North American Fungi Volume 10, Number 5, Pages 1-15 Published June 15, 2015 Leucoagaricus sabinae (Agaricaceae), a new species from the Dominican Republic Alfredo Justo1, Claudio Angelini2, Alberto Bizzi3, Alfredo Vizzini4 1Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México. Apartado Postal 70- 367, 04510-México Distrito Federal (México). e-mail: [email protected]; 2Investigador Asociado Herbario Jardin Botanico Nacional Dr. Rafael Ma. Moscoso. Santo Domingo (Dominican Republic). Via tulipifero, 9 – 33080 - Porcia (PN, Italy). e-mail: [email protected]; 3Via A. Volta, 31 – Alte di Montecchio Maggiore (VI, Italy). e-mail: [email protected]; 4Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Viale P.A. Mattioli 25, I-10125, Torino, Italy.e-mail: [email protected] Corresponding author: Alfredo Justo ([email protected]). Accepted for publication June 4, 2015. Justo, A., C. Angelini, A. Bizszi, and A. Vizzini. 2015. Leucoagaricus sabinae (Agaricaceae), a new species from the Dominican Republic North American Fungi 10(5): 1-15. http://dx.doi:10.2509/naf2015.010.005 http://pnwfungi.org Copyright © 2015 Pacific Northwest Fungi Project. All rights reserved. Abstract: Leucoagaricus sabinae is proposed as a new species based on material collected in the Dominican Republic. This taxon is characterized macroscopically by the relatively small size, the dull gray-pink pileus, yellow gills immediately turning pink when touched and the ever-present white velar residues on the margin of the pileus. Microscopically the vaguely metachromatic to non-metachromatic spores, the scarce, cylindrical-flexuous cheilocystidia and the piles covering elements with parietal pigment are diagnostic. Based on molecular data (nrLSU, nrITS) this species belongs in the Leucoagaricus/Leucocoprinus clade of the Agaricaceae. Key words: Lepiotaceaous fungi, Agaricales, Caribbean, phylogeny. 2 Justo et al. Leucoagaricus sabinae sp. nov. North American Fungi 10(5): 1-15 Introduction: Among the lepiotaceous fungi in Reagent and Cresyl Blue. Microscopical the Agaricaceae the Leucoagaricus/ observations were made with a Meiji optical Leucocoprinus clade is particularly diverse in the microscope. Descriptive terms for morphological tropics, with a lot of species awaiting discovery features follow Vellinga (1988, 2001). Color codes and formal description (Vellinga 2004a, b). The are from Munsell Soil Color Charts (Munsell major mycofloristic studies of this group in the Color 2009). The following abbreviations are American tropical region date back to before the used in the descriptions: avl for average length, molecular era (Dennis 1952, 1961, 1970; Pegler avw for average width, Q for quotient of length 1983), and most of the molecular data currently and width and avQ for average quotient. available for this area come from the studies on ant-associated fungi and their free-living relatives Protocols for DNA extraction, PCR and (Vo et al. 2009) that do not focus on taxonomy. sequencing. Genomic DNA was isolated from 10 mg dried herbarium specimens with the DNeasy Over the past ten years, one of the authors (C.A.) Plant Mini Kit (QIAGEN, Milan, Italy) according has collected and studied fungi in the Dominican to the manufacturer’s instructions. Universal Republic. More than 300 species of macrofungi primers ITS1F/ITS4 were used for ITS have been recorded, and almost all voucher amplification (White et al. 1990, Gardes and specimens are deposited in the herbarium of the Bruns 1993) and primers LR0R/LR6 (Vilgalys Jardin Botánico Nacional Dr. Rafalel Ma. and Hester 1990, Moscoso (Santo Domingo, Dominican Republic). www.botany.duke.edu/fungi/mycolab) for LSU Approximately 20% of the collections represent rDNA amplification. PCR was performed in a lepiotaceous fungi of different genera PE9700 thermal-cycler (Perkin-Elmer, Applied (Chlorophyllum, Cystolepiota, Lepiota, Biosystems) following the profiles given by Leucoagaricus, Leucocoprinus) and are currently Vizzini et al. (2011) for ITS and LSU. The PCR being studied and sequenced. products were purified with the AMPure XP kit (Beckman) and sequenced by MACROGEN This contribution is focused on the description of (Seoul, Republic of Korea). Sequence assembly a new species in the and editing were performed with Geneious 5.3 Leucoagaricus/Leucocoprinus clade (Drummond et al. 2010). The sequences are characterized by small size, the dull gray-pink deposited in GenBank and accession numbers are pileus, yellow gills immediately turning pink given with the collection information. when touched, and the ever-present white velar The newly generated nrLSU sequences were residue on the margin of the pileus. The included in a general Agaricaceae dataset phylogenetic position