Sequences of ITS and LSU gene regions of nrDNA from two collections of E. kumraticus; K-218 (LAH36945), K-219 (LAH36946), and E. swatica KU-74 (LAH36947), KU-75 (LAH36948), were obtained. The final dataset had an aligned length of 1625 characters, of which 939 were conserved sites, 653 are variable and 492 were parsimony-informative. In maximum likelihood analyses, (Fig. 1), Pakistani species E. kumraticus appeared as monophyletic and this species is found sister to the clade containing E. porphyrogriseum Noordel. and E. arcanum Reschke & Noordel. However, our species appeared as a distinct species with strong support (94%). Similarly, two collections of our second species E. swatica appeared as monophyletic with a high bootstrap support (96%) and is sister to E. cf. catalaunicum (Singer) Noordel. The phylogram (Fig. 1) inferred from combined nrITS+nLSU sequences revealed that both new species represented distinct monophyletic lineages with high statistical support.
Entoloma kumraticus A. Izhar, Kiran, Usman & Khalid sp. nov. (Figs. 2 and 3)
Diagnosis: Pileus bluish brown, centrally depressed; serrulate lamellar edge; bluish violet, fibrillose stipe; dense clusters of cheilocystidia; greyish brown intracellular pigments abundant in terminal cells of pileipellis and in some cheilocystidia.
Type: PAKISTAN. Khyber Pakhtunkhwa province, Dir Upper district, Kumrat, 2232 m asl, in grassy places on soil under Cedrus deodara, 25 July 2019, A. N. Khalid K-218, (LAH36945!; GenBank accessions for ITS: MZ157265; for LSU: MZ157269)
Etymology: The specific epithet kumraticus refers to the type locality.
Description: Pileus 1.8–2.5 cm in diameter, hemispheric, convex to plano-convex, deeply depressed at disc, deflexed towards margins, margins entire to sulcate-striate, dark aniline blue (X55) at center changing to Prout`s brown (XV15') to mummy brown (XV17') towards margin, pileal surface radially fibrillose, dry, velutinous, shiny when moist, slightly hygrophanous. Lamellae adnate, slightly adnexed, sub-distant, white (LI III) to pallid purplish grey (LIII 67'""), regular, some crisped, forked near margin, margins serrulate, lamellulae abundant, present in 2–3 tiers. Stipe 3.2–5 × 0.3–0.6 cm, cylindrical with slightly tapered base, equal, hollow, pale neutral grey ((LIII) to purplish grey (LIII 67"") at apex, pallid bluish violet (x57f) towards base, fibrillose, minutely pubescent all over, dry, some with white (LI III) tomentum at base. Annulus and Volva absent. Odor Mild.
Basidiospores [40/2/2], (8–)9–11(–12) × (6.1–)6.4–8 µm. avl × avw = 9.7 × 7.3 µm, Q = 1.25–1.38, avQ = 1.32, ellipsoid, heterodiametrical with 4–6 weak angles, thin-walled, olive yellow (5Y6/8) in KOH, inamyloid, mono-guttulate. Basidia (29–)33–47(47.3–) × (9–) 10.3–12.2(12.3–) µm. avl × avw = 38.9 × 11.6 µm, broadly clavate, hyaline in KOH, mostly 4-spored, some bi-spored, guttulate, clamp connections absent. Lamella edge sterile, serrulatum-type, made up of dense clusters of cheilocystidia, 12–34 × 4.9–8.4 µm, avl × avw = 19.5 × 6.5 µm, cylindrical to clavate, some flexuous, thin-walled hyaline in KOH, basal cells few aseptate others bi-tri septated, non-guttulate. Hymenophoral trama regular, composed of cylindrical elements, 80–125 × 4–9 µm. Pileipellis a cutis with transitions to a trichoderm, made up of inflated, clavate terminal elements, similar to pileocystidia, 30–93 × 15–25 µm, upper pileipellis cells 22–80 × 4–11 µm, a subpellis of relatively narrow septate, cylindrical hyphae, with 6–9 µm wide, avw = 7 µm, pigments intracellular as clusters, brown in water, relatively dark brown in KOH, clamp connections absent. Stipitipellis an intricate trichoderm, with cylindrical to clavate terminal elements, 1.6–5.8 µm wide, avw = 3.8 µm, mostly hyaline in KOH, others with dark brown brilliant granules, hyphae regular, septate, rarely branched, clamp connections absent. Caulocystidia absent.
