IMA FUNGUS · 7(1): 59–73 (2016) doi:10.5598/imafungus.2016.07.01.05 New species of Elaphomyces (Elaphomycetaceae, Eurotiales, Ascomycota) ARTICLE from tropical rainforests of Cameroon and Guyana* Michael A. Castellano1, Bryn T.M. Dentinger2, Olivier Séné3, Todd F. Elliott4, Camille Truong5, and Terry W. Henkel6 1United States Department of Agriculture, Forest Service, Northern Research Station, 3200 Jefferson Way, Corvallis, OR 97331, USA 2Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK 3Institute of Agricultural Research for Development, National Herbarium of Cameroon, PO Box 1601, Yaoundé, Cameroon 4Department of Integrative Studies, Warren Wilson College, Asheville, NC 28815, USA 5Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA 6Department of Biological Sciences, Humboldt State University, Arcata, CA 95521, USA; corresponding author email: Terry.Henkel@humboldt. edu Abstract: The seuestrate false trufes Elaphomyces favosus, E. iuppitercellus, and E. labyrinthinus spp. Key words: nov. are described as new to science from the Dja Biosphere Reserve, Cameroon. Elaphomyces adamizans biogeography sp. nov. is described as new from the Pakaraima Mountains of Guyana. The Cameroonian species are the ectomycorrhizal fungi rst Elaphomyces taxa to be formally described from Africa, occurring in lowland Guineo-Congolian tropical Gilbertiodendron rainforests dominated by the ectomycorrhizal (ECM) canopy tree Gilbertiodendron dewevrei (Fabaceae Guiana Shield subfam. Caesalpinioideae). The Guyanese species is the third to be discovered in lowland tropical South Guineo-Congolian rainforest America, occurring in forests dominated by the ECM trees Pakaraimaea dipterocarpacea (Dipterocarpaceae) Pakaraimaea and Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae). Macromorphological, micromorphological, seuestrate fungi habitat, and DNA seuence data are provided for each new species. Molecular and morphological data Uapaca place these fungi in Elaphomycetaceae (Eurotiales, Ascomycota). Uniue morphological features are congruent with molecular delimitation of each of the new species based on a phylogenetic analysis of the rDNA ITS and 28S loci across the Elaphomycetaceae. The phylogenetic analysis also suggests that a common ancestor is shared between some Elaphomyces species from Africa and South America, and that species of the stalked, volvate genus Pseudotulostoma may be nested in Elaphomyces. Article info: Submitted: 16 November 2015; Accepted: 2 March 2016; Published: 10 March 2016. INTRODUCTION Castellano et al. (2012) provided the rst report of Elaphomyces from the lowland South American tropics, Elaphomyces Nees 1820 (Elaphomycetaceae, Eurotiales, describing two new species associated with ECM Fabaceae Ascomycota) is a seuestrate, ectomycorrhizal (ECM) fungal hosts in Guyana. Subseuently, our continued collecting genus that associates with a broad range of primarily north efforts in the tropics of Africa and South America have or south temperate angiosperm and gymnosperm hosts uncovered four additional new Elaphomyces species. Here (Trappe et al. 2009, Castellano et al. 2011, Quandt et al. we describe Elaphomyces favosus, E. iuppitercellus, and E. 2015). Elaphomyces species generally fruit hypogeously labyrinthinus spp. nov. from the Dja Biosphere Reserve in and have relatively large cleistothecial ascomata with a thick Cameroon, and E. adamizans sp. nov. from the Pakaraima peridium, a powdery, hydrophobic gleba, and dark, globose, Mountains of Guyana. The Cameroonian species are the rst ornamented ascospores (Trappe 1979). Aside from new to be formally described from Africa, although Elaphomyces tropical Australian species recently described by Castellano et partial ITS root tip seuences have been reported from the al. (2011), there is a paucity of published Elaphomyces records African tropics (e.g. Tedersoo et al. 2010, 2011) and as yet from the tropics (e.g. Corner & Hawker 1955, Castellano et al. undescribed Elaphomyces ascomata have been collected in 2012). Unpublished and currently undescribed Elaphomyces Madagascar (Bart Buyck, pers. comm.). The Cameroonian collections have been reported from Costa Rica, Java, Papua species are currently only known from primary Guineo- New Guinea, and Thailand (Reynolds 2011, Nampia Sukarno Congolian tropical rainforests dominated by the ECM pers. comm., T.F.E., unpubl.data). canopy tree Gilbertiodendron