IMA FUNGUS · 7(2): 239–245 (2016) doi:10.5598/imafungus.2016.07.02.03 Kombocles bakaiana gen. sp. nov. (Boletaceae), a new sequestrate fungus ARTICLE from Cameroon* Michael A. Castellano1, Todd F. Elliott2, Camille Truong3, Olivier Séné4, Bryn T. M. Dentinger5, and Terry W. Henkel6 1United States Department of Agriculture, Forest Service, Northern Research Station, 3200 Jefferson Way, Corvallis, OR 97331, USA 2Department of Integrative Studies, Warren Wilson College, Asheville, NC 28815, USA 3Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA 4Institute of Agricultural Research for Development, National Herbarium of Cameroon, PO Box 1601, Yaoundé, Cameroon 5\M"O"jO]{jJ7*;5J{ZP5#O" University of Utah, Salt Lake City, UT 84112, USA 65#O"J&JG\G*++/'JGZ"LP7&¨ Abstract: Kombocles bakaiana gen. sp. nov. is described as new to science. This sequestrate, Key words: partially hypogeous fungus was collected around and within the stilt root system of an ectomycorrhizal ectomycorrhizas (ECM) tree of the genus Uapaca (Phyllanthaceae) in a Guineo-Congolian mixed tropical rainforest in ##\ Cameroon. Molecular data place this fungus in Boletaceae (Boletales, Agaricomycetes, Basidiomycota) Guineo-Congolian rainforest with no clear relationship to previously described taxa within the family. Macro- and micromorphological hypogeous fungi characters, habitat, and DNA sequence data are provided. Unique morphological features and a molecular Uapaca phylogenetic analysis of 304 sequences across the Boletales justify the recognition of the new taxa. Kombocles bakaiana is the fourth sequestrate Boletaceae described from the greater African tropics, and #\ Article info:JP';K/='UZGP/</='UZP''</='U INTRODUCTION al. 2016). Our recent collecting in the Dja Biosphere Reserve of southern Cameroon discovered a number of sequestrate fungi, Numerous genera of sequestrate fungi within Boletaceae "[G##Elaphomyces (Boletales) have been recognized from various regions of ascomata from native plant communities (Castellano et al. the world, including the widely distributed north temperate 2016). Included in the Dja Biosphere Reserve collections is a Chamonixia, Gastroboletus, and Octaviania, Australasian morphologically distinctive fungus that produces basidiomata Rossbeevera, South-East Asian Durianella, Spongiforma, gregariously in soil in lowland, mixed tropical rainforests in and Rhodactina, Australian Solioccasus and Royoungia, close proximity to ectomycorrhizal (ECM) trees of the genus and tropical South American Castellanea, Costatisporus, Uapaca (Phyllanthaceae). Molecular and morphological and Jimtrappea (Binder & Bresinski 2002, Desjardin et al. data indicate that this Cameroonian sequestrate fungus is a 2008, 2009, Halling et al. 2012, Lebel et al. 2012, Orihara et member of Boletaceae but is evolutionarily distinct from all al. 2012a, b, Moreau et al. 2013, Trappe et al. 2013, Smith other described epigeous and sequestrate genera and species et al. 2015). For the African tropics, despite a high diversity within the family. of non-sequestrate, epigeous Boletaceae known from some areas (e.g. Heinemann & Goossens-Fontana 1954), known sequestrate Boletaceae are currently limited to a single species MATERIALS AND METHODS each of Mackintoshia, Mycoamaranthus, and Octaviania (Dissing & Lange 1962, Castellano et al. 2000, Mueller et al. Collections 2007, Pacioni & Sharp 2000, Tedersoo & Smith 2013). Of late, Basidiomata were collected during the August–September the sequestrate Lactarius megalopterus (Russulaceae) was early rainy season of 2014 from Cameroon’s Dja Biosphere described from lowland rainforests of Cameroon (Beenken et Reserve, Northwest Sector near the village of Somalomo, Upper Dja River Basin, ~1.4 km west of a base camp located at 3o21’29.8” N 12o43’46.9” W, 650 m a.s.l., in mixed forest *7JG]# containing Uapaca species (Peh et al. 2014). time, and is therefore in the public domain and not subject to Descriptions of macromorphological features are from copyright. # [ \ # { © 2016 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: !""#$ Non-commercial: # No derivative works: # 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. 2 239 Castellano et al. Wanscher (1978) and are cited in parentheses (e.g. 5A4). 