ARTICLE 117
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g ) ) s 123 you get f mata 7– 1997 usin g cete , ( A 11 y u y : 2
steri ( O in sit N caceae resented here. · garicom i rITS rDN p axonom Key words: Basidiomycota A n t 3 ar E are s raphed UM tion and illustrations o g L Ag p
O , S – 9th decile (–maximum V the above conditions can be waived i
f · D , Bon & Caballero ) ongo red randomly selected S es + O l U C (2001), Vellinga (2001, 2006, (2001), Vellinga . G ic structures were observed in 1990 TH al ( p ca Leucoagaricus g ore measurements are based on !>! < UN i E t legalcode. Any o p / F e
% < 3.0 / ical features are described from fresh ar al g % t average value IMA . e , Contu Ag by-nc-nd a ital camera, with a tripod, and in natural / ) ore, a detailed descri and is compared with and is compared with i f g g AND M ( i undescribed th was measured from the apex di licenses / gy y LS 1990 g 0 ( tion below, s tion below, and of the main macro- and and of the main macro- A p Piloselli uu RI 4 n sit . The width of basidia was measured at the widest part, . E i i , and Vellin ) ) ant Cresyl blue, and Melzer’s reagent. Dried fragments T ecimens. The microsco Piloselli ht. Macromorpholo illi this species: its characteristics do not match any published this species: its characteristics do not match ll the studied collections were photo r Lanzoni 010 f p g creativecommons.org // Caballero (1997), Gennari & Migliozzi (1999), Migliozzi & Caballero (1997), Gennari & Migliozzi Resta (2001), Migliozzi this previousl from four collections. Only mature, normally developed and non-aberrant spores from spore prints were measured. Dimensions of the spores are given as follows: (minimum value–) 1st decile – both fresh and dried material, using several mountants \ 120 elements in ammoniacal a Nikon D5 li s and the len species. In addition, an ITS sequence analysis supported this species. In addition, an ITS sequence analysis statement. There MA Morpholo value treatments and papers by Bon (1981, 1993), Candusso treatments and papers by Bon (1981, A B & 2 were carried out with a ×1000 oil immersion objective. In the descri o : . , ound at http: e t. ) ) f belongs to section p r . turn sec osed
differ differ d ii p section section pain and Alfredo Vizzin and f , S 3 cus an i nct alepensis i variicolor d ollowing conditions h f ar s ar remnants rot g l se is described from a public park in Zaragoza, Spain, based on is described from a public i eucoagaricus b rr us L Leucocoprinus u ru / this work, which can be f R in b P eucoa ant ve ecies o ecies en L d ellis, and the sha h p p un , Marco Contu w 2 b Leucoagaricu . The species has been . s d ilei o y alahorra, La Rioja, p . s C e-re y g Leucoagaricus cu the i f mens, a i soil near a Parque José Antonio Labordeta Parque José ( y ar inger 1973, 1986, Vellinga 2010 inger 1973, 1986, Vellinga e 50 sq. m f n oran y i g uished from all other species in sect. (S oza g dcha. 26500 that encompasses species whose that encompasses species whose º g ) sta um spec e N i 2 , Agustín Caballer lly 1 ar . n z b i O . ical characters such as pileus colour, colour ical characters such as pileus colour, ical and molecular characters. Illustrations of fresh basidiomata ical and molecular characters. er ) s: eucoa a g g h k he park covers an area of 409 000 sq. m to the he park covers an area of 409 000 sq. : l its botanical biodiversit L p T f a n . rom an area o i i . f The new species Leucoagaricus variicolo garicacea c S r by the cream-ochre pileus which becomes entirely the city; it is regarded as an important green space the city; it is regarded as an important A e ve wor : , a section within the d w f i on ( i i i rom the copyright holder. Nothing in this license impairs or restricts the author’s Nothing in moral rights rom the copyright holder. a 2001 * f ll ree to share - to copy, distribute and transmit the work, under the ree to share - to copy, omata usua f e g vat mm i m -rose idi Our taxon is distin # co ecte er LUME 3 · N k o ose d n ll n- ia Marmilla, 12. 