Research Article |
Corresponding author: Alfredo Vizzini ( alfredo.vizzini@unito.it ) Academic editor: Thorsten Lumbsch
© 2019 Alfredo Vizzini, Alessia Tatti, Henk A. Huijser, Jun F. Liang, Enrico Ercole.
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:
Vizzini A, Tatti A, Huijser HA, Liang JF, Ercole E (2019) Looking for Lepiota psalion Huijser & Vellinga (Agaricales, Agaricaceae). MycoKeys 52: 45-69. https://doi.org/10.3897/mycokeys.52.34021
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Lepiota psalion is fully described based on a recent collection from Sardinia (Italy) and the holotype. NrITS- and nrLSU-based phylogeny demonstrates that sequences deposited in GenBank as “L. psalion” and generated from two Dutch and one Chinese collections are not conspecific with the holotype and represent two distinct, undescribed species. These species are here proposed as Lepiota recondita sp. nov. and Lepiota sinorecondita ad int.
Agaricomycetes, Basidiomycota, cryptic species, hymeniform pileus covering, taxonomy
Recent molecular analyses have indicated that the genus Lepiota (Pers.) Gray is a paraphyletic assemblage that is monophyletic only if it is considered together with species of Cystolepiota Singer, Echinoderma (Locq. ex Bon) Bon, Melanophyllum Velen., and Pulverolepiota Bon (
Species of Lepiota with a hymeniform pileus covering were distributed by
According to recent molecular analyses, the species with a hymeniform pileus covering do not form a monophyletic lineage (
During a 3-year survey of macrofungi in the Botanical Garden of Cagliari (Sardinia, Italy), a collection of a Lepiota with a hymeniform pileus covering was recorded. It showed striking morphological affinities with L. psalion Huijser & Vellinga. The present paper fully describes this collection using morphological features and molecular data, and infers, through sequencing of the holotype, the phylogenetic placement of L. psalion. Additionally, two morphologically allied taxa, Lepiota recondita sp. nov. and L. sinorecondita ad int. are described.
Macroscopic description was based on detailed field notes of fresh basidiomes. Colour terms in capital letters (e.g., Pale Cinnamon-Pink, Plate XXIX) are those of Ridgway (1912). HTML alphanumeric colour codes (https://html-color-codes.info/) were obtained using GIMP (GNU Image Manipulation Program, https://www.gimp.org/) with the “Color Picker” tool on photographs taken in natural light of fresh basidiomes. Micromorphological features were observed on dried material; sections were rehydrated in water or 5% KOH and mounted separately in ammoniacal Congo Red, Cotton Blue, Cresyl Blue, and Melzer’s reagent. Measurements of the microscopic features of Lepiota psalion and L. recondita were made by photographing all the elements occurring in the visual field of an Optika B-383 PLi light microscope. Measurements were performed using the Piximètre 5.9 R 1530 software (http://ach.log.free.fr/Piximetre/) at 1000× magnification. The microphotographs were taken by an Optikam B5, 5 MP× camera.
When possible, dimensions of the microscopic elements are given as: (minimum–) average minus standard deviation – average plus standard deviation (–maximum) of length × (minimum–) average minus standard deviation – average plus standard deviation (–maximum) of width. Spore dimensions do not include the hilar appendix. The width of each basidium was measured at the widest part, and the length was measured from the apex (sterigmata excluded) to the basal septum. The DNA fluorescent dye 4′,6-diamidino-2-phenyl-indoldihydrochloride (DAPI) was used to stain nuclei in spores following
Total DNA was extracted from seven dry basidiomes (Tab.
Taxa, vouchers and GenBank accession numbers used in the molecular analyses. Newly sequenced collections are in bold.