dewevrei (Fabaceae subfam. Caesalpinioideae) with additional scattered trees of the ECM *This paper was prepared by USA Govt. employees on ofcial time, genus Uapaca (Phyllanthaceae). The Guyanese species and is therefore in the public domain and not subject to copyright. occurs in primary forests co-dominated by the ECM trees © 2016 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specied by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 7 · NO. 1 59 Castellano et al. Pakaraimaea dipterocarpacea (Dipterocarpaceae) and dNTPs and excess primers by adding 1 L ExoSAP-IT (USB, Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae). Cleveland, OH) to 5 L PCR reaction mix and incubating for 15 Macromorphological, micromorphological, habitat, and min at 37 °C followed by 15 min at 80 °C. Unidirectional dye- DNA seuence data are provided for each new species. A terminator seuencing used BigDye3.1 (Applied Biosystems, molecular phylogenetic analysis assesses the relationships of Foster City, CA), by adding 2 L of cleaned PCR template to ARTICLE the new species within Elaphomycetaceae, and suggests that 3 L of solution containing 0.2 L BigDye, 1 L seuencing a common ancestor is shared between some species from buffer, 0.15 L 50mM MgCl2, 0.15 L of 10 M primer, and 1.5 Africa and South America, and that stalked, volvate species L of Milli-Q (Merck Millipore, Darmstadt, Germany) puried of Pseudotulostoma may be nested within Elaphomyces. water. Seuencing was performed with 60 cycles of 95 °C denaturation for 10 sec, 50 °C annealing for 10 sec, and 60 °C extension for 2 min. Seuencing reactions were cleaned MATERIALS AND METHODS using ethanol precipitation and resuspended in puried water before loading into an ABI 3730 DNA Analyzer (Applied Collections Biosystems, Foster City, CA). Complementary unidirectional In Guyana, ascomata were collected during the June rainy seuence reads were aligned and edited in Seuencher v. season of 2012 from Pakaraima Mountains, Upper Mazaruni 4.2 (Gene Codes, Ann Arbor, MI) and deposited in GenBank River Basin, near a base camp at 5o26’21.3” N 60o4’43.1” (Table 1). W, 800 m a.s.l., in savanna fringing forest dominated by P. dipterocarpacea and D. jenmanii (Smith et al. 2013). In Taxa used, sequence alignment, and Cameroon, ascomata and ECM root tips were collected phylogenetic analysis during the Aug.–Sep. early rainy season of 2014 from the All Elaphomycetaceae (e.g. Elaphomyces, Pseudotulostoma) Dja Biosphere Reserve, Northwest Sector near the village of ITS and 28S seuences derived from ascomata and ECM Somalomo, Upper Dja River Basin, within a two km radius of root tips available in GenBank were downloaded. Each a base camp located at 3o21’29.8” N 12o43’46.9” W, 650 m gene region was aligned separately with the seuences a.s.l., in forests dominated by G. dewevrei (Peh et al. 2014). from our new species using the RNA structure-based Descriptions of macromorphological features were made algorithm Q-INS-i implemented in MAFFT v7.023b (Katoh et from fresh material in the eld. Colours were compared with al. 2002, Katoh & Toh 2008, Katoh & Standley 2013). After colour plates from Kornerup & Wanscher (1978) and are correcting the orientations of four ITS seuences, removing cited in parentheses (e.g. 5A4). Fresh collections were dried one short seuence (GenBank accession AM087442) and with silica gel. Preserved specimens were later examined in one seuence with substantial numbers of ambiguous 3 % KOH, Melzer’s reagent, and Cotton blue. Microscopic bases (GenBank accession AB161194), the uneven ends descriptions are based on 3 % KOH mounts unless specied were trimmed and alignments rened with a second round otherwise. Twenty ascospores were measured from each of alignment in MAFFT, as above, and rened alignments type collection; dimensions reported include ornamentation. concatenated into a single dataset. Phylogenetic analysis Dried ascospores were mounted on aluminum pegs with under the maximum likelihood criterion was performed double-sided tape and coated with gold for scanning electron using the Pthreads parallelised version of RAxML v7.0.3 microscopy (SEM) with an AmRay 3300 FE eld emission (Stamatakis 2006, Ott et al. 2007) with a GTRGAMMA model, scanning electron microscope. Type and additional specimens allowing model parameters to be estimated for each gene are deposited in the following herbaria: BRG, University