07026, Olbia. Ital ia Marmilla, 12. 07026, vda. Valvanera 32, 5. vda. Valvanera antat n il n exhaustive search of the literature, including monographic ellin O as l i r lade o o heilocystidia, and subglobose to broadly ellipsoid spores. 2012 International Mycological Association 2012 International Mycological corresponding author Italy; corresponding author Torino, Mattioli 25, I-10125 P.A. Viale Torino, Sistemi, Università di e Biologia dei della Vita Dipartimento di Scienze C/ Andalucía 3, 4.º dcha. 26500 Calahorra, La Rioja, Spain Andalucía 3, 4.º dcha. 26500 Calahorra, C/ V A Submitted: 25 July 2012; Accepted: 14 October 2012; Published: 5 November 2012 Accepted: 14 October 2012; Published: Article info: Submitted: 25 July 2012; similar taxa both morpholo are added. micromorphological features Abstract: e-mail: [email protected] You are You Attribution Guillermo Muño Guillermo © 4 1 2 3 f A permission No N For any reuse or distribution, you must make clear to others the license terms o of the cheilocystidia and spores (Bon 1993, Vellinga 2010). of the cheilocystidia and spores (Bon 1993, Vellinga Species of the morphologically similar sect. a public park in Zara four collections of a remarkable species of four collections of a remarkable species green with ammonia to ammonia, the structure o p were recorded on cla on the pileus surface, mainly pyriform to sphaeropedunculate reactions of the basidiome surface when bruised or ex because o south o V p mainly in the immutable context (not changing colour when bruised) and the absence of a green reaction with ammonia on the basidiome surface (Singer 1948, 1986, Bon 1993, on morpholo INTRODUCTIO c A Piloselli V b P c c doi:10.5598/imafungus.2012.03.02.03 MuñozMño ettl al.
100 Leucoagaricus flammeotinctoides GU136173 100 Leucoagaricus flammeotinctoides GQ258476 78 Leucoagaricus flammeotinctoides AY243620 LE 98 100 Leucoagaricus pyrrhophaeus GU136199 C Leucoagaricus pyrrhophaeus GQ258473 100 Leucoagaricus pyrrhulus GQ258474 75 Leucoagaricus pyrrhulus GU136201 100 Leucoagaricus erythrophaeus GQ258468 97 Leucoagaricus erythrophaeus GQ258470 ARTI Lepiota roseifolia GQ203805 Leucoagaricus decipiens GQ203803 100 Leucoagaricus pardalotus GU136202 Leucoagaricus pardalotus GQ258479 100 Lepiota decorata GU136197 88 Lepiota decorata AY243645 68 Leucoagaricus ionidicolor AY176415 Leucoagaricus marriagei GQ329049 100 Leucoagaricus sp. Weber 6019 AY243633 100 Leucoagaricus brunnescens GQ203804 89 100 Leucoagaricus georginae AY176413 Leucoagaricus georginae GU136198 Leucoagaricus jubilaei AY243635 98 Leucoagaricus badhamii GQ329056 78 Leucoagaricus sp. ecv2484 GU136182 100 Leucoagaricus dyscritus GU136181 Leucoagaricus dyscritus GU136180 100 Leucoagaricus hesperius GU139789 Leucoagaricus hesperius GU139788 Lepiota fuliginescens GU136183 98 Lepiota fuliginescens GU136185 52 Lepiota fuliginescens GU136189 Lepiota flammeotincta GU136170 100 Leucoagaricus sp. Vellinga 2746 AY176440 95 Lepiota flammeotincta GU136167 54 Lepiota fuliginescens GU136187 Leucoagaricus sp. Vellinga 2619 AY243637 Lepiota fuliginescens GU136188 Leucoagaricus sp. Huijser s.n. AY243643 Leucoagaricus adelphicus GQ258478 100 Leucoagaricus adelphicus AY243622 Piloselli 68 Leucoagaricus adelphicus AY243624 100 Leucoagaricus pilatianus GQ329040 Leucoagaricus pilatianus GQ329057 85 93 Leucoagaricus cupresseus GQ258477 95 Leucoagaricus cupresseus GU136194 87 Leucoagaricus cupresseus AY243629 Leucoagaricus cupresseus AY243627 Leucoagaricus cupresseus AY243632 Leucoagaricus variicolor coll. GM-2485 JX880032 100 Leucoagaricus variicolor coll. AH 40328 (holotype) JX880030 Leucoagaricus variicolor coll. GM-2486 JX880033 Leucoagaricus variicolor coll. GM-2454 JX880031 89 Leucoagaricus rubrotinctus JN944081 58 Leucoagaricus rubrotinctus FJ481050 100 Leucoagaricus rubrotinctus JN944082 98 Leucoagaricus sublittoralis AY176442 Rubrotincti 100 Leucoagaricus littoralis GQ329041 Leucoagaricus wychanskyi AF482874 Cystolepiota seminuda AY176350