Species | Collection No. | Origin | GenBank accession No. | |
---|---|---|---|---|
nrITS | nrLSU | |||
Chamaemyces fracidus | Th.W. Kuyper 960 (L) | Belgium | AY176343 | AY176344 |
Cystolepiota cystophora | MCVE 56163 | Italy | GQ141550 | – |
Cystolepiota seminuda | 4-X-1989, H.A. Huijser s.n. (herb. Huijser) | The Netherlands | AY176350 | – |
MCVE 9247 | Italy | JF907983 | – | |
Lepiota aff. grangei | TENN 064380, ECV4063 | USA | – | MF797685 |
Lepiota acutesquamosa | DUKE-JJ177 | USA | – | U85293 |
Lepiota albogranulosa | LAH. NO. 10152012, Holotype | Pakistan | LK932284 | – |
LAH. NO. 9992012 | Pakistan | LK932285 | – | |
Lepiota apatelia | 26-IX-1990, H.A. Huijser (herb. Huijser) | The Netherlands | AY176462 | – |
04-X-1991, H.A. Huijser (herb. Huijser) | The Netherlands | GQ203819 | – | |
Lepiota aspera | E.C. Vellinga 2233 (L) | The Netherlands | AY176354 | – |
GLM 45944 | Germany | – | AY207219 | |
Lepiota bengalensis | Iqbal 825 GDGM 45684 Holotype | Bangladesh | KU563148 | KU563150 |
Iqbal 860 Paratype | Bangladesh | KU563149 | – | |
Lepiota brunneoincarnata | DB4157 | Hungary | – | MK278258 |
NL-5409 | Hungary | – | MK278260 | |
Lepiota castanea | TENN 064371, ECV4016 | USA | – | MF797675 |
NL-2980 | Hungary | – | MK278259 | |
Lepiota castaneidisca | E.C. Vellinga 2594 (UC) | USA | AF391055 | – |
E.C. Vellinga 2410 (UC) | USA | AF391064 | – | |
E.C. Vellinga 2805 (UC) | USA | GQ203808 | – | |
E.C. Vellinga 2756 (UC) | USA | GQ203816 | – | |
Lepiota cf. aspera | MFLU 09-0061 | Thailand | – | HM488788 |
Lepiota cf. cristata | E.C. Vellinga 2515 (UC) | USA | AF391052 | – |
E.C. Vellinga 2677 (UCB) | USA | AY176466 | – | |
E.C. Vellinga 2714 (UC) | USA | GQ203807 | – | |
Lepiota clypeolaria | E.C. Vellinga 1683 (L) | Germany | AY176361 | – |
TENN 064372, ECV4003 | USA | – | MF797684 | |
VPI-OKM22029 | South Korea | – | U85291 | |
CBS 146.42 | Sweden | – | MH867601 | |
Lepiota coloratipes | 9-X-1991, H.A. Huijser (herb. Huijser) | The Netherlands | AF391066 | – |
MCVE 16888 | Italy | FJ998406 | – | |
Zhu L. Yang 4790 | China | KC819621 | – | |
Zhu L. Yang 4951 | China | KC819622 | – | |
SAV F-3212 | Spain | KC900376 | – | |
SAV F-3213, Holotype | Spain | KC900377 | – | |
NL-5353 | Hungary | – | MK278270 | |
Lepiota cortinarius | NL-1602 | Hungary | – | MK278262 |
Lepiota cristata | 22-IX-1993, H.A. Huijser (herb. Huijser) | The Netherlands | AF391042 | – |
20-IX-1989, H.A. Huijser (L) | The Netherlands | AF391043 | – | |
9-VII-1998, Z.L. Yang 2238 (HKAS) | China | AF391044 | – | |
8-XII-2000, E.C. Vellinga 2611 (UC) | USA | AF391045 | – | |
30-I-1993, D.E. Desjardin 5658 (SFSU) | USA | AF391050 | – | |
24-IX-2000, S. Clark (coll. P.B. Matheny 1958) (WTU) | USA | AF391051 | – | |
AFTOL-ID 1625, ECV 2449 (UC) | USA | – | DQ457685 | |
E.C. Vellinga 2780 (UC) | USA | GQ203806 | – | |
E.C. Vellinga 2750 (UC) | USA | GQ203815 | – | |
DUKE1582 | USA | – | U85292 | |
420526MF0542 | China | – | MH141343 | |
420526MF0550 | China | – | MG712361 | |
Lepiota cristatoides | 5-IX-1996, H.A. Huijser s.n. (herb. Huijser) | The Netherlands | AY176363 | – |
Lepiota cystophoroides | E.C. Vellinga 2142 (L) | France | AF391031 | – |
