Research Article |
Corresponding author: Md. Iqbal Hosen ( iqbalpatho@gmail.com ) Corresponding author: Tai-Hui Li ( mycolab@263.net ) Academic editor: Maria-Alice Neves
© 2016 Md. Iqbal Hosen, Tai-Hui Li, Deborah Jean Lodge, Alan Rockefeller.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hosen MI, Li T-H, Lodge DJ, Rockefeller A (2016) The first ITS phylogeny of the genus Cantharocybe (Agaricales, Hygrophoraceae) with a new record of C. virosa from Bangladesh. MycoKeys 14: 37-50. https://doi.org/10.3897/mycokeys.14.9859
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This is the first internal transcribed spacer (ITS) phylogeny of the enigmatic genus Cantharocybe and includes ITS sequences from two out of the three holotype collections. Two species are reported from the Americas and only a single species from Asia. Additionally, a collection of Cantharocybe virosa collected from tropical Bangladesh was included in this study. This species is a new record for Bangladesh, and is characterized by its tawny gray or grayish brown pileus and stipe surface, smooth ellipsoid basidiospores, elongated necked lecythiform cystidia, a trichoderm pileipellis, and abundant clamp connections. Molecular phylogenetic analysis using ITS, and combined analyses of ITS with the large subunit of nuclear ribosomal RNA (nrLSU) showed that the collection from Bangladesh is conspecific with the Indian C. virosa. A large, previously unknown intron was found in the ITS of C. brunneovelutina and C. virosa, while the C. gruberi sequence was found to be truncated where the intron would have been inserted. The intron was not identical between Cantharocybe species, and may be phylogenetically informative. Morphological description, color photographs and line drawings are provided for Bangladesh collection C. virosa. A key to the genus Cantharocybe is provided.
Biogeographic distribution, tropical mushroom, molecular phylogeny, taxonomy
The genus Cantharocybe was introduced by Bigelow and Smith in 1973 to accommodate Clitocybe gruberi Smith, based on the large yellow basidiomata, oblong to subcylindrical to elongated basidiospores and the presence of lageniform to lecythiform cheilocystidia. However, other taxa in this genus do not have large-sized yellow basidiomata and oblong to elongated basidiospores. Therefore,
The phylogenetic relationships among the taxa of Cantharocybe are well resolved, based on partial nrLSU sequence analyses (
The first author has recently collected Cantharocybe material from tropical Bangladesh that is morphologically similar to C. virosa to some extent. Careful microscopic observation of the material from Bangladesh indicates that it could be conspecific with the Indian C. virosa, but the nrLSU sequence analysis suggested that it could be a new species or perhaps a variety of C. virosa. Therefore we attempted to obtain the holotype material of C. virosa from
Cantharocybe specimens (Iqbal568 and 693) were collected from Madhupur upazila of Bangladesh on ground near to or associated with the roots of Cocos nucifera, a tree of the plant family Arecaceae during the monsoon (June to August) of 2012–2013. Specimens examined are deposited in the Cryptogamic Herbarium of Kunming
The morphological description of the basidiomata is based on field notes and documented by photographs. Color codes are according to
The protocol for DNA extraction followed that of
Three sequences (nrLSU: KF303143 and KX452406, ITS: KX452403) were generated from the Bangladeshi Cantharocybe. Additionally, two ITS sequences were also obtained from the type materials of C. brunneovelutina (BZ-1883: KX452404) and C. virosa (
Both ML and BI analyses were conducted using RAxML v.7.2.6 (
Three datasets (ITS, nrLUS and ITS+nrLSU) are constructed and analysed separately. The ITS dataset includes 17 sequences of fungal taxa (Table
Name of the species | Voucher/isolate or collection number | Origin | GenBank accession number | |
---|---|---|---|---|
ITS, 5.8S | nrLSU | |||
Ampulloclitocybe clavipes | AFTOL-ID 542 | USA | AY789080 | AY639881 |
Cantharocybe brunneovelutina | BZ-1883* | Belize | KX452404 | HM588721 |
Cantharocybe gruberi | AH24539 | Spain | JN006422 | JN006420 |
Cantharocybe gruberi | DED6609 | USA | - | AF261530 |
Cantharocybe gruberi | AFTOL-ID 1017 | USA | DQ200927 | DQ234540 |
Cantharocybe virosa | Iqbal-568 | Bangladesh | KX452403 | KF303143 |
Cantharocybe virosa | Iqbal-693 | Bangladesh | - | KX452406 |
Cantharocybe virosa | TENN63483* | India | KX452405 | JX101471 |
Cuphophyllus acutoides var. pallidus |
|
USA | KF291096 | KF291097 |