Additional specimens examined. PAKISTAN. Khyber Pakhtunkhwa province, Dir Upper district, Kumrat, 2232 m asl, mostly solitary on moss covered soil under Cedrus deodara, 15 August 2018, M. Usman and A. N. Khalid K-219, (LAH36946!; GenBank accessions for ITS: MZ157266; for LSU: MZ157270)
Entoloma kumraticus sp. nov. is conspicuous on account of its bluish brown centrally depressed pileus, greyish, serrulate lamellar edge, bluish violet, fibrillose stipe, dense clusters of clavate to cylindrical, septate cheilocystidia at lamellar edge, greyish brown intracellular pigments abundant in terminal cells of pileipellis and in some cheilocystidia.
Phylogenetically E. kumraticus formed a distinct clade in phylogenetic analyses, and based on molecular data available, it can be distinguished from all other members of subgenus Cyanula. On Molecular analysis, E. kumraticus is the closest relative to E. porphyrogriseum in the phylogenetic tree (Fig. 1), presented here separating with 94% bootstrap support. E. porphyrogriseum is a common grass land species from Austria, differs in having comparatively large diameter of pileus (up to 3.7 cm), pileus surface violet brown to black, a reddish coloration is present from center towards edges throughout the pileus, dirty pink to greyish pink lamellae, longer stipe reaching up to 7 cm getting greyish red towards base, short basidia (22–35 µm in length) and bigger cheilocystidia 25–41 × 6–13 µm (Noordeloos et al. 1995b). When nrLSU sequence of these two species were compared, the sequence of E. porphyrogriseum (MK277960) showed differences at 25 nucleotide positions.
Phylogenetically, E. arcanum seems to be another closely related species to E. kumraticus but morphologically E. arcanum differs from E. kumraticus due to its non-translucently striate and non hygrophanous pileus with large pileus diameter (3.0–4.5 cm), adnate to slightly decurrent lamellae, fusiform cheilocystidia and much larger end cells of pileipellis (40–140 × 10.5–19.0 µm) and was found under Alnus- and Quercus-dominated forests (Reschke et al. 2022).
The combined nrITS and LSU sequences based phylogram (Fig. 1) shows that E. kumraticus is related to E. melleosquamulosum Reschke, Manz & Noordel., but the later one differs morphologically by its honey-colored pileus with scaly surface, distinct scales at disc, sinuate, segmentiform and ventricose lamellae, pale yellow to white stipe and its habitat in tropical submontane forest covered by Oreomunnea mexicana (Standl.) J.-F.Leroy and Quercus species. While looking towards micromorphology E. melleosquamulosum produces thickwalled, yellowish pink pigmented basidiospores, smaller basidia (26–35 × 8.0–10.0 µm), significantly longer (23–64 µm), lageniform cheilocystidia, hymenophoral trama with much larger cells (65–200 × 4.0–19.0 µm), a cutis type stipitipellis and presence of oleiferous hyphae in the trama (Reschke et al. 2022).
E. kumraticus is close to a sequence (KY744158) labelled as E. fuscosquamosum Hesler. This American species, E. fuscosquamosum differs by its mouse gray pileus, white to pinkish, arcuate to sub-decurrent, lamellae, somewhat larger basidiospores (9–13 × 6–7.5 µm), smaller basidia (34–41 × 8–10 µm), significantly larger capitate cheilocystidia (32–54 µm in length), pileocystidia bigger with size range of 40–80 × 10–15 µm or complete absence of cheilocystidia ((Helser 1967; Noordeloos 1988).
E. fuscosquamosum was reported from humus rich soil of Cades Cove, Tennesse, which is a flat valley with temperate climate, present between smoky mountains, the dominant vegetation of the valley is Quercus alba L., Tsuga canadensis L., and Pinus strobus L. (Helser 1967; Noordeloos 1988). E. kumraticus has been collected from moist temperate forests where vegetation cover was including Picea smithiana Boiss., Taxus baccata L., Cedrus deodara Roxb., Pinus walllichiana A. B. Jacks., scattered among mosses (Rajpar et al. 2020).