Lepiota erminea | NL-3095 | Hungary | – | MK278263 |
Lepiota felina | VPI-OKM20596 | USA | U85330 | U85295 |
NL-4207 | Slovakia | – | MK278264 | |
Lepiota geogenia | MEL 2358504 | Australia | – | JX179270 |
MEL:2358503 | Australia | – | JX179271 | |
Lepiota griseovirens | MCVE 13747 | Italy | FJ998403 | – |
Lepiota hymenoderma | E.C. Vellinga 2017 (L) | The Netherlands | AF391083 | – |
Lepiota laevigata | FP2012-11-02 | Hungary | – | MK278266 |
Lepiota lilacea | E.C. Vellinga 2451 (UCB) | USA | AY176379 | – |
E. Brown (coll. E.C. Vellinga 1873) (L) | United Kingdom | GQ203820 | – | |
Lepiota luteophylla | H.V. Smith 284 (MICH) | USA | AY176475 | – |
Lepiota maculans | TENN 064381 | USA | – | HQ832458 |
Lepiota mandarina | HKAS 50028 | China | – | KM214816 |
Lepiota neophana | E.C. Vellinga 2602 (UCB) | USA | AY176492 | – |
E.C. Vellinga 3947 (UC) | USA | GQ203812 | – | |
rh24 08/27/07 (ISC) | USA | GQ375546 | – | |
rh39 08/11/07 (ISC) | USA | GQ375547 | – | |
E.C. Vellinga ecv3955 (UC) | USA | – | HM488785 | |
Lepiota ochraceofulva | E.C. Vellinga 2267) (L) | The Netherlands | AF391032 | – |
E.C. Vellinga 2273 (L) | The Netherlands | AY176386 | – | |
Lepiota ochraceofulva | NL-2973 | Hungary | – | MK278267 |
Lepiota ochraceoumbonata | Murhula Cizungu 39 | Gabon | – | MK278268 |
Lepiota oreadiformis | FO 46679 | Germany | – | AF291344 |
Lepiota phaeoderma | E.C. Vellinga 3000 (UC) | USA | GQ203810 | – |
Lepiota psalion | WU 5152 Holotype | AUSTRIA | MG581687 | MG581699 |
Lepiota psalion basidiome a | CAG P.11_9/7.68 | Italy | MG581688 | – |
Lepiota psalion basidiome b | CAG P.11_9/7.68 | Italy | MG581689 | MG581700 |
Lepiota psalion (L. recondita) | 15-IX-1999, H.A. Huijser (herb. Huijser) hah6153 | The Netherlands | AY176390 | – |
3-VIII-1999, H.A. Huijser s.n. (herb. Huijser) | The Netherlands | – | AY176391 | |
H.A. Huijser (herb. Huijser) hah6177 | The Netherlands | GQ203823 | – | |
Lepiota psalion (L. sinorecondita ad interim) | HMJAU3799 | China | GU199362 | GU199355 |
Lepiota pseudohelveola | GLM 45945 | Germany | – | AY207220 |
Lepiota pyrochroa | E.C. Vellinga 2006 (L) | The Netherlands | AY176477 | – |
Lepiota recondita | TR gmb 01481, paratype | The Netherlands | MK508899 | MK508901 |
TR gmb 01482, holotype | The Netherlands | MK508900 | MK508902 | |
Lepiota rhodophylla | E.C. Vellinga 2610 (UCB) | USA | AY176480 | – |
Lepiota sanguineofracta | TO-HG2916, Holotype | Italy | KF879620 | MG581701 |
TO-HG2917 | Italy | KF879621 | MG581702 | |
Lepiota scaberula | E.C. Vellinga 2307 (UC) | USA | AF391029 | – |
E.C. Vellinga 2595 (holotype) (UC) | USA | AF391030 | – | |
UC1999143 | USA | – | MK278271 | |
Lepiota subcastanea | HKAS 45633 | China | – | KM214817 |
Lepiota subgranulosa | ANGE253 (JBSD, duplicate in MEXU) | The Dominican Republic | KR022007 | – |
Lepiota subalba | E.C. Vellinga 2242 (L) | The Netherlands | AY176489 | – |
Lepiota subincarnata | E.C. Vellinga 2234 (L) | The Netherlands | AY176491 | – |
VPI-OKM22153 | South Korea | – | U85294 | |
NL-2022 | Hungary | – | MK278273 | |
Lepiota thiersii | E.C. Vellinga 2590 (UCB) | USA | AY176485 | – |
E.C. Vellinga 2589 (UC) | USA | GQ203817 | – | |
