Sample collection
In this study, samples with morphological characteristics which were typical of the genus Anthracophyllum were collected from Son Tra Nature Reserve at an elevation of 368 m. The mushroom was found on rotten branches of the plant Mussaenda frondosa Linn with the shape of a skewed bell (Fig. 1). The pileus was 1−1.5 cm in diameters, dark blue to midnight blue, pinkish white lamellae. The color of the pileus surface became darker when drying (Fig. 1A). At the maturity stage, the pileus spread out to a half shell shape, pinker lamellae (Fig. 1B).
DNA barcoding analysis
In this study, we used two different molecular markers including ITS and LSU to confirm the result of morphological classification. Firstly, the ITS fragment containing ITS1-5.8S-ITS2 of approximately 900 bp was amplified by using the primer pairs ITS5/ITS4B (Fig. 2B). Secondly, the LSU fragment containing 28S rDNA of approximately 1100 bp was amplified by primer pairs LROR/LR6 (Fig. 2A). Those PCR products were then purified and sequenced by single-end sequencing method for the sequence alignment and species identification.
The ITS, LSU sequences deduced from sequencing were aligned with reference sequences of related species on GenBank by Basic Local Alignment Search Tool (BLAST). The BLAST similarity search analysis indicated that the ITS sequence of our specimen exhibited a low similarity level of only 87% to the ITS sequences of both A. lateritium voucher UOC-DIMIA-D26 (KP757737) and A. archeri isolate AFTOL-ID 973 (DQ404387), respectively. However, the LSU sequence of our specimen exhibited a higher similarity level of 98% to the LSU sequences of both A. lateritium strain CULTENN4419 (AF261324) and A. archeri isolate AFTOL-ID 973 (AY745709), respectively. The ITS, LSU sequences of Anthracophylum sontraense sp. nov. were deposited to GenBank (NCBI) with the accession numbers of OR990596 and OR978578, respectively.
Phylogenetic analysis
The sequences of ITS and LSU of the new species in this study were combined and used for the construction of the phylogenetic tree. The combined sequences of 1706 characters (containing gaps) including 808 bp of ITS and 898 bp of LSU from species in different genera of the family Omphalotaceae were used for phylogenetic analysis. As shown in Fig. 3, the new species A. sontraense sp. nov. formed a distinct clade with Anthracophyllum archeri AFTOL-ID 973 with strong bootstrap support of 94%. The consensus Maximum Likelihood tree based on all available ITS sequences of Anthracophyllum species in GenBank database indicated that A. sontraense was closely related to A. archeri and A. sinense than A. lateritium with strong bootstrap support of 90% (Fig. 4).
Taxonomy
Anthracophyllum sontraense P.T. Tran sp. nov.
MycoBank: MB852025
Figure: 1, 5, 6
Etymology: “sontraense” reffers to the name of Son Tra Nature Reserve, Danang, Vietnam.
Ecology: Living parasitically, then saprophytically causing brown rot on branches of Mussaenda frondosa Linn in Son Tra Nature Reserve at an evelation between 368–412 m with a typical tropical climate.
Holotype: VIETNAM, Danang City, Son Tra Nature Reserve, 16o05¢50²N 108°12′45²E, elevation 368 m, on small shrubs and rotten wood of Mussaenda frondosa Linn, May, 2022, P.T. Tran (Voucher: M34295).
Description:
Pileus 1.0–1.5 cm diam, skewed bell shape when young, half shell shape when mature, convex, smooth, non-viscid, velutinose, dark blue to midnight blue, darker when dry. Stipe absent, sessile. Basidiocarp attached directly to the substratum. Pileus trama thin and soft (Fig. 1).
Lamellae thin, 1 mm thick, pinkish white when young and pinker when mature, sparse, radiating from a basal point, occasionally branching, edge concolorous, serrated and breakable when dry. Lamellae pinker at the basal point and gradually faded to the edge of the cap. Lamellae unequal in length, 7–9 through lamellae and 4–8 branched lamellae (lamellulae).
Basidiospores broadly ovoid to subglobose, (5.5-)6-6.5-7(-7.5) × (5-)5.5-6-6.5(-7) µm, thin-walled, smooth, hilar appendix obvious, hyaline, some with oleaginous contents (Fig. 5, 6). Spore-print light brown. Basidia broadly cylindrical, (24-)28–34–39(–41) × (4-)5–7-9(-10) µm, with 2-4 sterigmata, some with oleaginous contents, di- to tetrasporic (Fig. 6). Sterigmata subulate (2–)3–5 × 1–1.5(–2) µm. Chailocystidia (26–)29–37–49(–54) × (3–)5–7–9(–10) µm, hyphoid cylindric to subfusoid, clavate, fusiform, with oleaginous contents, hyaline, thin-walled. Hyphae 1–3 µm diam, slightly inflating up to 3–5 µm, smooth and hyaline, thin-walled, blue in KOH.