0.06
Maximum Likelihood phylogram obtained from the ITS (ITS1-5.8S-ITS2) sequence alignment of Leucoagaricus spp. Cystolepiota seminuda $ {~ * * ! % * * +
excluded) to the basal septum. Microscopic pictures were XP kit (Beckman) and sequenced by MACROGEN (Seoul, taken on a Moticam 2500 digital camera connected to a Motic Republic of Korea). The sequences were submitted to BA300 microscope. Colour notations for the macroscopic GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and their descriptions are from Munsell (1994), hereafter shortened as accession numbers are reported in Fig. 1. Mu. Herbarium acronyms follow Index Herbariorum, except for GM and AC that refer to the personal herbaria of Guillermo Sequence alignment and phylogenetic Muñoz and Agustín Caballero. The type collection is housed analysis at AH. The name and description of the new species are deposited in MycoBank (Crous et al. 2004). The sequences obtained in this study were checked and assembled using Geneious v. 5.3 (Drummond et al. 2010) and compared to those available in the GenBank database sequencing by using the Blastn algorithm. Based on the Blastn results, sequences were selected according to the outcomes of Genomic DNA was isolated from 1 mg of a dried herbarium recent phylogenetic studies on Leucoagaricus (Vellinga specimen from four collections (AH-40328, GM-2454, GM- 2010, Vellinga et al. 2010, 2011). Alignments were generated 2485, and GM-2486), using the DNeasy Plant Mini Kit using MUSCLE (Edgar 2004) with default conditions for gap (Qiagen, Milan) according to the manufacturer’s instructions. openings and gap extension penalties. The alignment was Universal primers ITS1F/ITS4 were used for the ITS region then imported into MEGA v. 5.0 (Tamura et al. 2011) for manual et al. 1990, Gardes & Bruns 1993). adjustment. The phylogenetic analysis was performed using X!! the Maximum Likelihood (ML) approach. Following Vellinga cycler (Perkin-Elmer, Applied Biosystems) following Vizzini et (2010) and Vellinga et al. (2010), a Cystolepiota seminuda al. !>> # { sequence (AY176350) was used as outgroup. ML estimation
118 I MA FUNGUS A new Leucoagaricus species from Spain ARTI C LE
Fig. 2. Leucoagaricus variicolor. Macroscopic characters. A–C. Fresh basidiomata in situ. D. Herbarium specimens. A, D from AH 40328 (holotypus); B from GM-2454; C from GM-2485. Bars = A–B = 50 mm; C–D = 20 mm.
was performed through RAxML v. 7.0.4 (Stamatakis 2006) Etymology\ # Ovariicolor” refers to the highly with 1000 bootstrap replicates (Felsenstein 1985) using the variable colours of the pileus surface depending on fresh or GTRGAMMA algorithm to perform a tree inference and search dry conditions. for a good topology. Support values from bootstrapping runs (MLB) were mapped on the globally best tree using the “-f a” Diagnosis: Differs from every other described species of option of RAxML and “-x 12345” as a random seed to invoke Leucoagaricus sect. Pilosellii in having a cream to pink to egg- * < {~ * ! % yellow, or ochre pileus in fresh basidiomata that becomes are reported in the resulting tree (Fig. 1). dark rose in the herbarium, the presence of universal veil remnants on the pileus surface and stipe base, subglobose to broadly ellipsoid spores, pyriform to spheropedunculate RESULTS cheilocystidia, and a unique ITS sequence.
