ARTICLE 297 .,

et al. et

et al. 2015). et al. (2007) 2 et al. 2002, Binder Key words: Caesalpinioideae ectomycorrhizal fungi gasteroid fungi Guiana Shield 2004, Giachini Montecchi & Sarasini 2001, Sarasini & Montecchi et al. 2010) whereas other ) et al. et 6(2): 297–317 (2015) 297–317 s · 6(2): gu Fun IMA et al. 2006, Lebel et al. 2014), Mueller , and Terry W. Henkel W. Terry , and 4 , ), and Pakaraimaea Cabral et al. 2006, Henkel Costatisporus cyanescens Costatisporus Bougher & Lebel 2001, Lebel & Bougher et al. et 2009), but tropical sequestrate fungi remain gen. sp. and Costatisporus nov., Knowledge of the diversity and distributions of sequestrate of distributions and diversity the of Knowledge , , M. Catherine Aime , M. Catherine Papilionoideae 1 ., and estimated that ~30 species of hypogeous sequestrate taxa are taxa sequestrate hypogeous of species ~30 that estimated currently described from the Neotropics with approximately 200 species remaining unknown to science. Recent studies in the Guiana Shield region of northeastern South America have revealed a fungi in remote, primary diverse tropical rain forests dominated assemblage by of sequestrate Understanding of the multiple origins and taxonomic affinities taxonomic and origins multiple the of Understanding evolutionary the into insight provided has fungi sequestrate of forces that drastically alter basidioma form, dispersal (Thiers function, 1984, Kretzer & and Bruns 1997, 2007). Albee-Scott & Claridge 2005, Trappe Reijnders 2000, fungi has progressively advanced for some regions (e.g. of world the Trappe Trappe especially poorly known. While some epigeous sequestrate fungi have recently been documented Amazon from (e.g. the Brazilian sequestrate clades of earlier radiated across origin the globe (e.g. Grubisha have speciated and & Hibbett 2006, Hosaka 2006, Smith 2010, Gube & Dorfelt 2012, Lebel & Syme 2012, Ge & Smith & Ge 2012, Syme & Lebel 2012, Dorfelt & Gube 2010, isolated recent, from resulted fungi sequestrate Some 2013). evolutionary events that led to one species or within a a few sequestrate clade of Kretzer non-sequestrate & Bruns relatives 1997, (e.g. Martin subfam. (Boletales et al. et al. , Keisuke Obase 3 ( ) that are Castellanea pakaraimophila Aldina ) , Boletales ), , Todd F. Elliott F. Todd , 2 et al. 2001, Miller & Aime (Basidiomycota ) with the Gasteromycetes) Caesalpinioideae . (Boletaceae gen. sp. nov gen. sp. pakaraimophila

, Kevin R. Amses , Kevin R. subfam. 1 Agaricomycetes ). Molecular data place these fungi in fungi these place data Molecular ). et al. 2006, 2007, Lebel Henkel & Tonkin et al. 2000, Peintner et al. 1989, Mueller & 1994, Hibbett gen. sp. nov. are described as new to science. These sequestrate, hypogeous fungi were collected Jimtrappea guyanensis gen. sp. nov.,

(Fabaceae specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). must attribute the work in the manner You Application of molecular techniques in has Agaricomycetes, demonstrating that the sequestrate Department of Botany & Pathology, Purdue University, West Lafayette, IN 47907, USA Lafayette, West Purdue University, & Plant Pathology, Department of Botany Department of 95521, Arcata, Biological USA; CA Sciences, corresponding Humboldt author State email: University, Terry.Henkel@humboldt. USA Asheville, NC 28815, Wilson College, Studies, Warren Department of Integrative Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA 32611, University of Florida, Gainesville, FL Department of Plant Pathology, Submitted: 31 May 2015; Accepted: 19 September 2015; Published: 2 October 2015. Accepted: 19 Article info: Submitted: 31 May 2015; and inform their relationships to other known epigeous and micromorphological characters, sequestrate habitat, and taxa multi-locus DNA sequence within data are that provided for family. each new Macro- taxon. and Unique morphological features and a molecular phylogenetic analysis of 185 taxa across justify the recognition of the three new genera. the order (Dipterocarpaceae gen. sp. nov gen. sp. cyanescens in Guyana under closed canopy tropical forests in association Dicymbe with ectomycorrhizal (ECM) host genera Abstract: edu © 2015 International Mycological Association © 2015 International Mycological conditions: distribute and transmit the work, under the following are free to share - to copy, You Attribution: 2 3 4 Matthew E. Smith 1 gen. sp. nov gen. sp. guyanensis uyana: Jimtrappea from G fungi New sequestrate Castellanea Non-commercial: No derivative works: Any of the above conditions can be waived if you get 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. transform, or build upon this work. may not alter, You may not use this work for commercial purposes. You Nothing in this license impairs or restricts the author’s moral rights. permission from the copyright holder. functionally united in their enclosed hymenial development and lack of such ballistospory. charismatic This macrofungi as puffballs, informal earthstars, false group earthstars, includes earthballs, bird’s nest stinkhorns, and and false truffles cannonball (Ingold fungi, 1965,1988). Miller These fungi had & Miller once been treated taxonomic as a cohesive unit basidiomycete in lost rarely was ballistospory that (e.g. assumption class evolutionary history (e.g. Coker & Couch 1928) or that the sequestrate state was ancestral, predating the evolution of ballistospory (e.g. Singer 1971). Other sequestrate basidiomycetes authors as a regarded polyphyletic assemblage based on morphological and developmental evidence (e.g. Reijnders 1963, 2000, Heim 1971, Moore 1998). since corroborated lineages -level and the family numerous in taxa sequestrate latter view by in discovering new basidioma form has independently evolved multiple (e.g. times Bruns 1997, Miller 2001, Binder Gasteroid fungi comprise a diverse, of fungi within artificial assemblage INTRODUCTION doi:10.5598/imafungus.2015.06.02.03 volume 6 · no. 2 298 ARTICLE

08 20, Lebel 2009, 2008, al. pce i cran ein o te oln Norpc (e.g. Neotropics Singer lowland the of regions certain in species epigeous non-sequestrate, of diversity high a Moreau and African tropical other describedgeneraandspecieswithinthefamily. members of features, indicate that these Guyanese sequestrate fungi are rDNA, 28S Smith 2009, Degagne 2003, (Henkel Neotropics lowland the in type habitat infrequent an , ECM by dominated rainforests Guyana. These fungi were collected from closed-canopy, wet sequestrate of genera monotypic new three describing by situation this (Mueller sequestrate of reports few very South-East Asian ein, nldn: h wdl dsrbtd ot TemperateChamonixia North distributed widely the including: regions, world various from recognized been have fungi sequestrate 2010, 2012,Castellano and as Elaphomyces and genera suchasHysterangium ECM-forming including ascomycetes, and basidiomycetes diverse from taxa hypogeous and epigeous include These Papilionoideae ie wr dmntd y ECM by dominated were sites collection The a.s.l. m 710 W, 59°54’40.4” N 5°18’04.8” at Potaro River Basin, within Upper a 15 km the radius of a of permanent base camp forests from 2015 and 2012, 2009, of seasons rainy May–July the during made were Collections Collections MATERIALMETHODS S AND of subfam. species (ECM) ectomycorrhizal oiae b ECM by dominated insignis oiae b ECM by dominated co- were site this at Forests a.s.l. m 800 at W, 60°04’43.1” and N 5°26’21.3” at camp base a of radius km six a within 2011 and June of 2012 from the Upper Mazaruni River Basin 2010– of Dec.–Jan. during made were collections Guyana jenmanii microscope with light and phase contrast optics. Rehydrated optics. contrast phase and light with microscope BX51 Olympus an using imaged and examined Fresh later were parentheses. in noted specimens Preserved gel. silica using dried were collections plates colour with (1978), described were Wanscher & Kornerup Colours to according field. coded and subjectively the in material fresh from Protubera Within Descriptions of macromorphological features were made were features macromorphological of Descriptions Geastrum Tremellogaster Bne & rsnk 20, Desjardin 2002, Bresinski & (Binder 93 Henkel et al.1983, (Smith 2013, Trappe 2013, et al. et al.2007, Tedersoo & Smith 2013). Here we rectify (Smith etal.2013). Caesalpinioideae Boletaceae 1, and RPB1, andOctaviania Boletaceae Boletaceae 03. oeua dt fo te T and ITS the from data Molecular et al.2013). (Eurotiales ( ), and Pakaraimaea (Geastrales 01 Henkel 2011, et al. Mackintoshia Durianella, Spongiforma Durianella, (Boletales D. corymbosa Pakaraimaea dipterocarpacea Pakaraimaea 02 Orihara 2012, et al. 2 loci, along with morphological with along loci, RPB2 but are evolutionarily distinct from all et al.2012,Henkelunpubl.). (Boletales ), as well as non-ECM genera such genera non-ECM as well as ), rm h Pkria onan of Mountains Pakaraima the from ) (Miller (Miller ) ), 03. oee, despite However, 2013). et al. 02 21) hr are there 2015) et al.2012, Australasian ; ad Australian and ; (Hysterangiales ), ), and Pseudotulostoma Aldina Dicymbe Guyanagaster Boletaceae ), numerous generaof 2001, Henkel 2001, et al. , 2012). Additional et al. 2012). D. altsonii Dicymbe (Fabaceae (Dipterocarpaceae

corymbosa , andRhodactina fromtheregion 02, b, 2012a, et al. ), Rossbeevera , andAldina Scleroderma ( (Fabaceae Soliocassus Boletaceae subfam. and r co- or et al. et al. Smith etal. and D. et ), ). ; ;