Keys
1. Spores with hilar appendix prominent……………………………………………….……..2
1. Spores with hilar appendix not prominent………………………………………………….5
2. Spores broadly ovoid to subglobose………………………………………………………..3
2. Spores ellipsoid or elongate ellipsoid………………………………………………………4
3. Spores 6–7 ´ 5.5–6 µm, thin-walled. Basidia 28–39 × 5–9 µm. Lamellae sparse, occasionally branching, 7–9 through lamellae and 4–8 branched lamellae. Pileus 1–1.5 cm diam, skewed bell, half shell shape. Stipe absent, sessile..…..…Anthracophyllum sontraense
3. Spores 8–14 ´ 5–9 µm, thin-walled. Basidia 30–45 × 6–12 µm. Pileus 1.5–4 cm diam, flabelliform to semicircular, convex to applanate, surface rugulose, irregularly to radially sulcate. Stipe rudimentary or absent, sessile…………………….....Anthracophyllum sinense
4. Spores ellipsoid, 8.5–11 ´ 5.5–6.5 µm. Basidia 35–40 ´ 8–11 µm. Lamellae 6–9 through-lamellae, radiating from a basal point, fuscous brown to cocoa-brown. Pileus 1–2.5 cm diam, sessile, conchiform to semiorbicular convex………………………Anthracophyllum discolor
4. Spores elongate ellipsoid, 10–12.5 ´ 5.5–6.5 µm. Lamellae are quite strongly interveined. Pileus more than 1 cm diam……………………............................Anthracophyllum proximum
5. Spores hyaline or stained brownish………………………………………………………..6
5. Spores oleaginous contents ………………………………………………………………..9
6. Spores hyaline, ovoid to ellipsoid………………………………………………………….7
6. Spores hyaline or staining brown. Pileus convex to applanate ……………………………8
7. Spores 7.5–11 ´ 5–8 µm. Basidia 20–50 ´ 7–10 µm. Lamellae 5–9 through-lamellae. Pileus 1–3 cm, reniform to short flabelliform, convex; surface pale pinkish cream to reddish brown, smooth. Stipe rumidentary, up to 3 mm long………………………..Anthracophyllum archeri
7. Spores globose to ovoid, ellipsoide, 8–14 ´ 8–10 µm. Basidia 50–75 ´ 5–14 µm. Lamellae less than 5 through-lamellae. Pileus 0.6–0.8 cm diam, sometimes clustered, orbicular either kidney-shaped or shell-shaped. Stipe lateral, very short …………Anthracophyllum pallidum
8. Spores ovoid to ellipsoid, 8.5–11 ´ 6–7.5 µm. Lamellae 9–12 through lamellae. Basidia 35–45 ´ 8–11 µm. Pileus 1–3 cm diam, sessile, semiorbicular to reniform, surface brownish or brick-red…………………………………………………Anthracophyllum melanophyllum
8. Spores oblong ellipsoid to ovo-ellipsoid, 6.5–9.5 ´ 3.5–5 µm. Basidia 27–33 ´ 6–8 µm. Lamellae 11–12 through-lamellae, radiating from a basal point, greyish-brown to fuscous brown. Pileus 0.5–2 cm, subreniform to flabelliform or orbicular. Stipe rudimentary or absent………………………………………………………….......Anthracophyllum nigritum
9. Spore broadly ovoid to subglobose, 11.5–16 ´ 10–13 µm. Basidia 50–80 ´ 11–15 µm. Lamellae 12–15 through-lamellae. Pileus 2–3 cm diam, reniforrn, convex to applanate, surface pale buffy brown to cinnamon-brown, stipe 4–12 ´ 1–2 mm…………………………………………………………….......Anthracophyllum andinum
9. Spores ovo-ellipsoid………………..………………………………………………….....10
10. Spores 7–10 ´ 4.5–7 µm. Basidia 38–70 ´ 7 µm. Lamellae in 3 series with at least 9 principal ones in the bigger basidiomes. Pileus 1.5–2.5 cm diam, fan-shape. Stipe absent…………………….…………………………………Anthracophyllum glaucophyllum
10. Stipe small to rudiment, 0.5–3 mm………………………………………………………11
11. Spores 9–12 ´ 5.5–7 µm. Basidia 33–40 ´ 7.5–9 µm. Lamellae 15-17 through-lamellae, subdecurrent, purplish umber drying cocoa-brown. Pileus 0.5–3.5 cm diam, sessile or nearly so, conchoid to reniform, convex to applanate. Stipe small to rudiment 1–2.5 mm ………………………………………………………........................Anthracophyllum beteroi
11. Lamellae less than 13……………………………………………………………………12
12. Spores, 6.5–8 ´ 4–5 µm. Lamellae 8–10 through-lamellae. Pileus 0.5–2 cm diam. Stipe conspicuous up to 3 mm. Basidia 20–25 ´ 5.5–6.5 µm.................Anthracophyllum paxilloides
12. Spores 9.5–15 ´ 5.5–8 µm. Lamellae 9–12 through-lamellae, but not interveining. Basidia 35–40 ´ 8–9 µm. Pileus 0.5–2 cm diam, semiorbicular to reniform, surface reddish brown. Stipe absent or very reduced lateral, 0.5–1 mm…………………..Anthracophyllum lateritium