Cuphophyllus adonis |
|
Chile | KF291035 | KF291036 |
Cuphophyllus aff. pratensis | AFTOL-ID 1682 | USA | DQ486683 | DQ457650 |
Cuphophyllus aurantius |
|
Puerto Rica | KF291099 | KF291100 |
Cuphophyllus basidiosus | AFTOL-ID 1759 | USA | DQ486684 | DQ457651 |
Cuphophyllus borealis | BHS2009-104 | - | HM020684 | HM026552 |
Cuphophyllus canescens | AFTOL-ID 1800 | USA | DQ486685 | DQ457652 |
Cuphophyllus flavipes | Hattori-JP-6 | Japan | KF291044 | KF291045 |
Cuphophyllus fornicatus | Boertmann 2009/94 ( |
Denmark | KF291123 | KF291124 |
Cuphophyllus griseorufescens | PDD:27230 | New Zealand | GU233328 | GU233423 |
Phyllotopsis nidulans | HMJAU7272 | China | GQ142019 | GQ142039 |
The ITS sequences of C. brunneovelutina and C. virosa are longer than normal because a ca. 210 bp intron is inserted about 70 bp after the ITS1 primer. This intron was not previously known as Blast searches turned up no matches, but the intron sequences were similar between Cantharocybe species. The GenBank ITS sequence DQ200927 of C. gruberi was found to be truncated on the 3' end, where the intron was likely inserted. We infer that the partial ITS sequence of C. gruberi deposited in GenBank as part of the Assembling the Fungal Tree of Life (AFTOL) project was truncated because the first 70 bp of the ITS are missing beginning at a point which coincides with the position of the intron insertion in C. brunneovelutina and C. virosa.
Tree topologies obtained from both ML and BI methods of phylogenetic analyses are congruent, the ML trees are shown in Fig.
Phylogenetic relationships of the genus Cantharocybe inferred from nrLSU (a), ITS (b) and ITS+nrLSU (c) sequences using maximum likelihood (ML). RAxML bootstrap supports values (>50%) and Bayesian posterior probabilities (>0.95) are indicated on the branches at nodes (ML/PP). Newly generated sequences are highlighted in bold. GenBank accession or voucher numbers are provided after the species name.
Cantharocybe virosa (Manim. & K.B. Vrinda) T.K.A. Kumar, Mycotaxon 124: 235 (2013).
≡ Megacollybia virosa Manim. & K.B. Vrinda, Mycotaxon 111: 364 (2010).
Basidiomata medium-sized to large. Pileus 50–80 mm diam., convex at first then applanate, sometimes uplifted with cracked margin, tawny gray, dark brown (6E4–5) to grayish brown (5E3–5E4, 6E3–6F4), dry, pruinose or with fine appressed scales under lens, margin without striation. Hymenophore lamellate; lamellae adnate to decurrent, subdistant to crowded, white to pallid white (5A1, 6A1); lamellulae numerous, concolorous with lamellae. Stipe 50–80 × 10–15 mm, central, slightly curved, cylindrical, gradually thickening towards the base, at the apex ribbed by the subdeccurent lines of the hymenophore, upper half pale gray or brownish gray (5D2) to grayish brown (5E3) pruina or squamules and the remaining half nearly concolorous with the pileus, with cottony mycelium at the base, interior solid, milky white to white. Context 12 mm thick in the center of the pileus, milky white to white (6A1), firm, solid, unchanging when cut or bruised.
Basidiospores [40/2/2] (8–) 8.5–10(–11.5) × 5–6.2(–7.0) µm, [Q = 1.54–1.62, Qm = 1.58 ± 0.12] ellipsoid to broadly ellipsoid, hyaline, thin-walled, smooth, inamyloid, apiculus conspicuous. Basidia 45–60 × 8–11 µm, clavate, hyaline, thin-walled, 4-spored, sterigmata up to 8 µm long; basal septum usually clamped. Cheilocystidia 25–45 × 5–9 µm, abundant, lecythiform to lageniform, sometimes with a mucronate apex, basal portion usually clavate, the upper portion extending into an elongated neck up to 15 µm long with or without a rounded capitulum; basal septa often clamped. Pleurocystidia absent. Lamellar trama parallel to sub-regular composed of branching filamentous hyphae 4–10 µm wide, hyaline to pale yellow, thin-walled. Pileipellis a trichoderm, slightly interwoven, composed of 6–10 µm wide hyphae with often pale brown vacuolar to plasmatic pigments; terminal cells 20–65 × 6–10 µm, usually cylindrical to somewhat narrowly clavate, sometimes mucronate; pileocystidia with or without extending neck, occasionally with one or two short rounded capitula, elongated neck up to 15 µm long, clamp connection frequently present at septa. Stipitipellis composed of vertically arranged, branching, 7–10 µm wide filamentous hyphae, outer surface more or less covered with cylindrical to narrowly clavate cells (35–100 × 6–11 µm) with or without a rounded capitulum, mostly similar to cheilocystidia but sometimes double necked with an extending capitulum head, pale brown vacuolar to plasmatic pigments. Clamp connections common at septa.