Paragyrodon sphaerosporus, lividus, for pairs base 693 and RPB1, for pairs base 709 28S, for pairs base 729 of dataset aligned exclude ambiguous portions of the alignment, producing a final to used was 2007) (TalaveraCastresana package & software Gblocks The 2013). Standley & (Katoh v.MAFFT7 of aid the (RPB2) (Tamurasoftware pairs MEGA5 using base 903 and (RPB1), pairs base 978 (28S), compiled in separate nucleotide alignments of 1131 base pairs 28S, of Sequences region. gene one for only available were sequences if even included, also were Representatives fromnumeroussequestrateBoletaceaetaxa 28S, Wu (sensu possible as clades sequestrate non- epigeous, many as for included were taxa representative Henkel 2015, 2014, recent phylogenetic studies (e.g. Nuhn (e.g. studies phylogenetic recent family the across from clades species infrafamilial representative for GenBank from taxa sequences 185 additional of and data sequence original included additional with University; andNY, BotanicalGarden. HSU, Purdue Herbarium, Kriebel Guyana; PUL, University; State Humboldt of University BRG, herbaria: in following deposited the were specimens additional and Type kV. 20 using microscope electron scanning 250 Quanta FEI a with Scanning SD). ± obtained were values of (SEM) micrographs electron Q of (mean Qm basidiospores and “n” measured) over values are Q of basidiospores (range Qr for as reported values Q Length/width examined. specimen least each for at measured were in structures individual structures 20 other and features, hyphal basidia, basidiospores, For solution. Melzer’s and (KOH), hydroxide MI) anddepositedinGenBank(Table 1). Arbor,Ann Codes, (Gene 5.1 v. Sequencher in edited were Wu and (2011), Dentinger of primers the used and protocols the followed (RPB 2) subunit largest second and (RPB1) subunit largest II polymerase RNA for gene the (28S), domains D1–D2 rDNA 28S nuc (ITS), rDNA 5.8S the with along 2, and 1 ITS the encompassing rDNA region nuc the of sequencing DNA and PCR CA). Valencia, (QIAGEN, kit mini DNAeasy Plant a or 1993) CTAB Bruns & (Gardes modified method the using specimens additional and types from tissue basidioma on performed were extractions DNA sequencing DNA extraction,PCRamplification,and on 28S, on based dataset concatenated a of (ML) Likelihood Maximum used we affinities, phylogenetic their assess further to order relationships. phylogenetic in putative their GenBank explore to against order queries BLASTn to subjected initially were species new each from sequences DNA ribosomal ITS phylogenetic analysis Taxa used,sequencealignment,and ugl ise wr mutd n H in mounted were tissues fungal RPB1, and 1, and RPB1, Boletaceae castaneus, RPB2 sequences being available in GenBank. taxa as outgroups. The analysis analysis The outgroups. as taxa Boletales 21) Nwy eeae sequences generated Newly (2014). et al. 2015). The type species and/or key key and/or species type The 2015). et al. 2 sequences of diverse of sequences RPB2 genera or undescribed genus-level genus-level undescribed or genera aff. 2014), contingent on their their on contingent 2014), et al. RPB2. 2011) and aligned with with aligned 2011)and et al. 21) Smith (2010), et al. meruloides, 1, and RPB1, Phlebopus portentosus, based on on based Boletaceae 2 2013, Wu 2013, et al. granulatus, and , % potassium % 3 O, ima 2 were were RPB2 Boletaceae Boletaceae S. aff. et al. et al. In In

New sequestrate Boletaceae from Guyana ARTICLE s.s. clade s.s. clade s.s. clade s.s. clade outgroup taxa Collapsed clade in Fig. 1 Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Austroboletus Austroboletus Austroboletus Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Xerocomoideae Boletales Xerocomoideae RBP 2 — KF112741 KF112745 — KF112752 — — KF112768 KF112764 KF112765 — KM605179 — KF112760 — — — KF112757 — KF112755 KF112743 — KF112756 KF112753 KF112748 KF112744 KF112759 KF112758 KF112771 KF112747 — — RBP 1 KF030392 KF112557 KF112559 KF030381 KF112626 JX889721 KF030358 KF112573 KF112569 KF112570 KF030370 KJ184564 LC043079 — HQ161822 KF030383 HQ161821 KF112621 HQ161831 KF112625 KF112624 HQ161830 KF112562 — KF112567 KF112566 KF112623 KF112622 KF112612 KF112568 DQ435803 KF030388 G enBank accession number 28 S KF030238 KF112344 KF112421 KF030311 KF112420 JX889720 DQ534624 KF112487 KF112383 KF112485 KF030246 KJ184558 LC043078 JQ924336 HQ161853 KF030312 HQ161852 KF112426 HQ161862 KF112376 KF112360 HQ161861 KF112419 KF112363 KF112364 KF112365 KF112428 KF112427 KF112468 KF112413 AY684153 KF030302 Location Africa Brancciano, Lazio, Italy Brancciano, Lazio, Italy Chestnut Ridge, NY, USA Chestnut Ridge, NY, Dêqên, Yunnan, China Yunnan, Dêqên, China MA, USA Chenzhou, Hunan, China Fuzhou, Jiangxi, China Yunnan, China Yunnan, Chestnut Ridge, NY, USA Chestnut Ridge, NY, Shenlongjia, Yunnan, China Yunnan, Shenlongjia, Region 8 Potaro-Siparuni, Guyana Puntarenas, Costa Rica Region 8 Potaro-Siparuni, Guyana North Collins, NY, USA North Collins, NY, Region 8 Potaro-Siparuni, Guyana Sanming, Fujian, China Region 8 Potaro-Siparuni, Guyana Chenzhou, Hunan, China Lijiang, Yunnan, China Yunnan, Lijiang, Region 8 Potaro-Siparuni, Guyana Fuzhou, Jiangxi, China Dêqên, Yunnan, China Yunnan, Dêqên, Sanming, Fujian, China Sanming, Fujian, China Dali,Yunnan, China Dali,Yunnan, Baoshan, Yunnan, China Yunnan, Baoshan, Nujiang, Yunnan, China Yunnan, Nujiang, Baoshan, Yunnan, China Yunnan, Baoshan, MA, USA Cape San Blas, FL, USA Voucher ID Voucher 00-436 MG372a MG374a DS 626-07 HKAS 76655 HKAS 75207 112/96 HKAS 53450 HKAS 57756 HKAS 59624 MB 07-001 HKAS 75739 Henkel 8035 NY 815459 NY TH 8819 3838 TH 8840 HKAS 52678 TH 8809 HKAS 53398 HKAS 57776 TH 8077 HKAS 76852 HKAS 56317 HKAS 53375 HKAS 53376 HKAS 58713 HKAS 59536 HKAS 74783 HKAS 74888 AFTOL-ID 575 AFTOL-ID 4588 mutabilis aff. aff. sp. shichianus sp. aff. aff. sp. sp. sp. sp. emodensis aff. sp. sp. Taxa and GenBank accession numbers for sequences used in the phylogenetic analysis. If a taxon appeared in a collapsed clade in Fig.1, the collapsed clade is indicated on the right. Guyanese right. the on indicated is clade collapsed the Fig.1, in clade collapsed a in appeared taxon a If analysis. phylogenetic the in used sequences for numbers accession GenBank and Taxa 1. Table taxa described here are in bold. Unavailable sequences for individual indicated by —. Taxon Afroboletus luteolus Aureoboletus gentilis Aureoboletus moravicus Aureoboletus roxanae Aureoboletus thibetanus Austroboletus fusisporus Austroboletus gracilis Austroboletus Austroboletus Austroboletus Baorangia bicolor Baorangia pseudocalopus Binderoboletus segoi Boletellus ananas Boletellus ananas Boletellus chrysenteroides Boletellus dicymbophilus Boletellus Boletellus exiguus Boletellus longicollis Boletellus mirabilis Boletellus piakaii Boletellus shichianus Boletellus Boletellus Boletellus Boletellus Boletellus Boletellus Boletellus Boletinellus merulioides abruptibulbus

volume 6 · no. 2 299 300 ARTICLE

Table 1. (Continued).

Taxon Voucher ID Location GenBank accession number Collapsed clade in Fig. 1 28S RBP1 RBP2

Boletus aereus REH 8721 Redwood NP, CA, USA KF030339 KF030377 — Boletus clade Boletus aokii HKAS 59812 Wanling, Hainan, China KF112378 KF112597 — Boletus aff. aokii HKAS 52633 Yunnan, China KF112379 KF112598 KF112736 Boletus edulis HMJAU 4637 Kyrov, Russia KF112455 KF112586 KF112704 Boletus clade Boletus pallidus 179/97 Bavaria, Germany AF457409 KF030396 — Boletus projectellus AFTOL-713 MA, USA AY684158 AY788850 AY787218 Xerocomoideae Boletus pulchriceps DS 4514 Chiricahua Mtns, AZ, USA KF030261 KF030376 — Boletus punctilifer HKAS 52269 Kunming, Yunnan, China KF112385 KF112628 KF112773 Xerocomoideae Boletus reticuloceps HKAS 57671 Dêqên, Yunnan, China KF112454 KF112648 KF112703 Boletus clade Boletus roseopurpureus MB 06-059 Chestnut Ridge, NY, USA KF030262 KF030372 — Boletus rufomaculatus 4414 Chestnut Ridge, NY, USA KF030248 KF030369 — Boletus semigastroideus PBM 3076 Arataki VC, Auckland, NZ KF030352 KF030384 —

