ARTICLE 239

Elaphomyces 6 #O"

Key words: ectomycorrhizas ##\ Guineo-Congolian rainforest hypogeous fungi Uapaca IMA · 7(2): 239–245 (2016) 239–245 · 7(2): FUNGUS IMA (Peh et al. 2014). and Terry W. Henkel W. Terry and

) , 5 43’46.9” W, 650 m a.s.l., in mixed forest 43’46.9” W, species o , Uapaca Phyllanthaceae). Molecular and morphological ( Our recent collecting in the Dja Biosphere Reserve ofOur recent collecting in the Dja Biosphere 21’29.8” N 12 o Descriptions of macromorphological features are from , Bryn T. M. Dentinger M. T. , Bryn 4 # \ # { al. 2016). of sequestrate fungi,southern Cameroon discovered a number "G## (Castellano et al.ascomata from native plant communities collections is a2016). Included in the Dja Biosphere Reserve basidiomatamorphologically distinctive fungus that produces tropical rainforests ingregariously in soil in lowland, mixed trees of the genusclose proximity to ectomycorrhizal (ECM) Uapaca fungus is adata indicate that this Cameroonian sequestrate member of but is evolutionarily distinct from all other described epigeous and sequestrate genera and species within the family. MATERIALS AND METHODS MATERIALS Collections August–September Basidiomata were collected during the Dja Biosphere early rainy season of 2014 from Cameroon’s Reserve, Northwest Sector near the village of Somalomo, Upper Dja River Basin, ~1.4 km west of a base camp located at 3 containing ,

et justify the recognition of the new taxa. et al. et al. , Olivier Séné 3 described from the greater African tropics, and African tropics, described from the greater ) in a Guineo-Congolian mixed tropical rainforest in ) in a Guineo-Congolian mixed tropical rainforest Costatisporus, et al. 2013, Smith Russulaceae) was et al. 2012, Orihara , Camille Truong , Camille 2 et al. 2000, Mueller , , Castellanea, (Phyllanthaceae gen. sp. nov. is described as new to science. This sequestrate, science. is described as new to gen. sp. nov. Solioccasus and , * Mycoamaranthus, and Uapaca are currently limited to a single species , Todd F. Elliott F. Todd , 1 et al. 2012, Lebel

is the fourth sequestrate Boletaceae Boletaceae Kombocles bakaiana

!""#$ , Australian Jimtrappea (Binder & Bresinski 2002, Desjardin time, and is therefore in the public domain and not subject to copyright. ) have been recognized from various regions ofBoletales) have been recognized from various Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA 32611, University of Florida, Gainesville, FL Department of Plant Pathology, Cameroon Yaoundé, PO Box 1601, Development, National Herbarium of Cameroon, Agricultural Research for Institute of United States Department of Agriculture, Forest Service, Northern Research Station, 3200 Jefferson Way, Corvallis, OR 97331, USA Corvallis, OR 97331, Way, Research Station, 3200 Jefferson Agriculture, Forest Service, Northern of United States Department USA Asheville, NC 28815, Wilson College, Studies, Warren Department of Integrative 5#O"J&JG\G*++/'JGZ"LP7&¨ \M"O"jO]{jJ7*;5J{ZP5 7JG]# Abstract: partially hypogeous fungus was collected around and within the stilt root system of an ectomycorrhizal was collected around and within the partially hypogeous fungus (ECM) tree of the University of Utah, Salt Lake City, UT 84112, USA 84112, UT Lake City, University of Utah, Salt © 2016 International Mycological Association © 2016 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: * ), a new sequestrate fungus sequestrate ), a new (Boletaceae sp. nov. gen. bakaiana Kombocles from Cameroon 6 3 4 5 1 2 Michael A. Michael A. Castellano Cameroon. Molecular data place this fungus in Boletaceae ( Cameroon. Molecular data place this fungus Non-commercial:No derivative works:if you get Any of the above conditions can be waived 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. # # Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. with no clear relationship to previously described taxa within the family. Macro- and micromorphological taxa within the family. with no clear relationship to previously described are provided. Unique morphological features and a molecular sequence data characters, habitat, and DNA the Boletales phylogenetic analysis of 304 sequences across Kombocles bakaiana Article info:JP';K/='UZGP/</='UZP''</='U #\ 2007, Pacioni & Sharp 2000, Tedersoo & Smith 2013). Of late, Tedersoo 2007, Pacioni & Sharp 2000, the sequestrate Lactarius megalopterus ( described from lowland rainforests of Cameroon (Beenken et Numerous genera of sequestrate fungi within BoletaceaeNumerous genera of sequestrate fungi ( north temperatethe world, including the widely distributed , AustralasianChamonixia, , and Octaviania Asian Rossbeevera, South-East and American and tropical South INTRODUCTION and 2008, 2009, Halling (Dissing & Lange 1962, Castellano 2013, Trappe Trappe al. 2012a, b, Moreau et al. 2013, African tropics, despite a high diversity et al. 2015). For the of non-sequestrate, epigeous Boletaceae known from some areas (e.g. Heinemann & Goossens-Fontana 1954), known sequestrate each of Mackintoshia, doi:10.5598/imafungus.2016.07.02.03 VOLUME 7 · NO. 2 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