7[_7J#>'> Collections were dried with silica gel. Preserved specimens We also used ML analysis of a large dataset based on 28S E ; @ {<& R " sequences of diverse Boletaceae with additional Boletales \;@{<& taxa as outgroups. The 28S analysis included our original [ 7 sequence data, 288 sequences used in Wu et al. (2014), ARTICLE were measured from the type collection. Dried basidiospores seven sequences from sequestrate genera used in Smith et were mounted on aluminum pegs with double-sided tape and al. (2015), and eight sequences from the Heimioporus species coated with gold for scanning electron microscopy (SEM) used in Halling et al !/='+$ 7 [ # Gj ;;== MX [ " 304 sequences representing species from infrafamilial clades microscope using 5 kV. Type specimens are deposited in across the Boletaceae based on recent studies (e.g. Nuhn et the following herbaria: YA (Cameroon National Herbarium, al. 2013, Wu et al. 2014, 2015, Smith et al. 2015, Henkel et al. $Z &J\ !& J G \G$Z 2016) and outgroup taxa. Representatives from all available <J\!<"J\$Z{!$!M" sequestrate Boletaceae genera were included: Castellanea, jO]{$ Chamonixia, Costatisporus, Durianella, Gymnogaster, Heliogaster, Jimtrappea, Mackintoshia, Mycoamaranthus, 'Z[\]^_\ Neoboletus, Octaviania, Rossbeevera, Royoungia, Solioccasus, sequencing and Spongiforma. All DNA work was carried out in the Jodrell Laboratory, Royal Each dataset was aligned separately using default O ] { 5DG E # settings in PASTA (Mirarab et al. 2015). For both datasets, dried basidioma tissue using the Extract-N-Amp Plant PCR kit phylogenetic analysis under the ML criterion was performed (SIGMA-ALDRICH, Saint Louis, MO), followed or not by plate using the Pthreads parallelised version of RAxML v. 8.2.4 [!5"et al. 2010), or using a Plant DNeasy mini (ITS) or v8.2.9 (28S) (Stamatakis 2006, Ott et al. 2007) with kit (QIAGEN, Valencia, CA). Full ITS 1 and 2 regions, along a GTRGAMMA model, allowing model parameters to be +9J 5DG !_7J$ \jL estimated for each gene partition separately. Branch support ITS1F and ITS4 (White et al. 1990, Gardes & Bruns 1993), was assessed using nonparametric bootstrapping with and the nuclear 28S rDNA D1–D2 domains (28S) were PCR- the autoMRE option. The alignments and trees have been [ qj=jQqj+ !" & '**=$ #" deposited in TreeBASE and are available at http://purl.org/ the cycling conditions in Dentinger et al. (2010). PCR products phylo/treebase/phylows/study/TB2:S19818. L\#"/)q\j products in a 1 % agarose gel containing 0.005 % ethidium bromide. Prior to sequencing, amplicons were cleaned of RESULTS D7 E " ' )q XEJGL_7!JO\<&$+)q\jE BLASTn queries and phylogenetic analysis and incubating for 15 min at 37 °C followed by 15 min at 80 Using GenBank BLASTn queries of the new taxon’s ITS °C. Unidirectional dye-terminator sequencing used BigDye3.1 sequence, the top 100 best hits all belonged to Boletaceae !GOM\\G$"/)q# but were uninformative at the generic level. The sequence \j ; )q # " =/ )q O"5 from the new taxon was, however, nearly identical (99 % ')q"##='+)q+="\2='+)q#'= over 606 nucleotides) to a sequence (FR731937) from an )'+)q#L¡!5 ECM root tip of Uapaca guineensis from Gabon sampled by ]$[J"#U= Tedersoo et al. (2011). The BLASTn matches to remaining cycles of 95 °C denaturation for 10 sec, 50 °C annealing for sequences in GenBank covered only a maximum of 79 % of 10 sec, and 60 °C extension for 2 min. Sequencing reactions the query sequence with 86 % identity. Final alignments for were cleaned using ethanol precipitation and resuspended in phylogenetic analysis consisted of 717 sequences and 4009 [#"GO_;>;=5DGG aligned positions for the ITS (1476 parsimony informative, (Applied Biosystems, Foster City, CA). Complementary 2119 constant, 414 autapomorphic), and of 304 sequences unidirectional sequence reads were aligned and edited in and 1293 positions for the 28S (441 parsimony informative, Sequencher v. 4.2 (Gene Codes, Ann Arbor, MI) and deposited 634 constant, 140 autapomorphic). All characters were ]O!_7JP{¬9/>==?Z/9JP{¬9/>==;$ included in the analysis of both datasets. For the ITS dataset, RAxML rapid bootstrapping terminated after 350 replicates Taxa used, sequence alignment, and (WRF average of 100 random splits = 2.603297) and the phylogenetic analysis best ML tree had a likelihood score of –60202.046042. For The ITS ribosomal DNA sequence from the new taxon was the 28S dataset, RAxML rapid bootstrapping terminated initially subjected to a BLASTn query against GenBank in after 300 replicates (WRF average of 100 random splits = order to explore its putative phylogenetic relationships. To 2.696777) and the best ML tree had a likelihood score of # " # # #" –31601.477718 (Fig. 1). A second analysis of the 28S dataset used Maximum Likelihood (ML) analysis of an ultra-large # " " " " ]Oq<\{J alignment of all ITS sequences