Boletus semigastroideus CSAK004 North Island, NZ AY253721 — — Smith etal. Boletus aff. speciosus HKAS59467 Baoshan, Yunnan, China KF112331 KF112517 KF112672 Boletus subalpinus 27882 - KF030340 KF030379 — Boletus clade Boletus aff. subtomentosus HKAS 58865 Dali, Yunnan, China KF112389 KF112630 KF112784 Xerocomoideae Boletus variipes 4249 Cheboygan Co., MI, USA JQ327014 KF030378 — Boletus clade Boletus violaceofuscus HKAS 62900 Chuxiong, Yunnan, China JN563859 JN563876 KF112762 Boletus sp. HKAS 52525 Ning’er, Yunnan, China KF112337 KF112514 KF112671 Boletus sp. HKAS 55373 Yunnan, China KF112362 KF112588 KF112804 Boletus sp. HKAS 57774 Lijiang, Yunnan, China KF112330 KF112513 KF112670 Boletus sp. HKAS 59660 Yunnan, China KF112358 KF112503 KF112664 Boletus sp. HKAS 59814 Baisha, Hunan, China KF112336 KF112546 KF112699 Borofutus dhakanus HKAS 73789 Gazipur, Bangladesh JQ928616 JQ928586 JQ928597 Bothia castanella MB 03-053 MA, USA DQ867117 KF030382 — Buchwaldoboletus lignicola HKAS 76674 Yichun, Heilongjiang, China KF112350 KF112642 KF112819 Butyriboletus appendiculatus Bap1 Bavaria, Germany AF456837 KF030359 — Butyriboletus roseoflavus HKAS 54099 Kunming, Yunnan, China KF739665 KF739741 KF739703 Caloboletus aff. calopus HKAS 74739 Dêqên, Yunnan, China KF112335 KF112507 KF112667 Caloboletus clade ima fUNGUS Caloboletus firmus MB 06-060 Chestnut Ridge, NY, USA KF030278 KF030368 — Caloboletus clade Caloboletus inedulis MB 06-044 Erie Co., NY, USA JQ327013 KF030362 — Caloboletus clade Caloboletus panniformis HKAS 55444 Dêqên, Yunnan, China KF112334 KF112506 KF112666 Caloboletus clade Caloboletus yunnanensis HKAS 74864 Nujiang, Yunnan, China KF112415 KF112508 KF112679 Caloboletus clade New sequestrate Boletaceae from Guyana ARTICLE clade s.s. clade s.s. clade s.s. clade clade clade clade clade clade outgroup taxa outgroup taxa Collapsed clade in Fig. 1 Caloboletus Xerocomoideae Xerocomoideae Boletales Boletales Xerocomoideae Imleria clade Imleria clade Imleria clade Lanmaoa Lanmaoa Lanmaoa Lanmaoa Lanmaoa Leccinum Leccinum RBP 2 LC053664 KF112668 — — KF112710 — — JN205455 KF112732 — — KF112675 — LC043083 — GU187786 KF112827 KF112792 KF112806 — — KF112707 KF112706 — KM605177 KF112682 KM605176 — KF112680 — KF112724 KF112723 KF112722 RBP 1 LC053663 KF112518 — — KF112533 GU187453 KF030364 — KF112552 — HQ161824 — HQ161843 LC043082 — GU187461 KF112644 KF112580 KF112618 — KF112609 KF112608 KF112607 LC053661 KM605166 KF112522 KM605165 KF030363 KF112521 KF030389 KF112593 KF112592 KF112590 G enBank accession number 28 S – KF112410 LC053662 KC155381 KF112435 DQ534648 KF030313 KF112440 KF112469 EU293063 HQ161855 JQ924342 HQ161874 LC043081 JX889673 AF098378 KF112478 KF112437 KF112347 EF183541 KF112375 KF112374 KF112373 LC053660 KM605139 KF112353 KM605143 JQ327001 KF112322 KF030347 KF112444 KF112443 KF112442 Location Region 8 Potaro- S iparuni, G uyana Sanming, Fujian, China Region 8 Potaro- S iparuni, G uyana Region 7 Cuyuni-Mazaruni, G uyana Chenzhou, Hunan, China MA, USA West Newton, MA, USA West Qiongzhong, Hainan, China Yunnan, China Yunnan, Malaysia San Gerardo, José, Costa Rica San Gerardo, José, Costa Rica Anoka, MN, USA Region 8 Potaro-Siparuni, Guyana SE Queensland, Australia SE Queensland, Bavaria, Germany Harbin, Heilongjiang, China Dêqên, Yunnan, China Yunnan, Dêqên, Chuxiong, Yunnan, China Yunnan, Chuxiong, Kyoto, Japan Marburg, Germany Ninger, Yunnan, China Yunnan, Ninger, Lijiang, Yunnan, China Yunnan, Lijiang, Region 8 Potaro- S iparuni, G uyana Gongshan, Yunnan, China Yunnan, Gongshan, Kunming, Yunnan, China Yunnan, Kunming, Nanhua, Yunnan, China Yunnan, Nanhua, Erie Co., NY, USA Erie Co., NY, Nujiang, Yunnan, China Yunnan, Nujiang, unknown Kunming, Yunnan, China Yunnan, Kunming, Yanbian, Jilin, China Yanbian, Qamdo, Tibet, China Tibet, Qamdo, Voucher ID Voucher Henkel 9061 HKAS 53353 Henkel 9067 Henkel 9514 HKAS 53424 MB 04-001 9606 HKAS 63126 HKAS 52601 REH 8692 BDCR 0418 NY 815462 NY BD391 Henkel 8848 REH 9455 REG Gl1 HKAS 76672 HKAS 50527 HKAS 52237 TNS-F-11696 HKAS 74714 HKAS 52557 HKAS 74712 Henkel 9163 HKAS 74752 HKAS 54094 HKAS 63603 MB 06-061 HKAS 74765 Buf 4507 HKAS 63502 HKAS 76669 HKAS 57266 1 scabrum sp. sp. aff. aff. Taxon Caloboletus Costatisporus caerulescens Castellanea pakaraimophila Crocinoboletus rufoaureus piperatus Cyanoboletus pulverulentus Corneroboletus indecorus Cyanoboletus Costatisporus caerulescens Durianella rambutanispora Exsudoporus frostii Exsudoporus frostii Frostiella russellii Guyanaporus albipodus boletoides Gyrodon lividus Gyroporus castaneus chromapes Heimioporus japonicus Heliogaster columellifer Imleria sp. Imleria sp. Jimtrappea guyanensis Lanmaoa angustispora Lanmaoa asiatica Lanmaoa asiatica Lanmaoa carminipes Lanmaoa flavorubra Leccinellum corsicum Leccinum aurantiacum Leccinum monticola Leccinum Table 1. (Continued). Table

volume 6 · no. 2 301 302 ARTICLE

Table 1. (Continued).

Taxon Voucher ID Location GenBank accession number Collapsed clade in Fig. 1 28S RBP1 RBP2

Leccinum subglabripes 72206 Jefferson, NH, USA KF030303 KF030374 — Xerocomoideae Leccinum variicolor HKAS 57758 Lijiang, Yunnan, China KF112445 KF112591 KF112725 Leccinum s.s. clade Mackintoshia persica Trappe 28216 Zimbabwe KC905034 — — Mucilopilus castaneiceps HKAS 75045 Nujiang, Yunnan, China KF112382 — KF112735 Mycoamaranthus congolensis v99-105 Mashonaland, Zimbabwe LC053665 — — brunneissimus HKAS 52660 Kunming, Yunnan, China KF112314 KF112492 KF112650 Neoboletus brunneissimus Neoboletus brunneissimus HKAS 57451 Jianchuan, Yunnan, China KM605137 KM605161 — Neoboletus brunneissimus Neoboletus magnificus HKAS 54096 Kunming, Yunnan, China KF112324 KF112495 KF112654 Neoboletus aff. luridiformis HKAS 55440 Dêqên, Yunnan, China KF112315 KF112499 KF112652 Neoboletus sinensis HKAS 53369 Sanming, Fujian, China KF112323 KF112509 KF112659 Neoboletus sinensis HKAS 76851 Changjiang, Hainan, China KF112321 KF112493 KF112651 Neoboletus thibetanus HKAS 57093 Nyingchi, Tibet, China KF112326 KF112496 KF112655 Octaviania japonimontana KPM-NC-0017812 Okayama Prefecture, Japan JN378486 — — Smith etal. Octaviania tasmanica OSC 132097 Tasmania, Australia JN378494 — — Paragyrodon sphaerosporus MB 06-066 Iowa, USA GU187593 — GU187803 Boletales outgroup taxa Parvixerocomus pseudoaokii HKAS 77032 Longnan, Jiangxi, China KP658467 KP658471 — Phlebopus portentosus php1 Africa AF336260 FJ536606 FJ536646 Boletales outgroup taxa Phlebopus aff. portentosus HKAS 52855 Yunnan, China JQ928622 KF112647 KF112822 Boletales outgroup taxa imbricatus HKAS 68642 Nujiang, Yunnan, China KF112398 KF112637 KF112786 Xerocomoideae Phylloporus luxiensis HKAS 75077 Chuxiong, Yunnan, China KF112490 KF112636 KF112785 Xerocomoideae Phylloporus pelletieri Pp1 Bavaria, Germany AF456818 KF030390 — Xerocomoideae Phylloporus rubrosquamosus HKAS 52552 Ninger, Yunnan, China KF112391 — KF112780 Xerocomoideae Porphyrellus holophaeus HKAS 74894 Baoshan, Yunnan, China KF112474 KF112554 — Pseudoboletus parasiticus xpa1 Bavaria, Germany AF050646 KF030394 — Pulveroboletus aff. ravenelii HKAS 53351 Sanming, Fujian, China KF112406 KF112542 KF112712 Pulveroboletus clade Pulveroboletus sp. HKAS 57665 Dêqên, Yunnan, China KF112409 KF112544 KF112715 Pulveroboletus clade Pulveroboletus sp. HKAS 58860 Dali,Yunnan, China KF112408 KF112543 KF112714 Pulveroboletus clade Pulveroboletus sp. HKAS 74933 Baoshan, Yunnan, China KF112407 KF112545 KF112713 Pulveroboletus clade Retiboletus griseus HKAS 63590 Dali, Yunnan, China KF112417 KF112537 KF112691 Retiboletus clade ima fUNGUS Retiboletus nigerrimus HKAS 59699 Chuxiong, Yunnan, China JQ928627 JQ928592 JQ928603 Retiboletus clade Retiboletus aff. ornatipes HKAS 63548 Lijiang, Yunnan, China KF112416 KF112536 KF112689 Retiboletus clade Rossbeevera vittatispora OSC 61484 New South Wales, Australia JN378506 — — Rossbeevera yunnanensis HKAS 70601 Gejiu, Yunnan, China KC552051 — KF112729 New sequestrate Boletaceae from Guyana ARTICLE clade clade clade clade clade clade clade clade clade clade clade outgroup taxa outgroup taxa Collapsed clade in Fig. 1 Rubroboletus Rubroboletus Rubroboletus Rubroboletus Rugiboletus Rugiboletus Rugiboletus Rugiboletus Xerocomoideae Strobilomyces Strobilomyces Strobilomyces Suillellus amygdalinus Suillellus amygdalinus Boletales Boletales eximius Sutorius eximius Sutorius eximius RBP 2 — KF112740 — — — KF112661 KM605168 KF112720 KM605170 KF112719 LC043089 — KF112754 — — KF112815 AY786065 KF112813 — KF112660 KF112823 KF112824 — — KF112803 KF112802 KF112737 KF112798 KF112797 KF112718 KF112688 KF112738 — RBP 1 — KF112619 KF030361 KP055026 KJ619482 KF112504 KM605158 KF112535 KM605159 KF112534 LC043088 LC043094 KF112561 — KF030387 KF112606 — KF112604 KF030360 KF112501 KF112645 KF112646 — — KF112585 KF112584 KF112575 KF112614 KF112613 KF112611 KF112539 KF112576 KF030395 G enBank accession number 28 S DQ534663 KF112458 KF030251 KP055023 KJ605673 KF112319 KM605134 KF112403 KM605135 KF112402 LC043087 LC043093 KF112361 JQ287643 EU685108 KF112479 AY684155 KF112461 JQ326996 KF112316 KF112429 KF112430 JQ327005 JQ327004 KF112400 KF112399 KF112411 KF112450 KF112449 KF112482 KF112405 KF112431 KF030350 Location Victoria, Australia Victoria, Chuxiong, Yunnan, China Yunnan, Chuxiong, Butner, NY, USA NY, Butner, Chongqing, China Deqin, Yunnan, China Yunnan, Deqin, Nujiang, Yunnan, China Yunnan, Nujiang, Linzhi, Xizang, China Chuxiong, Yunnan, China Yunnan, Chuxiong, Neixiang, Henan, China Dali, Yunnan, China Yunnan, Dali, Region 8 Potaro-Siparuni, Guyana Region 8 Potaro-Siparuni, Guyana Ninger, Yunnan, China Yunnan, Ninger, Fraser Island, Australia Fraser Island, Khao Yai Nat. Park, Thailand Nat. Park, Yai Khao Baoshan, Yunnan, China Yunnan, Baoshan, MA, USA Ninger, Yunnan, China Yunnan, Ninger, Mendocino Co., CA, USA Qamdo, Tibet, China Tibet, Qamdo, Chuxiong, Yunnan, China Yunnan, Chuxiong, Lijiang, Yunnan, China Yunnan, Lijiang, Fraser Island, Qld, Australia Fraser Island, Qld, Ulster County, NY, USA NY, Ulster County, Chuxiong, Yunnan, China Yunnan, Chuxiong, Kunming, Yunnan, China Yunnan, Kunming, Marburg, Germany Yunnan, China Yunnan, Chenzhou, Hunan, China Yanbian, Jilin, China Yanbian, Sanming, Fujian, China Yunnan, China Yunnan, Chestnut Ridge, NY, USA Chestnut Ridge, NY, Voucher ID Voucher AWC 4137 AWC HKAS 59700 JAM 0607 HKAS 80358 HKAS 56304 HKAS 68620 HKAS 83209 HKAS 63635 HKAS 76663 HKAS 68586 Henkel 9199 Henkel 9585 HKAS 50498 REH 9417 DED 7873 HKAS 59461 AFTOL-716 HKAS 55389 112605ba HKAS 57262 HKAS 57622 HKAS 57748 REH 9280 REH 9400 HKA S56291 HKA HKAS 52672 HKAS 54926 HKAS 59661 HKAS 53401 HKAS 76671 HKAS 53388 HKAS 50210 MB 06-056 verruculosus extremiorientalis seminudus aff. aff. aff. balloui eximius aff. aff. granulatus luteus aff. aff. rigens aff. aff. aff. aff. aff. Taxon Royoungia boletoides Tylopilus Rubroboletus dupainii Rubroboletus latisporus Rubroboletus sinicus Rubroboletus sinicus Rugiboletus brunneiporus Rugiboletus extremiorientalis Rugiboletus extremiorientalis Rugiboletus Singerocomus inundabilis Singerocomus rubriflavus Sinoboletus duplicatoporus Solioccasus polychromus Spongiforma thailandica Strobilomyces Strobilomyces strobilaceus Strobilomyces Suillellus amygdalinus Suillellus aff. amygdalinus Suillus Suillus Sutorius australiensis Sutorius eximius eximius Sutorius aff. aff. Sutorius aff. Tylopilus felleus Tylopilus Tylopilus microsporus Tylopilus Tylopilus otsuensis Tylopilus Tylopilus porphyrosporus Tylopilus Tylopilus Tylopilus Tylopilus plumbeoviolaceoides Tylopilus Tylopilus plumbeoviolaceus Tylopilus Table 1. (Continued). Table