')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 from the UNITE general (Castresana 2000) with the option to allow gap positions in release v. 7 (release date 31 Jan. 2016) (Nilsson et al. 2015) !?U9 $ # Boletaceae#L"_7JE monophyletic Boletaceae, evidence that phylogenetically (Bengtsson-Palme et al /=';$ E" informative regions were lost (data not shown). In both the ITS

240 IMA FUNGUS Kombocles, a new genus of sequestrate Boletaceae

44 KF112346 Heimioporus aff. japonicus HKAS52236 ARTICLE 15 KF112347 Heimioporus japonicus HKAS52237 55 KP327643 Heimioporus fruticicola REH8958 10 KP327644 Heimioporus fruticicola REH8962 KP327664 Heimioporus cooloolae REH9817 25 15 KP327652 Heimioporus australis REH9288 24 20 KP327617 Heimioporus retisporus REH8074 KP327624 Heimioporus mandarinus FRIM4636 57 32KP327630 Heimioporus ivoryi REH8620 22 KP327635 Heimioporus mandarinus REH8785 KF112345 Heimioporus sp. HKAS53451 KX827003 Kombocles bakaiana TH-DJA136

Fig. 1. Phylogenetic placement of Kombocles bakaiana within Boletaceae using Maximum Likelihood (ML) analysis of 28S ribosomal gene sequences. Left is the best ML tree from a RAxML search using the GTRGAMMA model of substitution on a PASTA-aligned dataset of 288 Boletaceae sequences and 16 outgroup taxa from other Boletales. The tree is rooted at the node leading to Suillus spp. The putative clade containing Kombocles bakaiana is highlighted in red and shown on the right as an enlargement. Numbers on branches are percentage support based on 300 nonparametric bootstrap replicates. Terminal labels are composed of the GenBank accession number followed by taxon name and collection code.

and 28S datasets, the new Cameroonian taxon was placed Etymology: Baka and –iana !q#E6$O#OPZ within Boletaceae, but was not nested within, or supported as referring to the Baka indigenous people inhabiting the type related to, any previously described epigeous or sequestrate locality. genera. Although the 28S analysis recovered the new taxon in a clade with the epigeous bolete genus Heimioporus, this Diagnosis: Differs from other known sequestrate taxa by !//@Z the combination of the pale cream to light yellowish brown Fig. 1), as did other nodes deeper than the terminal clades peridium, gleba with brown locules unevenly surrounded by in the tree. bright white to off-white tramal tissue, lack of a columella, yellow-brown basidiospores that appear smooth under light microscopy but rugulose under SEM, are dextrinoid when young with a distinct hilar appendage, uniquely long sterigmata, and absence of cystidia. Kombocles Castellano, T.W. Henkel & Dentinger, gen. nov. Type: Cameroon: East Province: Dja Biosphere Reserve, MycoBank MB818594 Northwest Sector near the village of Somalomo, Upper Dja (Fig. 2) River Basin, ~1.4 km west of Dja base camp located at 3°21’29.8” N 12°43’46.9” W, in mixed forest, around and in Etymology: Komba and –cles !] #E $ O{ between the stilt roots of Uapaca sp., near Gilbertiodendron renowned”, referring to the spiritual protector and provider of dewevrei monodominant plot 3, 25 Sept. 2014, Henkel the forest as recognized by the Baka indigenous people of THDJA 136 !GUU*''MZ&J\]'/=; the type locality of the Dja Biosphere Reserve. {!$/=+;U'M$]O_7JP {¬9/>==?Z/9JP{¬9/>==; Diagnosis: Distinguished from other Boletaceae by a combination of the following characters: Basidiomata Description: Basidiomata irregularly globose to subglobose, emergent, sequestrate, globose to subglobose. Peridium '=M/= '+M/+ " various shades of brown, unchanging. Gleba brown with Z # " irregular off-white veins, unchanging, loculate. Columella (4A2–5C4), inherently smooth but with numerous adherent absent. Basidiospores asymmetrical, variously shaped soil particles, occasionally with irregularly spaced cracks from fusoid to allantoid to unevenly ellipsoid, rugulose, revealing the underlying gleba, unchanging with handling but yellow-brown, dextrinoid when young, with a distinct hilar drying rugose to ruminate with numerous shades of brown appendage. Cystidia and clamp connections absent. and darker, humic-stained patches. Peridium two-layered in LZ¯=+ Type species: Kombocles bakaiana Castellano et al. 2016. the surface but somewhat mottled due to uneven distribution of humic stains, underlain by an inner, pale yellowish (1A2– Kombocles bakaiana Castellano, T.W. Henkel & 1A3) layer up to 1 mm thick that becomes cottony near the Dentinger, sp. nov. gleba. Gleba initially marbled, then with increasingly well- MycoBank MB818595 ##L" (Fig. 2) EZ