Pakistani species E. kumraticus are close to E. violaceoserrulatum Noordel., but E. violaceoserrulatum contrasts by its violet black to greyish brown pileus, adnate to emarginate lamellae occasionally with bluish black spots, relatively longer but narrow (20–50 × 2.5–7.0 μm) cheilocystidia, pileipellis just a trichoderm and no significant clamp connections observed (Dima et al. 2021).
Japanese species: E. nipponicum is closer to our species but it differs in its light orange to greyish red with occasionally umbilicate centre, radially splitting with age, stipe pale orange or whitish to grey towards base, smaller basidia (25–39 × 7–10 μm) and much larger (32–63 × 7–18 μm) sublageniform or subfusiform cheilocystidia (Crous et al. 2019).
Hesler (1967) placed E. serrulatum (Fr.) Hesler, under the synonymy of Leptonia serrulata (Fr.) P. Kumm. originally described from North Carolina, Tennesse, France, Sweden, Scotland and England. In comparison with the Pakistani species, E. serrulatum differs by its non-hydrophanous pileus, crowded, emarginate lamellae, green to olivaceous or brown stipe, ellipsoid 5–7 angled basidiospores, larger basidia (24–53 × 8–15 µm), bigger cheilocystidia (25–100 × 3.5–20 µm) with blue intracellular pigment, much wider trama hyphae (7–16 µm) as well as terminal elements of pileipellis (8–40 µm) having bluish intracellular pigment (Hesler 1967; Pegler 1977).
Entoloma swatica A. Izhar and Khalid sp. nov. (Figs. 4 and 5)
Diagnosis: Bluish brown centrally depressed pileus, greyish, serrulate lamellar edge, bluish violet, fibrillose stipe, dense clusters of clavate to cylindrical, septate cheilocystidia at lamellar edge, greyish brown intracellular pigment abundant in swollen terminal cells of pileipellis and in some cheilocystidia.
Type: PAKISTAN. Khyber Pakhtunkhwa province, Dir Upper district, Kumrat, 2232 m asl, in groups on grassy spots under Cedrus deodara, 30 July 2019, A. N. Khalid KU-74, (LAH36947!; GenBank accessions for ITS: MZ157271; for LSU: MZ157267)
Etymology: The specific epithet swatica refers to district Swat, the type locality.
Description: Pileus 1.5–3.2 cm in diameter, hemispherical to plano-convex, depressed at center, deflexed then involute margins, margins entire, translucently striate, initially light brownish olive (XXX 19") to Saccardo’s umber (XXIX 17") dark dull violet-blue (XXIV 53*) near margins, pileal surface wholly covered with minute squamules, crowded thin fibrils at disc, dry, dull, slightly hygrophanous. Lamellae emarginate with decurrent tooth, ventricose, moderately distant, white to pale lobelia violet (XXXVII 61") with warm blackish brown (XXXIX 1"'), uneven, finely crenulate lamellar edge, lamellulae abundant, mostly in 1–2 tiers. Stipe 4.5–5.5 × 0.4 – 0.7 cm, cylindrical, slightly swollen at base, hollow, Vanderpoel’s violet (XXXVI 55") at apex, dark Tyrian blue (XXXIV 47") towards base, fibrillose to twisted, finely pruinose in upper portion, scaly all over, not shiny, dry, white (LI III) tomentum at base. Odor and taste not recorded.
Basidiospores [40/2/2], (7.3–)9.6–10.9(–11) × (6.5–)6.9–8(–8.5) µm. avl × avw = 10.15 × 7.26 µm, Q = 1.1–1.3, avQ = 1.2, ellipsoid, heterodiametrical, with 5–7 angles, thick-walled, hyaline in KOH, inamyloid, mono- multiguttulate. Basidia 37–38.6 × 9–10 µm. avl × avw = 38 × 9.5 µm, clavate, hyaline in KOH, frequently 4-spored, rarely 2-spored, guttulate, clamp connections absent. Lamella edge heterogeneous, made up of dense clusters of cheilocystidia, (13–)14–21.8(–23) × (6.1–)6.8–11.7(–15) µm. avl × avw = 20.9 × 8.8 µm, clavate to subvesiculose, thin-walled hyaline, some with brownish intracellular pigment in KOH, basal cells septate, guttules present. Hymenophoral trama regular to intermixed, made up of cylindrical to swollen elements, 73–100 × 5–9 µm. Pileipellis a cutis with transition to a trichoderm, composed of cylindrical to clavate end cells, 15–55 × 6–17 µm, upper pileipellis cells 12–18 × 3–7 µm, a subpellis with narrow, septate hyphae 2.8–6.4 µm, avw = 4.5 µm, frequent intracellar pigments as clumps, pale yellowish brown in water, brown in KOH, clamp connections absent. a cutis, hyphae composed of narrow cylindrical to clavate cells, 15–27 µm, avw = 19 µm, terminal cells relatively inflated, hyaline in KOH, parallel, septate, frequently branched, clamp connections absent. Caulocystidia absent.