volume 6 · no. 2 303 304 ARTICLE

Table 1. (Continued).

Taxon Voucher ID Location GenBank accession number Collapsed clade in Fig. 1 28S RBP1 RBP2

Tylopilus violatinctus HKAS 50208 Jinghong, Yunnan, China KF112472 KF112620 KF112799 Tylopilus virens HKAS 76678 Liangshan Yi, Sichuan, China KF112438 KF112582 KF112793 Tylopilus sp. HKAS 46334 Dêqên, Yunnan, China KF112471 KF112581 KF112795 Tylopilus sp. HKAS 50229 Yunnan, China KF112423 KF112574 KF112769 Tylopilus sp. HKAS 53367 Sanming, Fujian, China KF112439 KF112615 KF112790 Tylopilus sp. HKAS 55438 Dêqên, Yunnan, China KF112404 KF112538 KF112687 Tylopilus sp. HKAS 74925 Baoshan, Yunnan, China KF112473 KF112577 KF112739 Tylopilus sp. HKAS 74928 Baoshan, Yunnan, China KF112483 KF112583 KF112794 Veloporphyrellus alpinus HKAS 57490 Lijiang, Yunnan, China KF112380 KF112555 KF112733 Xanthoconium affine BD217 Giles, VA, USA HQ161854 HQ161823 — Xanthoconium clade Xanthoconium purpureum BD228 Macon, NC, USA HQ161864 HQ161833 — Xanthoconium clade Xanthoconium separans DPL 2704 TX, USA KF030329 KF030385 — Xanthoconium stramineum 3518 Gainesville, FL, USA KF030353 KF030386 — Xanthoconium clade Smith etal. Xerocomellus chrysenteron xch1 Bavaria, Germany AF050647 KF030365 — Xerocomellus clade Xerocomellus cisalpinus AT2005034 Upsala, Uppland, Finland KF030354 KF030367 — Xerocomellus clade Xerocomellus aff. rubellus HKAS 51239 Nyingchi, Tibet, China KF112425 KF112550 KF112695 Xerocomellus zelleri REH 8724 Redwood Nat. Park, CA, USA KF030271 KF030366 — Xerocomellus clade cyaneibrunnescens TH 8821 Region 8 Potaro-Siparuni, Guyana HQ161866 HQ161835 — Xerocomus aff. macrobbii HKAS 56280 Chuxiong, Yunnan, China KF112418 KF112541 KF112708 Xerocomus magniporus HKAS 58000 Qamdo, Tibet, China KF112392 KF112632 KF112781 Xerocomoideae erythrocephala HKAS 75046 Nujiang, Yunnan, China KF112414 KF112579 KF112791 1For the new taxon Costatisporus caerulescens, original data for 28S was derived from specimen Henkel 9067, and for RBP1 and RBP2 from Henkel 9061, and concatenated prior to analysis. These two specimens are conspecific morphologically and have identical ITS sequences. ima fUNGUS New sequestrate Boletaceae from Guyana

luteus served as Boletales outgroup taxa for the phylogenetic Type species: Jimtrappea guyanensis T.W. Henkel et al. ARTICLE analysis. Maximum likelihood analysis was performed on the 2015. concatenated 28S+RPB1+ RPB2 dataset, with inclusion of the taxa in which one or more of those loci were missing, with RAxML Jimtrappea guyanensis T.W. Henkel, M.E. Smith & on the CIPRES Science Gateway (www.phylo.org, Stamatakis Aime, sp. nov. 2006, Stamatakis et al. 2008). For this analysis the three codon MycoBank MB812360 positions were partitioned and evaluated separately and the (Figs 2–3, 4A) GTRGAMMA setting was used to determine the best ML tree and for rapid bootstrapping with 1000 replicates. Note that for Etymology: Guyana and –ensis (Latin adj. B) = adjectival the new taxon Costatisporus cyanescens, specimens Henkel suffix indicating origin or place; referring to the country of 9067 and Henkel 9061 had identical ITS rDNA sequences and known occurrence of the species. morphology. For the phylogenetic analysis we used 28S rDNA from Henkel 9067 and for RBP1 and RBP2 from Henkel 9061 Diagnosis: Reminiscent of a Tylopilus species, but lacking but treated them as a single terminal taxon. For the new taxon a and with a loculate gleba. Peridium pale, delicate. Castellanea pakaraimophila only ITS and 28S sequences were Basidiospores pinkish to reddish brown, smooth, and cystidia successfully obtained. For this taxon, only 28S was included in strongly dextrinoid in Melzer’s solution. the phylogenetic analysis. Type: Guyana: Region 8 Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, ~10 km southeast of RESULTS a base camp at 5°18’04.8” N 59°54’40.4” W, near Tadang camp, 20 cm deep within lateritic soil under Dicymbe BLASTn queries and phylogenetic analysis corymbosa, D. altsonii, and Aldina insignis, 29 Dec. 2009, ITS BLASTn queries of each of the new taxa on GenBank Henkel 9163 (BRG 41210 – holotype; HSU G1115, NY indicated affinities with Boletaceae at the family level, 02460742 – isotypes). GenBank accession numbers ITS and but were uninformative at the genus level, with none 28S: JN168684, LC053660; RPB1: LC053661. of the searches exceeding 89 % similarity with any ITS sequences in GenBank. The ML analysis of the combined Description: Basidiomata subglobose to ovate and irregularly 28S, RPB1, and RPB2 dataset produced a phylogram (-ln lobed, occasionally appearing fused, (6–)11–21 mm tall, = 57979.037507) with overall topology similar to that of (3–)8–29 mm broad, subfirm to soft and gelatinous with previously published studies (e.g. Wu et al. 2014) (Fig. 1). age; surface off-white to pale cream (4A1–4A2, 5A2) The new Guyanese taxa were placed within Boletaceae, throughout, unchanging with pressure or slightly browning, but none were nested within previously described genera, with occasional humic stains, glabrous macroscopically, including the boletoid sequestrate genera , under hand lens a tightly appressed hyphal mat; base Durianella, , Gastroleccinum, Heliogaster, subtended by delicate white hyphal cords and occasionally Mackintoshia, Mycoamaranthus, Octaviania, Rossbeevera, concolourous ectomycorrhizas. Peridium in longitudinal Royoungia, Solioccasus, or Spongiforma. section extremely thin (< 0.25 mm), light creamish white, single-layered, delicate, separable. Gleba nearly white (6A1) initially, with age light pink (6A2–6A3) to greyish pink (6B2– 6B3), eventually variably darker pink (6C4–7C4, 7D5, 8B3– 8B4), unchanging with exposure, moist, spongy, under hand Jimtrappea T.W. Henkel, M.E. Smith & Aime, gen. lens of compact, folded locules that gelatinize with maturity; nov. in longitudinal section columella a short basal structure 1–4 MycoBank MB812359 mm wide, off-white, gelatinous, opaque; upward-radiating (Figs 2–3, 4A) sterile veins short (1–2 mm), less evident at maturity. Odour faintly fragrant, clay-like. Taste slightly bitter, astringent. Etymology: The genus is named in honour of Dr. James “Jim” Macrochemical reactions not obtained. Trappe, the world’s foremost authority on sequestrate fungi. Peridium 94–200 μm thick, single-layered, of interwoven repent hyphae, these laterally branching, uninflated to Diagnosis: Distinguished from other Boletaceae by a inflated, 1–7 μm wide, occasionally swollen at septa, with combination of the following characters: Basidiomata irregular extracellular encrustations, granulose-guttulate, hypogeous to partially emergent, sequestrate, subglobose hyaline in KOH and H2O, interspersed with golden brown, to ovate. Peridium off-white, unchanging, glabrous, thin. opaque conductive hyphae; terminal cells cylindrocapitate, Gleba variously pink at maturity, unchanging, moist, infrequently subventricose, or rarely cylindrical or with loculate. Columella short, pad-like, with short sterile veins. distinct angles, occasionally with short side branches, 7–81 Basidiospores statismosporic, subfusiform, smooth, pinkish μm long, 2–16 μm wide at apex, 2–7 μm centrally, 2–6 μm to reddish brown, inamyloid, pedicellate. Basidia clavate. at base, with brownish yellow, densely granulose contents Hymenial cystidia cylindrical, lanceolate or ventricose, hyaline in KOH, and occasionally with globose, hyaline extracellular in KOH, dextrinoid in Melzer’s solution. Clamp connections encrustations and swollen at the basal septum. Glebal trama absent. hyaline, of tightly packed, parallel to slightly interwoven hyphae diverging toward ; hyphae cylindrical, volume 6 · no. 2 305 306 ARTICLE