Lepiota xanthophylla | TUB 011553 | Germany | – | DQ071712 |
Uncultured Basidiomycota | Environmental sample, man22_soil_G02 | USA | GU328508 | – |
Sequences obtained in this study were compared to those available in the GenBank (http://www.ncbi.nlm.nih.gov/) and UNITE (http://unite.ut.ee/) databases by using the Blastn algorithm (
Based on the BLASTn results (sequences were selected based on the greatest similarity) and outcomes of recent phylogenetic studies incorporating Lepiota sequences (
Phylogenetic hypotheses were constructed with Bayesian inference (BI) and Maximum likelihood (ML) criteria. The BI was performed with MrBayes v. 3.2.6 (
ML estimation was performed with RAxML v. 7.3.2 (
Bayesian phylogram obtained from the general nrITS sequence alignment of Lepiota spp. Here there are included Lepiota species with a hymeniform pileus covering, eight species representative of the major clades in Lepiota (indicated by *), and Chamaemyces fracidus as an outgroup taxon. Support values in either the Bayesian (Posterior Probabilities values [BPP]) or Maximum likelihood (ML Bootstrap percentage [MLB]) analyses are indicated. Only BPP values over 0.70 (in bold) and MLB values over 50% are given above clade branches. Newly sequenced collections are in bold.
The PCR product was 476–729 bp (nrITS) and 894–1128 bp (nrLSU). The nrITS data matrix comprised 68 sequences (including 63 from GenBank). This dataset was 814 bp long and contained 545 (66.9 %) variable sites. The nrLSU data matrix comprised 45 sequences (including 39 from GenBank). This dataset was 953 bp long and contained 335 (35.2%) variable sites.
As both Bayesian and Maximum likelihood analyses produced a consistent topology, only the Bayesian trees with both BPP and MLB values are shown (Figs
In both the nrITS and nrLSU analyses (Figs
Bayesian phylogram obtained from the general nrLSU sequence alignment of Lepiota spp. Chamaemyces fracidus was used as an outgroup taxon. Support values in either the Bayesian (Posterior Probabilities values [BPP]) or Maximum likelihood (ML Bootstrap percentage [MLB]) analyses are indicated. Only BPP values over 0.70 (in bold) and MLB values over 50% are given above clade branches. Newly sequenced collections are in bold.
Both the nrITS and nrLSU analyses (Figs
Macrocharacters (Fig.
Microcharacters (Figs
Gregarious on bare soil, in gardens and parks; so far known only from the type locality (Austria) and Sardinia (Italy).
Italy, Sardinia, Cagliari, Botanical Garden, 6 basidiomes growing among the Searsia/Rhus sp. litter, calcareous soil, 17 January 2017, Alessia Tatti and Giacomo Calvia (CAG P.11_9/7.68). Austria, Wien-Lobau, N. Uferhaus, 23 August 1985, Anton Hausknecht (WU 5152, holotype) (Fig.
The Netherlands, prov. Limburg, Valkenburg, Schaelsberg, 02 September 2004, Henk A. Huijser (TR gmb 01482).
From the Latin “reconditus”, meaning hidden, forgotten, which refers to its resemblance with L. psalion with which it was confused.
It is distinguished from Lepiota psalion by larger spores (3.7–)4.4–5.4(–5.9) × (2.4–)2.9–3.6(–4.3) μm, versiform cheilocystidia and different nrITS and nrLSU sequences.
Macrocharacters (Fig.