Phylogenetic analysis Type: Spain: Aragón: Zaragoza, “José Antonio Labordeta” # # (antea: “Parque Grande”) park, UTM 30TXM7511, N 41.634 W four specimens, yielding a PCR product of about 700 bp. The 0.894, alt. 225 m, on argillose-sandy soil, in Pinus halepensis ITS data matrix comprises a total of 59 sequences (including litter, 3 Dec. 2011, G. Muñozz (AH 40328 – holotype; GM- 55 from GenBank). In the obtained ML phylogram (Fig. 1), our 2453 and AC-4896 – isotypes). four sequences of the new Leucoagaricus clustered together and are distinct and basal to all the existing sequences of Description: Pileuss 40–100 mm wide, when young previously sequenced species of section Piloselli. hemispherical-convex to hemispherical, expanding to Taxonomy slightly exceeding margin, involute or incurved in young } _ Leucoagaricus variicolor G. Muñoz, A. Caball., Contu * & Vizzini, sp. nov. cream or egg-yellow to ochre (Mu 2.5Y 8/1-2; Mu 5Y 8/1 MycoBank MB801565 “White”; Mu 2.5Y 8/2-4 “Pale yellow”) to orange-pink (Mu 5YR (Figs 2–4) 6/6-8 “Yellowish red”; Mu 2.5YR 5/6-8 “Red”); in adult stages
V O LUME 3 · NO . 2 119 MuñozMño ettl al. LE C ARTI
Fig. 3. Leucoagaricus variicolor. Microscopic characters. A–B. Elements of the pileipellis. C–D. Spores (in ammoniacal Congo red). E–F. Cheilocystidia (in ammoniacal Congo red). A, C, E from AH 40328 (holotype); B, D, F from GM-2485. Bars: A–B, E–F = 20 μm; C–D = 10 μm.
these tinges are mixed, in dried herbarium material the main slightly beige, darker towards the margin, not reddening when tinge is pink-rose (Mu 10R 5/3-4; 6/3-4) (Fig. 2D); surface * \ not reddening but tardily darkening on handling, covered, Stipe 40–100 × 12–18 mm, stout, solid, cylindrical, in most mainly toward the disc, but often up to the antimarginal zone, specimens with a napiform to submarginate bulb and then * ^ } _\ patches, as remnants of the universal veil. Lamellae not at the apex, at times slightly browning on handling or due to reaching the stipe, attached to a pseudocollarium, crowded, the environmental conditions, in young stages with minute to 6 mm broad, with (0–)1–3 lamellulae, white to cream or * * \
120 I MA FUNGUS A new Leucoagaricus species from Spain ARTI C LE
Fig. 4. Leucoagaricus variicolor. Line drawings of microscopic characters (from AH 40328, holotype). A. Spores. B. Basidia. C. Cheilocystidia. D. Pileipellis. Bars: A–C = 20 μm; D = 100 μm.
Annulus thin, simple, membranous, persistent, not movable, Pleurocystidia absent. Pileipellis a trichoderm consisting of usually descending (rarely ascending), entirely white or erect, long cylindrical to fusiform, not gelatinized elements, ochre towards margin. Contextt \ occasionally septate, 150–350(–400) × 8–16 μm, without or slightly turning ochre-pink towards the base of the stipe. a subtending (basal) hymeniform layer (Figs 3A, B, 4D); Smelll and taste not distinctive, fungoid, pleasant. Edibility pigment brownish, usually parietal, smooth, but sometimes unknown. Spore-print: white. Chemical reactions: surface of also intracellular in some terminal elements. Velar patches the pileus, stipe, annulus, and lamellae green in ammonia. of the pileus surface composed of hyaline, tightly interwoven, Sporess (5.6–)6.0–6.8–7.6 (–8.2) × (4.5–)4.8–5.2–5.6(–5.7) cylindrical, 3–7 μm wide hyphae. Clamp-connections absent. μm, Q = (1.1–)1.2–1.3–1.4 (–1.6) (n = 120), subglobose to broadly ellipsoid or slightly ovoid, smooth, without a germ- Habitat and distribution: Terrestrial, on clayey soil, in the litter pore, dextrinoid, metachromatic in Cresyl Blue (Figs 3C, D, of a Pinus halepensiss plantation, in a park with considerable 4A). Basidia 20–30 × 7–9 μm four-spored, clavate, without public pressure. Basidiomes produced in winter (December). basal clamp connection (Fig. 4B). Lamella edge sterile. Known only from the province of Aragón, Spain, at this time. Cheilocystidia abundant, 20–45 × 10–16 μm, pedicellate, pyriform to sphaeropedunculate, rarely with a very short Additional collections examined: Spain: Aragón: Zaragoza, “José mucro, nearly hyaline or with light brown contents (diluted Antonio Labordeta” (antea: “Parque Grande”) park, N 41.634 W and homogeneous cytoplamatic pigment) (Figs 3E, F, 4C). 0.894, alt. 225 m, near Pinus halepensis, on argillose-sandy soil,
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basidiomata nearly covered by the substrate, 3 Dec. 2011, G. Muñoz Bon M (1981) Clé monographique des “Lépiotes” d’Europe. (GM-2454); ibid., in closeby neighbourhoods, on soil, 10 Dec. 2011, Documents Mycologiques 11 (43): 2–77. LE G. Muñozz (GM-2485, GM-2486). Bon M (1993) Flore Mycologique d’Europe 3: Les Lépiotes. C Lepiotaceae Roze. Documents mycologiques, Mémoire hors série 3: 1–153.