non-Boletaceae with multiple species from the same higher taxon are collapsed into triangles for visual simplification as is a clade of outgroup taxa from several shown above the nodes. Other sequestrate taxa are indicated in bold with solid black dots preceding their binomials. Previously identified clades logenetic relationships of the of relationships logenetic on based 57979.037507) = (-ln phylogram (ML) likelihood Maximum Fig. 1. 77 0.05 lineages of 75 70 Boletales 83 71 Boletaceae 99 94 . 89 97 98 96 98 93 95 99 96 98 85 98 94 96 97 and new sequestrate Guyanese taxa (in red bold). ML bootstrap support values greater than 70 are 70 than greater values support bootstrap ML bold). red (in taxa Guyanese sequestrate new and 89

95

Baorangia bicolor bicolor Baorangia

Boletus 100 93 Boletus rufomaculatus rufomaculatus Boletus

Buchwaldoboletus lignicola lignicola Buchwaldoboletus Boletus 100

● amygdalinus Suillellus 100

Rubroboletus Baorangia pseudocalopus pseudocalopus Baorangia

100 frostii Exsudoporus

Pseudoboletus parasiticus parasiticus Pseudoboletus 100

N

Neoboletus

Lanmaoa Butyriboletus appendiculatus appendiculatus Butyriboletus Boletus Xerocomellus Neoboletus magnificus magnificus Neoboletus

e 88 Butyriboletus roseoflavus roseoflavus Butyriboletus Neoboletus 100 ● o sp.HKAS59660

Boletus roseopurpureus roseopurpureus Boletus Caloboletus

99 Neoboletus sinensis sinensis Neoboletus 100 b sp.HKAS57774 Neoboletus sinensis sinensis Neoboletus

87 o 98 H

98 Tylopilus

l Boletus e e Retiboletus

Chalciporus piperatus piperatus Chalciporus

100 Veloporphyrellus alpinus alpinus Veloporphyrellus l t Boletus sp.HKAS52525

Xerocomus cyaneibrunnescens cyaneibrunnescens Xerocomus i u 100

94 Mucilopilus castaneiceps castaneiceps Mucilopilus o

Tylopilus

Boletus pulchriceps pulchriceps Boletus Crocinoboletus rufoaureus rufoaureus Crocinoboletus Xerocomellus clade verulentus verulentus l pu Cyanoboletus s Austroboletus gracilis gracilis Austroboletus g ● t

clade a aff. 100 Gymnogaster boletoides boletoides Gymnogaster

99 ●

h Bothia castanella castanella Bothia 100 s Rugiboletus

MB 07-001

C

97 i brunneissimus

Binderoboletus segoi Binderoboletus t aff. b

Cyanoboletus Imleria Tylopilus felleus felleus Tylopilus 93 clade e

C 99

sp.HKAS55438

Boletus e 71

a Xerocomus

clade Parvixerocomus pseudoaokii pseudoaokii Parvixerocomus Singerocomus rubriflavus Singerocomus sp.HKAS59814 r

Pulveroboletus luridiformis Singerocomus inundabilis inundabilis Singerocomus Boletus aokii aokii Boletus c t Xanthoconium o ● s a

●

Tylopilus t s clade Boletus o 4414 aff. ● speciosus n 97

e t S 100

100 99 l Smith etal. u 74 u M a l

l

o M s

clade m a Harrya chromapes chromapes Harrya

Boletus t ● y

l

i Boletales Boletales

i rigens H aff. s n a Sutorius australiensis australiensis Sutorius

c

o e eximius Sutorius 87 aff. J Porphyrellus holophaeus holophaeus Porphyrellus HKAS 76674 c p

o

e K HKAS 54096 c l HKAS 53369 100 clade HKAS 75739 i l HKAS 76851 k

Xerocomoideae i a m c o

a A

f

Xpa1 Tylopilus plumbeoviolaceoides plumbeoviolaceoides Tylopilus Rubinoboletus 99 i s.s. clade e Leccinum

a m aff. sp.HKAS50229 n HKAS55440 r

p aokii Boletus violaceofuscus violaceofuscus Boletus porphyrosporus Tylopilus S t HKAS 54099 Zangia erythrocephala Zangia r u MB 06-059 rubellus s sp.HKAS52601 t r

HKAS59467

a o HKAS53388 5 Tylopilus sp.HKAS55373 u T a s a r HKAS 59812 DS 4514 Bap1 s

MB 03-053 MB04-001 ● s

c N a

p Tylopilus virens virens Tylopilus k 7

clade clade HKAS 54926 h Tylopilus macrobii Austroboletus p ● Xanthoconium separans separans Xanthoconium

HKAS52633 n a 0 p ● Afroboletus luteolus luteolus Afroboletus S 112/96

i y outgroup taxa a B

t HKAS 57490 9 o r

e Tylopilus - h

a R p

a Leccinellum corsicum corsicum Leccinellum o F 3 l HKAS 75045 S a Henkel8035 u y n RPB1, l o i HKAS51239

-

e

e c s.clade s p m g 1 Tylopilus e REH 9455 HKAS 53424

s

r h c t o ● Borofutus dhakanus dhakanus Borofutus Guyanaporus albipodus albipodus Guyanaporus 1 sp.HKAS53367 s u s s ● 9606 o u r

n

6 o i b s o Tylopilus microsporus microsporus Tylopilus Tylopilus otsuensis otsuensis Tylopilus c

c HKAS56280 p y g sp.HKAS74925

B 9 n s e TH8821 m HKAS 50527

e a R Henkel9585

a i h aff

6 g e o f

Boletus pallidus pallidus Boletus e n Henkel 9199

o o u sp.HKAS46334

T n i

o v l Strobilomyces l m e ● s y r

s . a r e e l 2, and 28S ribosomal DNA sequences depicting phy - depicting DNAsequences ribosomal 28S and RPB2, ● m e t a o

● REH9280 HKAS 77032 s.clade balloui i

r u R n

sp.HKAS74928 H O g n p H u a D a

s HKAS 76678

s a E s e p n v R t e s u 100 c i i s n e

g H s h s n r o i t HKAS 74894 t e t k 2 a i i

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HKAS75046 k 9 t a a s v m HKAS 62900 o H e a v e i 4 s b n HKAS59700 9 8 l i l t i a e l d 9 00-436 b i a 1 i e 9 2 9 HKAS 76671 g s o n n e e n 7 l - 1 p a 0 l l d 1 5 k u a e

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v t DPL 2704 Buf 4507 c

t r t e 7 o clade HKAS 50210 l r 5 4

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i Tylopilus plumbeoviolaceus plumbeoviolaceus Tylopilus HKAS 73789 179/97

● r d Tylopilus violatinctus violatinctus Tylopilus

HKAS 53401 h i s a O B D 1 d e m i

y 6 M n e E S s O HKAS 59661 3 Henkel 8848 u u a

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K P M - N C - ima fUNGUS 0 0 1 7 8 1 2

New sequestrate Boletaceae from Guyana ARTICLE

Fig. 2. Basidiomata of Jimtrappea guyanensis. A. Holotype (Henkel 9163). B–C. Longitudinal sections. B. Off-white immature gleba (Henkel 9540). C. Pink mature gleba (Henkel 9689). Bars = 10 mm.

volume 6 · no. 2 307 308 ARTICLE

H and KOH in maturity at brown reddish more brown, pinkish 0.25; ± 2.34 = Qm 1.88–2.83(–3.25), = Qr μm; 0.6 ± 6.9 × 1.6 ± 16.0 = (13–)14– (mean μm 6–8 × 18(–20) views, all in symmetrical bilaterally amygdaloid, statismosporic, smooth, subfusiform to fusiform, occasionally n uhmna rgo, –(9 μ wd, golden-brown, wide, μm opaque. 2–7(–9) region, subhymenial in frequent hyphae conductive guttulate-granulose; sparsely or evident not contents specimens; mature in separating and 2–8 μm, hyaline in KOH, thin-walled, often heavily gelatinized × 8–82 cells points; branch or septa at swollen infrequently ie, oiay o ueos seimt to tre o four μm. 1–2 × 1.5–2 even, straight, or basidium, per three, two, sterigmata numerous; to solitary sized, variably- refractive, guttules granulose-guttulate; opaque or H at μm and KOH 6–9(–12) in hyaline thin-walled, base, at μm (3–)4–6 apex, centre, at broad μm 7–12 long, μm 23–50 cylindrical, rarely or base, toward evenly tapering clavate, to subclavate age, with absent to rare increasingly specimens, branching subhymenial hyphae. dichotomously from arising cystidia and basidia basidioles, Microscopic features of features Microscopic Fig. 3. contrast. Bars=10µm. subcylindrical cystidia (in Melzer’s). cystidium. 2 2 O, inamyloid; sterigma detaching irregularly from basidium evident not contents solution; Melzer’s in unreactive O, Hymenium Four-sterigmate basidium with developing basidiospores. developing with basidium Four-sterigmate C. lining locules composed of a palisade of palisade a of composed locules lining Jimtrappea guyanensis Jimtrappea E. Dextrinoid, sublanceolate cystidium (in Melzer’s). Basidia n abundant in younger = 120), initially light initially 120), = Basidiospores (holotype; Smith etal. Henkel 9163 Henkel