Additional specimens examined. PAKISTAN. Khyber Pakhtunkhwa province, Dir Upper district, Kumrat, 2232 m asl, in meadows under Cedrus deodara, 12 August 2018, M. Usman and A. N. Khalid KU-75, (LAH36948!; GenBank accessions for ITS: MZ157272; for LSU: MZ157268)
This species can be recognized by the light brownish olive to violet-blue finely fibrillose, translucently striate pileus, blackish brown lamellar edge, scaly, fibrillose to twisted stipe, clusters of cheilocystidia, often septate and lack of clamp connections in all tissues.
In our combined ITS+LSU analyses (Fig. 1), E. swatica formed a monophyletic lineage with European species E. catalaunicum previously known as Leptonia catalaunica Singer. However, E. catalaunicum differs from E. swatica by having non-translucently striate and non-hygrophanous pileus, pink lamellae, 6-9 angled basidiospores, larger cheilocystidia (25–100 x 7–20 µm), relatively broader terminal elements of pileipellis (12–30 µm wide) and stipitipellis a trichoderm with frequent caulocystidia (Singer 1936; Noordeloos 1982). E. norlandicum is another closer species and the major differences of E. norlandicum from E. swatica include dark brown, non-translucently striate pileus, lamellar edge white to pinkish concolorous to lamellae, terminal elements of pileipellis bigger (80–120 × 6–20 μm) and presence of caulocystidia (Noordeloos et al. 2021).
Our phylogenetic dataset shows that E. swatica is closer to E. roseotinctum Noordel. & Liiv, but E. roseotinctum is different on account of its greyish pink, non-hygrophanous and non-translucently pileus, pink-colored free lamellae and grey stipe. Microscopic features that make E. roseotinctum different from E. swatica are relatively slender and much longer lageniform cheilocystidia (25–60 × 8–13 µm) and bigger terminal cells of pileipellis (30–70 × 12–20 µm) (Noordeloos and Liiv 1992).
Entoloma swatica has a morpho-anatomical resemblance to phylogenetically closer species E. largentii Courtec., previously named Leptonia convexa Largent based on a fibrillose violet pileus covered with squamules, ventricose lamellae, purplish fibrillose stipe with a white base and ellipsoid basidiospores. However, E. largentii is easily distinguished from our species because of the sinuate lamellae, smaller basidiospores (8–10 × 5–6 µm), absence of cystidia and pale to violet pigmentation in the pileus hyphae (Courtecuisse 1986). When nr ITS sequences were compared, our DNA sequences were different at 47 nucleotide positions when compared with sequences of E. largentii.
E. holmvassdalenense Eidissen, Lorås & Weholt, differs due to non-striate margins of the pileus, a white lamellae edge, large size of basidiospores (10.2–13.7 × 7.8–10.9 µm), (1–)2-spored basidia, cheilocystidia somewhat lageniform, others with a swollen base and attenuated, mucronate or rostrate. E. holmvassdalenense has been reported from calcareous spruce forests of Holmvassdalen, Nature Reserve in Norway, where soils are roofed with tall to low herbs and some localities are covered with mosses like Sphagnum L. spp. (Weholt et al. 2014). In contrast to E. holmvassdalenense, our species was collected from moist temperate forests, from soil covered with coniferous trees.
When morphology was compared E. caesiellum Noordel. & Wölfel, appeared closer to our new species but E. caesiellum is different due to its brown to beige pileus covered with blackish blue granules, longer (up to 7.5 cm), glabrous stipe and pale yellowish intracellular pigments in the terminal cells of the pileipellis (Noordeloos 1995a; He et al. 2016).
A SriLankan species, E. gnophodes (Berk. & Broome) E. Horak, can be confused with E. swatica but E. gnophodes produces bigger basidiospores (10–13 × 7.5–10 µm), lack of cystidia and pileipellis repent cutis type (Pegler 1977).