ARTI DISCUSSION Bon M, Caballero A (1997) Le genre Leucoagaricus dans “La Rioja” (Espagne). Documents Mycologiques 27 (106): 27–42. According to morphological data and phylogenetic analyses Caballero A (1997) Flora Micológica de La Rioja, 1: Lepiotaceae. CD- of ITS sequences (Fig. 1), the collections studied merit ROM. Calahorra-La Rioja: A Caballero. recognition as an independent species within Leucoagaricus Candusso M, Lanzoni G (1990) Lepiota s.l. [Fungi Europaei vol. 4.] sect. Piloselli. No similar species could be found in the literature Saronno: Giovanna Biella. since all the previously described species are distinguished Contu M (1990) Nuovi taxa di Agaricales (Basidiomycetes) dalla by different tinges in the pileus, the absence of pyriform to Sardegna. Boletim da Sociedade Broteriana 63(2): 379–386. spheropedunculate cheilocystidia, or more elongated and Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) differently shaped spores (Candusso & Lanzoni 1990, Bon MycoBank: an online initiative to launch mycology into the 21st 1993, Caballero 1997, Gennari & Migliozzi 1999, Migliozzi & century. Studies in Mycology 50: 19–22. Resta 2001, Migliozzi et al. 2001, Vellinga 2001, 2006, 2010). Drummond AJ, Ashton B, Cheung M, Heled J, Kearse M, Moir R, Among the macromorphologically most similar species, Stones-Havas S, Thierer T, Wilson A (2010) Geneious. Version Lepiota decorata (Leucoagaricus idae-fragum Vellinga 5.3.
122 I MA FUNGUS A new Leucoagaricus species from Spain
Vellinga EC (2001) Leucoagaricus (Locq. ex) Sing. In: Flora Agaricina Vizzini A, Contu M, Musumeci E, Ercole E (2011) A new taxon in ARTI Neerlandica (ME Noordeloos, TW Kuyper & EC Vellinga EC, the Infundibulicybe gibba complex (Basidiomycota, Agaricales, eds) 5: 85–108. Lisse: AA Balkema Publishers. Tricholomataceae) from Sardinia (Italy). Mycologia 103: 904–
Vellinga EC (2006) Lepiotaceous fungi in California, U.S.A. – 3. Pink 911. C
and lilac species in Leucoagaricus sect. Piloselli. Mycotaxon 98: # # ~ # >XX! LE 213–224. sequencing of fungal ribosomal RNA genes for phylogenetics. In: Vellinga EC (2010) Lepiotaceous fungi in California, U.S.A. PCR Protocols: method and applicationss (MA Innis, DH Gelfand, Leucoagaricus sect. Piloselli. Mycotaxon 112: 393–444. JJ Snisky & TJ White, eds): 315–322. San Diego: Academic Vellinga E, Contu M, Vizzini A (2010) Leucoagaricus decipiens Press. and La. erythrophaeus, a new species pair in sect. Piloselli. Mycologia 102: 447–454. Vellinga E, Sysouphanthong P, Hyde KD (2011) The family Agaricaceae: phylogenies and two new white-spored genera. Mycologia 103: 494–509.
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