Hymenium section showing basidia, basidiospores, and dextrinoid, and basidiospores, basidia, showing section Hymenium D. Mazaruni RiverBasinsofGuyana. dipterocarpacea n O ad H refractive and highly KOH and in grey faintly thin-walled, 4–6(–8) base, centre, at at μm μm 6–12 apex, at broad μm 5–9 long, or μm sublanceolate, (30–)34–72(–88) cylindroclavate, cylindrical, rarely subventricose, palisade, hymenial above projecting not subhymenium, lower from arising maturation, light layers. Cystidia irregular under short, of smooth surface nearly SEM under thick, microscopy, μm 0.3–0.9 wall base; and leaving a pedicel (0.5–)1–3.5(–5) μm long at basidiospore Dicymbe altsonii Dicymbe under soils sand white or lateritic on forests on in soil, interface mineral within deeper hypogeous layer or floor, forest the soil/humic mineral on semi-emergent Habit, habitat,anddistribution age. advanced Clamp connections with locules into deliquescing cytoplasm, refractive highly uniform, a later granulose-guttulate, initially ). Basidiospores. A. bnat n on seies ls feun with frequent less specimens, young in abundant F. Opaque, hyaline cystidium (in KOH). B–C,F = phase 2 , strongly O, kon rm h Upr oao n Upper and Potaro Upper the from known ; , D.corymbosa absent. Bisterigmate basidium, basidioles, and basidioles, basidium, Bisterigmate B. etiod n ezrs contents Melzer’s; in dextrinoid : , D. jenmanii oiay r n ml groups small in or Solitary , or Aldina insignis Aldina Pakaraimaea ima fUNGUS , New sequestrate Boletaceae from Guyana ARTICLE

Fig. 4. Scanning electron micrographs of basidiospores of new sequestrate taxa from Guyana. A. Jimtrappea guyanensis (holotype; Henkel 9163). B. Castellanea pakaraimophila (holotype; Henkel 9514 ). C. Costatisporus cyanescens (holotype; Henkel 9061). Bars A–B = 5 µm, C = 10 µm.

Additional specimens examined: Guyana: Region 8 Potaro-Siparuni: Jimtrappea guyanensis is micromorphologically most Pakaraima Mountains, Upper Potaro River Basin, ~1.5 km southwest similar to the tropical African monotypic sequestrate of base camp at 5°18’04.8” N 59°54’40.4” W, on Cathie’s Hill, in genus Mackintoshia, originally described as a member of lateritic soil under D. corymbosa, 12 June 2012, Aime 4891 (BRG Agaricales (Pacioni & Sharp 2000), but now known to belong 41211; PUL F2833; HSU G1118; GenBank accession number ITS: to Boletaceae based on ITS and 28S rDNA data (Fig. 1; KR261060); 100 m south-east of base camp near Dicymbe plot JP5, Nuhn et al. 2013, Tedersoo & Smith 2013). Mackintoshia in alluvial sand soil under D. corymbosa, 12 June 2015, Henkel 10077 persica is characterized by prominent cystidia, smooth (BRG 41221; HSU G1128). Region 7 Cuyuni-Mazaruni: Pakaraima basidiospores, and a putative symbiotic association with ECM Mountains, Upper Mazaruni River Basin, ~10 km west of Mt Caesalpinioideae (Pacioni & Sharp 2000). The subfusiform Ayanganna in vicinity of Pegaima savanna base camp at 5°26’21.3” basidiospores and dextrinoid cystidia of J. guyanensis contrast N 60°04’43.1” W, vicinity of base camp, in white sand soils under P. with the ellipsoid basidiospores and non-dextrinoid cystidia of dipterocarpacea and D. jenmanii, 25 Dec. 2010, Henkel 9540 (BRG M. persica (Castellano et al. 2000, Pacioni & Sharp 2000). 41212; HSU G1119); 27 Dec. 2010, Henkel 9555 (BRG 41213; HSU Although there is no bootstrap support for the placement of G1120); ~200 m south of base camp, in white sand soils under P. either M. persica or J. guyanensis in the phylogenetic analysis, dipterocarpacea and D. jenmanii, 1 June 2012, Henkel 9661 (BRG they were resolved in highly divergent clades and on relatively 41214; HSU G1121); 2 km south-west of base camp in Pakaraimaea long branches, suggesting no close relationship (Fig. 1). plot 2, in white sand soils under P. dipterocarpacea and D. jenmanii, Basidiospores of the Asian and Australasian genus 5 June 2012, Henkel 9689 (BRG 41215; HSU G1122). Rossbeevera are nearly smooth except for broad longitudinal ridges that give them a slight to distinct polar angularity that is Commentary: Jimtrappea guyanensis is recognized in lacking in J. guyanensis (Lebel et al. 2012, Orihara et al. 2012b). the field by the white peridium, unchanging tissues, pink, Rossbeevera species also lack cystidia and have basidiomata loculate gleba, and short columella. Micromorphologically that turn blue with exposure (Lebel et al. 2012, Orihara et al. J. guyanensis is distinguished by the smooth, subfusiform, 2012b). Additionally, Rossbeevera is phylogenetically distant reddish brown basidiospores and prominent dextrinoid from J. guyanensis (Fig. 1), resolving in a well-supported, cystidia. Smooth basidiospores are relatively rare among previously recovered “leccinoid” clade with other sequestrate sequestrate Boletaceae, and the dextrinoid cystidia of J. and non-sequestrate taxa (Nuhn et al. 2013, Wu et al. 2014). The guyanensis are unprecedented among sequestrate Boletales fusoid, smooth, pedicellate basidiospores of J. guyanensis also with smooth basidiospores (e.g. Dodge 1931, Smith & Singer resemble those of species of Hysterangium (Hysterangiales) 1959, Pegler et al. 1989, Pacioni & Sharp 2000, Lumyong which otherwise differ in having a dendroid columella, a dark et al. 2003, Nouhra et al. 2005, Yang et al. 2006, Desjardin greenish or brown gleba with gel-filled locules, and in lacking et al. 2008, Moreau et al. 2011, Moreau et al. 2013, Lebel cystidia (Castellano et al. 1989). et al. 2012, Orihara et al. 2012a, b, Trappe et al. 2013, Species in a few other temperate sequestrate genera of Hayward et al. 2014). In the phylogenetic analysis reported Boletales have large, fusoid, smooth basidiospores that could here, J. guyanensis was putatively related to the South- potentially be confused with those of J. guyanensis, but differ, East Asian sequestrate Durianella echinulata, albeit without in addition to lacking cystidia, in the following ways: Alpova bootstrap support (Fig. 1). These two species are distinct species have gel-filled locules and are associated primarily morphologically as D. echinulata is characterized by highly with Alnus; Melanogaster species have a black gleba with ornamented , a blue colour change upon exposure, gel-filled locules; Rhizopogon species have an olivaceous and a rough, warted peridium (Desjardin et al. 2008). to dark brown gleba and are associated with Pinaceae; and Additionally, J. guyanensis is putatively related to a cluster of species have a greenish brown gleba, a Tylopilus species, including the type species of that genus (T. dendroid columella, and are associated with Pinaceae hosts felleus), but without bootstrap support (Fig. 1). (Trappe et al. 2009). volume 6 · no. 2 309 310 ARTICLE

ture basidiospores. Microscopic features of features Microscopic Fig. 6. peridium ( 9514 of Basidiomata Fig. 5. Diagnosis Castellano, aworldauthorityonsequestratefungi. Etymology: (Figs 4A,5–6) MycoBank MB812361 Castellanea T.W. Henkel&M.E.Sm., yoeu t prily mret sqetae ovate, sequestrate, stipe. short emergent, a characters: partially with following to the hypogeous of combination M.E. Sm.2015. Typepakaraimophila species:Castellanea Cystidia pedicellate. dextrinoid, often brown, Basidiospores Columella thin. subglabrous, ). ogtdnl eto soig ihy odd lb, aal tiknd eiim ad hr sie (Henkel9670 stipe short and peridium, thickened basally gleba, folded highly showing section Longitudinal B. andclampconnectionsabsent. Henkel 9670 : hr, a-ie wt a ige trl vein. sterile single a with pad-like, short, The genus is named in honor of Dr. Michael A. Michael Dr. of honor in named is genus The itnuse fo other from Distinguished statismosporic, D. Glebal trama showing distinct mediostratum and strongly diverging lateral stratum. B–C = phase contrast. Bars = 10 µm. ). Bars=10mm. Castellanea pakaraimophila Castellanea rw, nhnig loculate. unchanging, brown, Gleba Peridium Castellanea pakaraimophila Castellanea subfusiform, smooth, yellowish rnebon unchanging, orange-brown, Basidia Boletacaeae gen. nov. . .. ekl & Henkel T.W. Dorsal view (left) and ventral views (middle, right) showing short stipe (holotype; stipe short showing right) (middle, views ventral and (left) view Dorsal A. subclavate. Basidiomata (holotype; y a by Smith etal.

Henkel 9514 11, Y 2673 ioye) Gnak accession GenBank isotypes). number ITSand28S:KC155381. – 02460743 NY G1116, 2010, Dec. 22 under soil sand white in camp, base of vicinity northern W, 60°04’43.1” N 5°26’21.3” of at camp west base savanna Pegaima of vicinity in km Mt Ayanganna ~10 Basin, River Mazaruni Upper Mountains, Type: often are that basidiospores dextrinoid andreleasedintetrads,absenceofcystidia. gleba, unchanging brown peridium, orange-brown to grey-orange the of combination Diagnosis with and basidiomata as species the of occurrence to reference Etymology: (Figs 4A,5–6) MycoBank MB812362 Castellanea pakaraimophila T.W. Henkel & M.E. Sm., sp. nov. Guyana: ). Basidiospores. A. : Differs from other known sequestrate taxa by the by taxa sequestrate known other from Differs Pakaraimaea BG 11 – ooye HSU holotype; – 41216 Henkel 9514(BRG Pakaraimaea dipterocarpacea Region 7Cuyuni-Mazaruni P. dipterocarpacea and Four-sterigmate basidia with ma- with basidia Four-sterigmate B–C. and –philus G. = oig in loving; = (Gk.) ). rnih brown Orangish C. Pakaraima : . ima fUNGUS D. jenmanii Henkel