VOLUME 7 · NO. 2 241 Castellano et al.

locules initially off-white to yellowish orange (4B7) to brown phylogenetic analysis, K. bakaiana was putatively sister to the !>X9M>M9$ Z Australasian epigeous bolete genus Heimioporus albeit with elements and basidiospores, irregularly spherical, up to ±1 very low bootstrap support (Fig. 1). While obviously different mm broad. Dried gleba Z macromorphologically (epigeous and non-sequestrate in to irregularly polygonal, variably-sized up to 1.1 mm broad, HeimioporusZ"#†Kombocles),

ARTICLE !/ /= )$ the basidiospores of H. cooloolae have a somewhat similar yellow-brown to medium brown, often with a paler central shape to those of K. bakaiana and are also dextrinoid (though core, contiguous to irregularly-spaced. Odour mild, pleasant, rarely), but differ in having a pitted surface ornamentation somewhat sweet. Taste mild, indistinct. compared to the non-pitted basidiospore surface of K. Peridium //=M;/=)LZ//= bakaiana (Halling & Fechner 2011, Halling et al. 2015). μm thick but non-uniform in thickness and sometimes absent Macromorphologically, K. bakaiana is reminiscent of in small portions of section, of pale yellow-brown tissues that species of and (), but the are distinctly and heavily encrusted with minute particles of dextrinoid, large, yellow-brown basidiospores allow easy unknown origin, otherwise of similar structure as underlying separation from these two genera (Moreau et al. 2011). Z /==M;== ) Microscopically K. bakaiana basidiospores are similar to mix of textura globosa and textura intricata appearing as the smooth, subfusiform basidiospores of Castellanea and interwoven tissue with abundant hyaline, elongate, clavate Jimtrappea (Boletaceae) from tropical forests in Guyana, /UM;'&';M'U!M/?$) but these two genera have symmetrical, pedicellate #"6"Z basidiospores compared to those of K. bakaiana that have /)Glebal trama similar in structure to the inner a distinct hilar appendage and are variably-shaped and peridium but with less elongate hyphae and more clavate asymmetrical (Smith et al. 2015). In addition, Jimtrappea or irregularly-shaped cells, variable in thickness to nearly has dextrinoid hymenial cystidia, which are absent in K. absent, with no apparent color differentiation near the outer bakaiana. Solioccasus (Boletaceae) from Queensland, locule edge. Basidia"?=M?+&UM>) Australia, has variably shaped, pale yellow basidiospores "Z"#?M9+&/') that are nondextrinoid (Trappe et al. 2008). hyaline. Basidiospores variably-shaped, fusoid, allantoid Among the few sequestrate putatively ECM fungi known to unevenly ellipsoid, asymmetrical in side view, with slight from the African tropics, Mackintoshia persica from Zimbabwe " is characterized by smooth, dextrinoid, thick-walled or faintly roughened with Nomarski imaging, but distinctly basidiospores that are very similar to those of K. bakaiana " JX {<& L " (Castellano et al. 2000, Pacioni & Sharp 2000). Mackintoshia mass, many distinctly and instantly dextrinoid, particularly persica was initially assigned to Cortinariaceae (Pacioni those towards the center of some locules, (12.5–)13.5–16.5 & Sharp 2000) but recent analyses have revealed that it is &!+M$++MU=)!'