Microcharacters (Figs
Gregarious on rich in nutrients and lime (marl) bare soil, in a mixed deciduous forest; so far known only from the type locality.
The Netherlands, Limburg province, Valkenburg, Schaelsberg, man-made (anthropized) hilly grove with mainly deciduous trees (Quercus, Fagus, Corylus, Fraxinus, Robinia, Prunus, Sambucus), together with Lepiota tomentella, L. poliochloodes, Melanophyllum eyrei, and Limacella ochraceolutea, 22 September 2001, Henk A. Huijser (TR gmb 01481, paratype); ibidem, 02 September 2004, Henk A. Huijser (TR gmb 01482, holotype).
The specific epithet is a combination of Medieval Latin “sino” (which means Chinese) and “recondita”, referring to the strong affinity of the Chinese taxon to the European L. recondita.
Basidiomata small (Fig.
Spores [60,3,1] (4.0–)4.5–5.5 × 2.5–3.0(–3.5) µm, Q = 1.50–1.80(–1.83), Qav = 1.64 (Fig.
China, Jilin Province, Changchun City, Jinyuetan Park, 7 July 2005, Wang Jianrui (HMJAU 3799).
The morphological differences among the Lepiota species with hymeniform pileus covering are often subtle (
Lepiota psalion is distinguished by having a non-smooth pileus with concentric non-uplifted squamules, a distinct annulus, and mostly clavate cheilocystidia (
Lepiota “cf. rufipes f. phaeophylla” sensu
The phylogenetically closest species are L. coloratipes (= L. rufipes ss. Auct. europ. non ss. orig.) and L. sanguineofracta (Fig.
The other morphologically allied species of Lepiota with a hymeniform pileus covering, ellipsoid spores, and a well-formed annulus, phylogenetically far from L. psalion (Figs
Lepiota psalion was established by
When the Sardinian specimens were collected, they were morphologically attributed to L. psalion, but when they were sequenced to obtain molecular evidence, they did not cluster either with the Dutch collections or with a collection named L. psalion from China (herb. HMJAU3799;
Lepiota psalion, L. recondita, L. “sinorecondita”, L. apatelia, and L. thiersii constitute a homogeneous morphology-based but not monophyletic group, here named the “L. psalion complex”, which is circumscribed by a set of shared characters: a pileus surface breaking into small squamules, well-formed white partial veil (usually forming an annulus, but see L. apatelia), hymeniform pileus covering, and ellipsoid spores.
An identification key for the taxa belonging to this complex is proposed below.
1 | Cheilocystidia absent | 2 |
– | Cheilocystidia present | 3 |
2 | Smell farinaceous, annulus often adhering to pileus margin (as velar remnants), spores weakly dextrinoid | L. apatelia (Europe) |
– | Smell L. cristata-like, annulus usually ascending on stipe, spores non-dextrinoid | L. thiersii (North America) |
3 | Spores ellipsoid, on average = 3.9 μm long, Qav = 1.36 | L. psalion (Europe) |
– | Spores ellipsoid to oblong, on average > 4.0 μm long, Qav > 1.4 | 4 |
4 | Cheilocystidia versiform, spores ellipsoid, Qav = 1.5, annulus entirely smooth | L. recondita (Europe) |
– | Cheilocystidia mainly clavate, spores oblong, Qav = 1.64, annulus covered by minute yellowish brown squamules on lower surface | L. sinorecondita ad int. (China) |
We thank Irmgard Greilhuber and Walter Till (University of Vienna) for sending us photographs and part of the holotype collection of Lepiota psalion, Giacomo Calvia (University of Cagliari) for his assistance in collecting specimens in the Botanical Garden of Cagliari, Marco Floriani (Pergine Valsugana, Trento) for depositing the collections of the new species in TR, and Else Vellinga (University of California - Berkeley) for her suggestions. AT also thanks the University of Cagliari and, in particular, Gianluigi Bacchetta, director of the Hortus Botanicus Kalaritanum, for allowing sampling of the studied material and Annalena Cogoni, the person in charge of the Herbarium CAG, for allowing us access to fungarium material.