, New sequestrate Boletaceae from Guyana

Description: Basidiomata irregularly flattened-ovate, 7–12 Commentary: Castellanea pakaraimophila is recognized in ARTICLE mm tall, 12–16 mm broad, subfirm, softer with age; surface the field by the ovate basidiomata, orange-brown peridium, light greyish orange (5A5–5B5–5B6) to orange-brown (7C8– dark brown loculate gleba, short stipe, unchanging tissues 7D8–7E8) with occasional darker humic stains, unchanging upon exposure, and association with P. dipterocarpacea. with pressure, glabrous macroscopically, under hand lens a Micromorphologically C. pakaraimophila is characterized dense repent mat of light orange hyphae, with age viscid to by the smooth, yellowish brown, frequently dextrinoid nearly glutinous; base subtended by a short, concolourous basidiospores that abscise in tetrads, and well- stipe, this 1.5 × 1.5 mm, with a single concolourous hyphal defined mediostratum of the glebal trama. Castellanea cord. Peridium in longitudinal section extremely thin over pakaraimophila has been confirmed as an ECM symbiont apical ¾ (< 0.25 mm), concolourous with the surface, over of P. dipterocarpacea based on analysis of ITS rDNA basal ¼ thickening to 0.75 mm and there off-white, single- sequences from ECM roots (Smith et al. 2013). In the layered, separable. Gleba dark brown (6E7–6F7, 7E7–7F7) phylogenetic analysis reported here, C. pakaraimophila is throughout, unchanging with exposure, of irregularly shaped putatively related to a cluster of Tylopilus species, including locules with interior surfaces minutely brownish hispid under the type species of the genus T. felleus, but without bootstrap hand lens; locule walls translucent-gelatinous; columella support (Fig. 1). arising from the thickened basal peridium, with a single Castellanea pakaraimophila is similar to Mackintoshia narrow gelatinous vein extending to apex. Odour slightly persica because both have dextrinoid or partially dextrinoid, of iodine; taste not obtained. Macrochemical reactions not light yellowish brown or ochraceous-yellow basidiospores obtained. (Castellano et al. 2000, Pacioni & Sharp 2000). However, Peridium 25–190 μm thick, single-layered, of tightly C. pakaraimophila differs from M. persica in its subfusiform interwoven, repent hyphae, yellowish brown in KOH and basidiospores released in tetrads and lack of cystidia (Pacioni

H2O, becoming more parallel and hyaline toward gleba; & Sharp 2000). Additionally, the glebal trama in M. persica individual hyphae 2–5 μm wide, thin-walled; terminal cells ranges from 200–330 μm wide, is gelatinous, and lacks a cylindrical to subcapitate, 19–36 × 2–3 μm. Glebal trama with distinct mediostratum. The protologue description of M. persica a distinct mediostratum and lateral stratum; mediostratum notes that basidium morphology changes with basidioma age hyaline in H2O and KOH, 12.4–29.6 μm wide, of parallel, in a manner similar to that seen in C. pakaraimophila (Pacioni slightly interwoven hyphae; individual hyphae 2–8 µm wide; & Sharp 2000). However, with C. pakaraimophila the basidia lateral stratum divergent at a right angle from mediostratum, become smaller and more angular with age, whereas the hyaline in H2O and KOH, grading imperceptibly into the initially clavate basidia of M. persica become long-utriform to densely interwoven subhymenium. Hymenium a palisade fusiform (Pacioni & Sharp 2000). The two species are also of tightly packed basidia and basidioles. Basidia faintly unrelated phylogenetically (Fig. 1). grey in H2O and KOH, changing in shape with maturity; in Some Rossbeevera species can nominally resemble developing basidiomata (e.g. Henkel 9670) subclavate, C. pakaraimophila because they have nearly smooth infrequently cylindro-clavate, rarely cylindrical, 36–54 μm basidiospores and lack cystidia. However, the basidiospores long, 6.0–12.5 μm broad at apex, 5.0–11.5 μm at centre, 5.0– of Rossbeevera are non-dextrinoid, individually abscised, 8.5 μm at base, thin-walled; sterigmata four, straight, 4–7.5 × slightly to distinctly longitudinally ridged, and barely 0.9–1.5 μm; in fully mature basidiomata (e.g. Henkel 9514) angular to stellate in polar view. In contrast, the dextrinoid consistently clavate, 20–25 μm long, with four short (~1 μm), basidiospores of C. pakaraimophila lack angularity and are highly reduced sterigmata. Basidiospores statismosporic, frequently abscised in tetrads. Basidiomata of Rossbeevera smooth, subfusiform, bilaterally symmetrical in all views, 12– species also undergo a blue or blackish colour change upon 18 × 5.5–8(–10) μm (mean = 14.7 ± 1.20 × 7.1 ± 0.91 μm; bruising or exposure (Lebel et al. 2012, Orihara et al. 2012b). Qr = (1.5–)1.9–2.7, Qm = 2.1 ± 0.25; n = 61), light yellowish Based on the basidiome colour and basidiospore shape, brown in H2O and KOH, often with one dextrinoid guttule, with C. pakaraimophila bears some resemblance to species of a short pedicel ± l μm long, frequently released in tetrads. Alpova () and Mycoamaranthus (Boletaceae). Cystidia and clamp connections absent. However, Alpova species have smaller basidiospores, a pseudoparenchymatous peridium, abundant clamp Habit, habitat and distribution: Solitary or in a small group connections, and are usually associated with Alnus (Dodge partially emergent on mineral soil/humic layer interface on 1931, Nouhra et al. 2005, Moreau et al. 2011, Moreau et al. 2013, the forest floor under P. dipterocarpacea, or immersed in Hayward et al. 2014). Species of Mycoamaranthus, though decaying wood humus at base of dead P. dipterocapacea; similar in peridial micromorphology to C. pakaraimophila, known only from the type locality in the Upper Mazaruni River have finely ornamented to spinulose basidiospores, a bright Basin of Guyana. yellow peridium, and are currently only known from Africa, South-East Asia, and Australasia (Castellano et al. 2000, Additional specimen examined: Guyana: Region 7 Cuyuni-Mazaruni: Lumyong et al. 2003). The fusoid, smooth, pedicellate Pakaraima Mountains, Upper Mazaruni River Basin, ~10 km west basidiospores of C. pakaraimophila also resemble those of of Mt Ayanganna in vicinity of Pegaima savanna base camp at species of Hysterangium (Hysterangiales) which otherwise 5°26’21.3” N 60°04’43.1” W, 150 m northeast of base camp, in wood differ in having a dendroid columella, a dark greenish or humus at base of dead P. dipterocarpacea, 3 June 2012, Henkel brown gleba, and in lacking cystidia (Castellano et al. 1989). 9670 (BRG 41217; HSU G1123). GenBank accession number ITS: Species in a few other temperate sequestrate genera of LC054831. Boletales have large, fusoid, smooth basidiospores that could volume 6 · no. 2 311 312 ARTICLE

M.E. Sm.2015. Type species:Costatisporuscaerulescens infrequent. pedicel discontinuous, clavate. Cystidiaandclampconnectionsabsent. or entire these pole, to pole ridges longitudinal of ornamentation costate with inamyloid, brown, light oblong, to subglobose statismosporic, with associated are and Pinaceae columella, dendroid a colours, brown, unchanging, loculate, sterile veins absent. Diagnosis: ridged ornamentationofthebasidiospores. distinctively the to reference in –spored; = adj. A) (L. sporus and with associated are and colours gleba brown dark 2009, under interface layer soil/humic mineral lateritic on solitary 1, plot Lance W,in 59°54’40.4” N 5°18’04.8” at camp base of southeast Mt km of 2.5 east Ayanganna, km ~15 Basin, River Potaro Upper Mountains, Type: and largebasidiosporeswithcostateornamentation. blue, brown acolumellate gleba, dark strong chocolate stains nutty odour, that peridium yellow greyish to off-white the by Diagnosis the of reaction auto-oxidation blue bruised peridium. dark the to referring Etymology: Costatisporus cyanescens Etymology: (Figs 4C,7–8) MycoBank MB812363 Costatisporus T.W. Henkel&M.E.Sm., with gel-filled locules; the following ways: Melanogaster in basidiospores, non-dextrinoid having to addition in differ, of those with confused be potentially tiig ak le garu t sboets, thin. subtomentose, to glabrous blue, dark staining sequestrate. emergent, partially to combination of the following characters: ih ml, osby netbae yohgs excavations, mycophagist invertebrate possibly in areas, small, with softer 5–10 firm, bruised, over or squeezed progressively where minutes and slowly stains 23D8–23E8) where 4B3) (23C8– blue deep increasingly developing soil, by unstained (4A3–4A4, yellow initially greyish surface to light broad; to mm off-white 12–33 tall, mm 12–26 lobed, Description KT447439; RPB1:LC053663; NY 02460744 – isotypes). GenBank accession numbers ITS: (Figs 4C,7–8) MycoBank MB812364 sp. nov. Truncocolumella Henkel 9061 Henkel Guyana: : (Trappe etal.2009).

: Basidiomata Easily differentiated from other sequestrate taxa sequestrate other from differentiated Easily Cyanescens itnuse fo other from Distinguished L aj A = ibd r igd and ridged or ribbed = A) adj. (L. Costatus Region 8 Potaro-Siparuni Region BG 11 – ooye HU G1117, HSU holotype; – 41218 (BRG Rhizopogon Rhizopogon pce hv gens bon gleba brown greenish have species L aj ) bcmn dr blue; dark becoming = A) adj. (L. ugooe o vt ad slightly and ovate to subglobose RPB2: LC053664. T.W.Sm., M.E. & Henkel species have a black gleba species have olivaceous to D. corymbosa rys yellow, greyish Peridium Basidiomata hypogeous C. pakaraimophila by a a by Boletacaeae