Solitary or in clusters, associated with roots of Cocos nucifera (collection Iqbal 568) or along the roadside on ground (collection Iqbal 693) near C. nucifera.
Known from tropical South Asia, Bangladesh and India.
Bangladesh, Dhaka division: Tangail, Madhupur, Bangladesh Agricultural Development Corporation (BADC) campus, 24°37'35"N, 90°03'33"E, 05 Aug 2012, 20–25 m, Iqbal 568 (
The Bangladeshi C. virosa is characterized by its gray to grayish brown basidiomata, moderately crowded lamellae, fine squamules on stipe surface formed from clusters of lecythiform caulocystidia, ellipsoid to broadly ellipsoid basidiospores, and a trichoderm pileipellis.
Based on molecular analyses, C. virosa, a species recently described from India is conspecific with the Bangladeshi collection (Fig.
Surprisingly, when we blasted the newly generated ITS sequences from the holotypes of C. virosa (
Cantharocybe brunneovelutina and C. gruberi can also be separated from C. virosa morphologically. Cantharocybe brunneovelutina differs from C. virosa by its velutinous basidioma, unusual cheilocystidia with multiple prong-like appendages at the apex resembling a basidia-like structure, and a trichoderm pileipellis (
Molecular phylogenetic analyses indicated that the genus Cantharocybe is monophyletic, with strong bootstrap values (Fig.
Cantharocybe is an uncommon genus that only consists of three species. Cantharocybe gruberi has wide distribution from America (New Mexico, western North America and British Columbia) to Europe (Spain). Cantharocybe brunneovelutina is reported from tropical Central America (Belize) whereas C. virosa is from tropical South Asia (Bangladesh and India). Based on the branching order with strong bootstrap support at all nodes in our phylogeny in which C. gruberi is basal, we infer that the genus Cantharocybe may have originated in America or Europe and then migrated independently to Central America and South Asia.
The south Asian species usually occurs with the roots of Cocos nucifera (
1 | Basidiomata medium (up to 75 mm broad), with dark brown pileus, cheilocystidia resembling basidia with apical appendages, basidiospores elliptical to oblong or subcylindrical, Belize (Central America, tropical) | C. brunneovelutina |
– | Basidiomata medium to large (45–200 mm broad), yellow or pale grayish brown to grayish brown, basidiospores oblong to cylindrical or ellipsoid to broadly ellipsoid or rarely subglobose, cheilocystidia lecythiform | 2 |
2 | Basidiomata large (up to 200 mm broad), yellow, basidiospores 11–16(–17.5) × (4.5–) 6–7.5 µm, elliptical, oblong to cylindrical, western North America (temperate zone) and Spain (Europe) | C. gruberi |
– | Basidiomata medium (up to 100 mm broad), pale grayish brown to tawny gray to grayish brown, basidiospores 6.5–11(–12) × 5.5–6.5(–7) µm, ellipsoid to broadly ellipsoid or rarely subglobose, Bangladesh and India (South Asia, tropical) | C. virosa |
The first author is grateful to Rezaul Karim (BADC, Madhupur, Tangail, Bangladesh) for his hospitality and field work opportunities. Profs. Drs. Zhu L. Yang (China), Michael Weiß (Germany) and Egon Horak (Austria) are acknowledged for the comments and suggestions on an earlier version of this manuscript. The authors are indebted to Drs. Karen W. Hughes (USA) and Tom Bruns (USA) for sharing information and the sequence data of the holotype material of C. virosa (TENN63483), Drs. Karen W. Hughes (USA) and Karen Nakasone (USA) are acknowledged for their suggestions, comments and improving the manuscript. This study was supported by the National Natural Science Foundation of China (Project nos. 31470155 and 31370071).