?;&+>)$¡‚/+= a member of Boletaceae (Smith et al. 2015). In contrast to " ‚ '9UM/U>!M;'>$ E" "Z K. bakaiana, basidia of M. persica lack distinct sterigmata, " '+ ) " Z hymenial cystidia are present, and the basidiospores are walls 1.0–1.5 μm thick. uniformly ellipsoid to broadly ellipsoid. Mycoamaranthus congolensis, known from the Congo, Malawi, and Zimbabwe, Habit, habitat, and distribution: Scattered and subhypogeous "" Boletaceae in leaf litter within and around the stilt-root system of an (Smith et al. 2015), differs from K. bakaiana primarily in ectomycorrhizal (ECM) Uapaca E #Z the spinose basidiospore ornamentation (Castellano et al. only from the type locality in the Dja River Basin of southern 2000). Octaviania ivoryana, while widespread in Africa, Cameroon. A nearly identical ITS sequence originating from "#]{J"´ an ECM root tip of U. guineensis from Gabon suggests that differs fundamentally from K. bakaiana in the cone-like the fungus may have a wider distribution in the Guineo- ornamentation of its basidiospores (Castellano et al. 2000). Congolian rainforest. While the phylogenetic position of O. ivoryana is currently unknown, other members of the genus are members of Commentary: Kombocles bakaiana"‡ Boletaceae (Smith et al. 2015, Orihara et al. 2012a). The " sequestrate Corditubera staudtii was originally described pale cream to light yellowish brown peridium, gleba with well- from Cameroon, but differs from K. bakaiana in the reddish " (vs. brown loculate in K. bakaiana) gleba and basidiospores orange and eventually to brown, lack of a columella, and mild that are globose with spinose ornamentation (Hennings odour and taste. Currently basidiomata of K. bakaiana are 1897). only known in close spatial association with Uapaca trees in forest stands lacking ECM Gilbertiodendron dewevrei _7J† ACKNOWLEDGEMENTS from a Uapaca root tip from Gabon (Tedersoo et al. 2011), so it may be presumed that Uapaca is the ECM host. We thank the following funding sources: The National Geographic Micromorphologically, K. bakaiana is distinguished by the Society’s Committee for Research and Exploration grant 9235- variably shaped, asymmetrical, yellow-brown, dextrinoid, 13 and National Science Foundation (NSF) DEB-1556338 to smooth basidiospores with a distinct hilar appendage. In the T.W.H., and a grant to C.T. from the Basler Stiftung für Biologische

242 IMA FUNGUS Kombocles, a new genus of sequestrate Boletaceae ARTICLE

Fig. 2. Kombocles bakaiana !GUU*''MZ$A. Basidiomata in section showing peridial surface, peridium, and gleba. B. Cross-section of outer (i.e. the thin layer of dark cells) and inner peridium. C. Cross-section showing tramal structure. D. Basidium with attached basidiospores and long sterigmata. E. Basidiospores in surface and cross-sectional view. FJ""#" detail of the ornamentation. Bars A = 10 mm, B = 50 μm, C = 25 μm, D = 6 μm, E = 10 μm, and F = 5 μm.

VOLUME 7 · NO. 2 243 Castellano et al.

M" _ \ # j J species and new combinations in , Innovation issued research permits. Jean Michel Onana, Head of The and . Australian Systematic Botany 28: 122. National Herbarium of Cameroon (Institute of Agricultural Research Heinemann P, Goossens-Fontana M (1954) Flore iconographique des for Development, IRAD) provided much logistical assistance. The champignons du Congo. Fascicle 3. . Brussels: Jardin Conservator of the Dja Biosphere Reserve, Mengamenya Goue Botanique de l’Etat.

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244 IMA FUNGUS Kombocles, a new genus of sequestrate Boletaceae

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