of the species, based on following the sampling of Vellinga et al. (2011). A molecular data, is also analyzed and discussed. very broad nrITS dataset with 323 sequences of Leucoagaricus/Leucocoprinus was also Materials and Methods: The basidiomata constructed to narrow down the closest relatives were photographed fresh, in habitat, using a of our sequences (data not shown). Based on the digital camera Nikon coolpix 8400 and results of these preliminary analyses and taking subsequently dried. Notes on fugacious into account the results of BLAST searches for characters (e.g. odor, taste) were made before our sequences, a smaller nrITS dataset with drying. Collections were studied using standard representatives of the Leucoagaricus / procedures for morphological examination of Leucocoprinus clade was constructed and lepiotaceous fungi (Candusso & Lanzoni 1990; analyzed. For all datasets sequences were aligned Vellinga 2001). Microscopical preparations were using MAFFT version 7.110 (Katoh & Toh 2008) mounted in 5% KOH, Congo Red, Melzer´s and alignments were manually corrected using Justo et al. Leucoagaricus sabinae sp. nov. North American Fungi 10(5): 1-15 3 MacClade 4.08 (Maddison & Maddison 2002). A appendiculate, commonly triangular, white veil Maximum Likelihood analysis was run in the remnants. Lamellae free, rather sparse, RAxML servers, version 7.2.8 (Stamatakis & et al. ventricose, with numerous lamellulae, usually 2008), under a GTR model with one hundred between 1 and 3 between lamella, white at first, rapid bootstrap replicates. Two species of then yellow, immediately turning pink when Macrolepiota (M. procera and M. rhodosperma) touched. Stipe 20–50 × 3–5 mm, cylindrical, a were used as outgroup taxa for the ITS analysis bit curved or flexuous in profile; surface white, and Pseudobaeospora pyrifera for the LSU turning pink-reddish when touched; in young analysis. specimens the surface smooth in the upper part and covered with veil floccules in the lower part; Results in older specimens the surface is almost completely smooth, sometimes with fugacious Leucoagaricus sabinae Angelini, Justo & ring-like remnants; with thin, white rhizomorphs Vizzini, sp. nov. (Figures 1–5) at base, that turn immediately pink when touched; fistulose. Context white to pink on Mycobank # 810631 pileus, and white to pink-reddish on stipe. Odor strong and unpleasant, like burnt rubber and/or Holotypus. Dominican Republic. Puerto Plata, fish. Taste similar to smell, bitter. Sosúa, on man-made litter in a deciduous forest, 30 November 2013, ANGE 306 (JBSD), nrITS Spores 5.5–8 × 3.7–4.5 µm, avl × avw = 6.4 × KM983667, nrLSU KM983669. 4.0, Q = 1.38–1.95, avQ = 1.62, ellipsoid to oblong, some phaseoliform, thick-walled, with Diagnosis. Characterized macroscopically by evident and eccentric apiculus, without germ the relatively small size for the genus, the dull pore under an optical microscope, slightly gray-pink pileus, yellow gills immediately turning dextrinoid, not or only vaguely metachromatic, pink when touched and the persistent white velar acetic-ammonium reaction negative. Basidia residues on the margin of the pileus. tetra-sterigmate, clavate or cylindrical with a Microscopically the most distinctive characters square base, 18–30 × 6.5–10 µm. Cheilocystidia are the vaguely metachromatic to non- 13.5–21 × 3–6 µm, cylindrical-capitulated, some metachromatic spores, the scarce, cylindrical- with flexuous outline, infrequent and unevenly flexuous cheilocystidia and the pileus covering distributed along the lamella edge. Pleurocystidia elements with incrusting, parietal pigment are absent. Pileus covering an intricate cutis, with diagnostic. septate, cylindrical, hyphae, some diverticulate; terminal elements cylindrical, up to 20 × 7.5 µm; Etymology. The epithet is a dedication to hyphae with vacuolar grayish pigment and Sabine Woght, for her logistic support of the brown, incrusting parietal pigment, often field-work that has made possible the discovery appearing as a zebra-like pattern on the surface. of this new taxon. Stipitipellis a cutis, with septate hyaline hyphae, 2–8.5 µm in diameter; at apex with some Pileus 10–25 mm in diameter, convex when diverticulate, bifurcated hairs. Clamp- young, then flat without any hint of umbo; connections absent in all tissues. surface gray-pinkish, pinkish-brown if handled (Munsell 10R 6/1-6/4, 5/1-5/4), with a matte Habit, habitat and distribution. Solitary or aspect, not hygrophanous, covered with whitish gregarious, on deciduous forest litter. So far, this squamules and fibrils; margin not striate or only species has been commonly found every year in slightly in older specimens, with copious, the rainy autumn and winter period (November