnan Province, Menghai County, Mengzhe Town, Mannong Village, 21°54′17.80″ N, 100°17′23.32″ E, 1242 m asl., 10 August 2016, MH2016-83 (RITF4462).
Notes: Both morphology and phylogeny place R. albolutea clearly in subsect. Virescentinae. In our phylogenetic tree, R. subpunicea is the sister taxon to R. albolutea but differs from it by the yellowish white to pinkish to dark pink pileus, often with smaller hymenial cystidia on the lamellar sides and larger hymenial cystidia on the lamellar edges.
Given the pileus coloration, the present species resembles four taxa (Russula pseudocrustosa G.J. Li & Chun Y. Deng, R. pallidula and R. indoalba A. Ghosh et al., and R. albidogrisea J.W. Li & L.H. Qiu) of this subsection. However, R. pseudocrustosa, originally described from China, possesses a cracked pileus with a radically disrupted cuticle and typically larger hymenial cystidia (67–88 × 8–12 μm, Deng et al. 2020). The Chinese species R. pallidula can be distinguished by the typically smaller basidiomata (40–55 μm) and pale turtle green to pale olive-buff pileus with minute white to cream scales at the margin (Chen et al. 2019). Russula indoalba (originally reported from India) is distinct in its wavy to interrupted pileus margin, adnexed to almost free lamellae, yellowish-cream spore print, larger basidia (45–60 × 9–13 μm) and hymenial cystidia on the lamellae sides (47–90 × 8–16 μm, Hyde et al. 2016). Russula albidogrisea, originally reported from China, has larger basidia (41–48 × 9–11 μm) and smaller hymenial cystidia on the lamellar sides (35–50 × 5–11 μm, Das et al. 2017).