.. ekl & Henkel T.W. gen. nov. Basidiospores Pakaraima : 1 June 19 , Pinaceae Basidia Gleba , but , Smith etal. – ;

n H in hyaline mediostratum stratum; lateral and mediostratum of thin-walled. wide, μm 3–8 these hyphae, parallel to interwoven loosely of hyaline, thick, 50–250 μm layer inner encrustations; spiraled extracellular with ring-like frequently to thin-walled, µm, 5–10 × 20–92.5 clavate, hyaline to faintly grey in H in grey faintly to hyaline clavate, basidioles. and basidia of palisade a thin-walled. wide, μm yellow,5–9 pale to hyaline walled, grading into interwoven subhymenium hyphae, these yln i KH n H and KOH these in hyphae, hyaline interwoven loosely to parallel of junctions, 4.5–5.5 × 1–2 μm. four,or three sterigmata thin-walled; base, the at μm 2.5–5.8 centre, the at μm 3–9 apex, at broad μm 7.5–12.2 long, μm aaam Mutis Upr oao ie Bsn ~5 m at of east km ~15 Basin, River Potaro Upper Mountains, Pakaraima examined specimens Additional tau mdrtl t srnl dvrig 1–9 m thick; individual hyphae hyaline µm in H lateral 10–49 diverging, thin-walled; strongly to wide, moderately stratum µm 2–8 pigments, cytoplasmic ~8 kmdistantintheUpperPotaro RiverBasinofGuyana. soil/humic insignis mineral on floor, under forest the on emergent interface layer partially to hypogeous Habit, habitatanddistribution Cystidia andclampconnections 0.17; ± 1.69 = Qm 1.36–2.21, = Qr µm; 0.65 ± 10.0 × 1.32 ± 16.8 = (mean × 8–11.514–21 excluded, with ornamentation acuminate µm 0.20; ± 1.27 Qm = 1.00–1.80(–2.30), = Qr μm; 1.96 ± 15.9 × 1.41 ± 19.9 = (mean µm (10–)13–20 × 17–25 included, ornamentation nearly perpendicular cross-ridges, subglobose to oblong with discontinuous narrow,numerous with bifurcating, occasionally and shallow, or entire ridges pole; to pole running ridges main spiraled somewhat longitudinal, 5–10 of ornamentation n H in pigment cytoplasmic yellow with hyphae individual hyphae; interwoven tightly of brownish, to yellow dark thick, μm 415 and gleba;NH4OHnegativeonallsurfaces. Macrochemical reactions putty-like, or of mushroom buillon; strong, variously described as chocolate-nutty, musty, soapy, mm. 5 × 1 pad basal sterile gelatinous greyish, a with stains evident on glebal trama under hand lens, acolumellate, blue and down breaking structure locule with 1/6 outer over brown (9F4–9F5–9F6), with advanced age violet brown darker (10F5) and gelatinizing age with throughout, 8F8) 7F7–7F8, under hand lens, initially brown (7E8) to reddish brown (7F6– Gleba separable. specimens, younger in exposure on intensely but slowly bluing initially, white macroscopically, single-layered Peridium matted cords. hyphal several to lens one by subtended base viscid; hand a and brown dark to areas in gelatinizing age with tomentose, under macroscopically, glabrous light brown in H Peridium 2 2 ad O, 54 µ wd, fe sltig t locule at splitting often wide, µm 25–45 KOH, and O ad O, cainly yln; neclr cells intercalary hyaline; occasionally KOH, and O esl lclt wt bonhsi itro surfaces interior brown-hispid with loculate densely ; known only from the type locality and a second site second a and locality type the from only known ; in longitudinal section thin, 0.3–0.7 mm, appearing mm, 0.3–0.7 thin, section longitudinal in 150–615 μm thick, two-layered; outer layer 50– layer outer two-layered; thick, μm 150–615 = 100); pedicel infrequent, 0.5–4 × 1–2 µm. 1–2 × 0.5–4 infrequent, pedicel 100); = n 2 18, vt t sbuiom n basally and subfusiform to ovate 118), = n O and KOH, inamyloid, with complex costate Basidiospores Basidiospores 2 o ifeunl wt pl yellow pale with infrequently or O : KOH reddish brown on peridium peridium on brown reddish KOH : : Guyana: 2 O and KOH, 4–7 µm wide, thin- : absent. statismosporic, yellowish to oiay o ctee and scattered to Solitary taste Glebal trama composed Glebal 2 O and KOH, 39.0–66.4 KOH, and O Region 8 Potaro-Siparuni: Region Basidia indistinctive, fungoid. D. corymbosa or subclavate to subclavate ima fUNGUS Hymenium Odour A.

New sequestrate Boletaceae from Guyana ARTICLE

Fig. 7. Basidiomata of Costatisporus cyanescens. A. Unsectioned basidioma showing blue stains on bruised peridium (holotype; Henkel 9061). B. Longitudinal section showing mature dark brown gleba with gelatinization around margins (holotype; Henkel 9061). C. Longitudinal sections of three basidiomata showing dark blue peridial stains, mycophagist excavations (left) and glebal maturation (left to right) (Henkel 10100). Bars = 10 mm.

Mt Ayanganna, within 10 km radius of base camp at 5°18’04.8” N Guyanagaster plot 40, 21 June 2015, Aime 5850 (BRG 41224; HSU 59°54’40.4” W, ~8 km southeast of base camp on lateritic soil-leaf G1127; PUL F2871). litter interface under A. insignis, 25 July 2009, Henkel 9067 (BRG 41219; HSU G1124). GenBank accession number ITS and 28S: Commentary: Costatisporus cyanescens is recognized LC053662; ~1 km southeast of base camp immersed hypogeously in in the field by the blue-bruising peridium with occasional decaying wood humus under D. corymbosa on lateritic soils, 11 June mycophagist excavations, and dark brown to violet- 2015, Henkel 10060 (BRG 41222; HSU G1125); GenBank accession brown, finely loculate, acolumellate gleba that gelatinizes number ITS: KT380011; 16 June 2015, Henkel 10100 (BRG with maturity. Micromorphologically, the basidiospore 41223; HSU G1126); vicinity of base camp, under D. corymbosa, in ornamentation of longitudinal main and lateral secondary volume 6 · no. 2 313 314 ARTICLE

to that seenin of C.cyanescens ornamentation & Singer 1959). (Smith subridges intervening lack and margins rounded cyanescens of cyanescens Furthermore, cyanescens 2012b), contrasting with the 5–10 spiraled, acute ridges of (Lebel angularity polar stellate to slight their to contribute which ridges longitudinal broad Rossbeevera Lebel 1959, Orihara Singer 2012, upon & change (Smith colour exposure blackish or bruising or green, blue, a often undergo and basidiospores the of ridging longitudinal feature Asia, andChamonixia Australasia) al. 2008). exoperidial warts, and echinulate basidiospores (Desjardin exposure, but also have a well-developed columella, fibrillose upon change colour blue deep genus a undergo that basidiomata Asian tropical The with thatofC.cyanescensdid not indicate a close relationship. comparison of the ITS1 sequence from or (Pegler in observed cyanescens ridges secondary intervening the lack but occurs intheXerocomoideae with relationship phylogenetic close no has Boletellus of ornamentations basidiospore similar Fig. 8. Microscopic features of Sne 18, Mayor 1986, (Singer genus is bolete ornamentation epigeous the of species in basidiospore that to similar remarkably The distinctive. is ridges µm. basidiospores. oping RPB2 sequences were available for the genus Chamonixia lhuh h lniuial rde basidiospore ridged longitudinally the Although pce o te sequestrate the of Species h bsdoprs f ot-at sa sequestrate Asian South-East of basidiospores The , Boletellus which has no known sequestrate members et al. 1989, Yang r aprnl cicdna, as coincidental, apparently are bt hi lniuia rde ae tagt with straight are ridges longitudinal their but , (Fig. 1). The ridged basidiospore ornamentation basidiospore ridged 1). The (Fig. ae upe n ae mut, n dextrinoid and mounts, water in purple are , (Boletaceae ht mat cnitnl selt plr shape. polar stellate consistently a impart that Rossbeevera lacks intervening subridges and has 4–5 short, 2012b). Basidiospore ornamentation in ornamentation Basidiospore 2012b). et al. species superficially resembles that of that resembles superficially species Four-sterigmate basidium. Four-sterigmate C. 20, Halling 2008, et al. et al. 2006). Although no 28S, seis r lniuial ridged longitudinally are species ) ( is phylogenetically distant from Costatisporus cyanescens Costatisporus clade Durianella

genera 02 Orihara 2012, et al. er sm resemblance some bears (mostly North Temperate) (Fig.1; Table 1). ) andAustrogautieria Rhodactina incarnata Rhodactina Rossbeevera Costatisporus 05. The et al.2015). has sequestrate Boletellus C. cyanescens Peridium hyphae with ring-like external encrustations. B–D = phase contrast. Bars = 10 = Bars contrast. phase = B–D encrustations. external ring-like with hyphae Peridium D. Rhodactina (holotype; , which RPB1, (East et al. et al. Smith etal. and C. C. C. C. et ,

Henkel 9061 Henkel species ofNeoboletus (Wu 1) (Fig. genus epigeous mostly characterized, the recently to sister as supported is here indicated clade Sutorius features micromorphological (Fulgenzi different other many among basidiospores, fusiform smooth, and basidia, ballistosporic cinnamon-brown, bruise that hymenophores exposed with Sutorius cyanescens putatively the between on relationships light strong shed to clade emerging this within species from loci additional sequence to necessary be will it future the In peridium. yellow bright and stipe notable its from distinguished easily is it exposure, Americas, Australasia, and sympatrically with the in occurring species bolete epigeous purple, to maroon Costatisporus molecular-based analysis may reflect evolutionary reality, but (Fulgenzi Guyana in with overlaps Australian the species, one only margins, subacute with ridges basidiospore convergent apically have While 1934). Zeller & (Dodge peridium the in cells globose and columella, dendroid persistent a poles, the before terminate that ridges from Stewart & Trappe 1985). Additionally, colour change upon bruising or exposure (Zeller & Dodge (Hysterangiale 1918, the cyanescens Hosaka etal.2006). from distant phylogenetically intervening and lack Additionally, 1985). Trappe that & (Stewart basidiospores subridges ellipsoid more µm), differs fromC.cyanescens ridges (Stewart & Trappe 1985). of species other all (5–10); ridges n h pyoeei aayi peetd here, presented analysis phylogenetic the In Tylopilus eximius , as members of the C. caerulescens ). A. Basidiospores. etal. 2007, Halling pce hv rbs, iet-tptt basidiomata pileate-stipitate robust, have species . is well supported as sister to sister as supported well is 2014, 2015). While the sole sequestrate sole the While 2015). et al.2014, C. caerulescens Austrogautieria and s), no species of these s), nospecies Sutorius 07 Halling 2007, et al. in having gently rounded basidiospore rounded gently having in ope) wih s gns f dark of genus a is which complex), , B. Three-sterigmate basidium with devel san bu upon blue stains N. thibetanus, are very different morphologically. in the smaller (14–20 × 8–13 8–13 × (14–20 smaller the in et al. 2012). The pce ae clmlae and acolumellate are species Boletales Sutorius, Neoboletus in the number of basidiospore of number the in Austrogautieria manjipumana Austrogautieria Austroguatieria Gautieria genera undergo a blue (Giachini Phallomycetidae C. cyanescens (formerly Sutorius 02. The et al.2012). A. manjimupana C. cyanescens Costatisporus species differspecies ima fUNGUS Neoboletus et al. 2006, have 8–14 have Gautieria , andC. , are by C. - - ,

New sequestrate Boletaceae from Guyana

(Boletus section Boletus). Molecular Phylogenetics and Evolution ACKNOWLEDGEMENTS ARTICLE 57: 1276–1292. We thank the following funding sources: National Science Foundation Desjardin DE, Wilson AW, Binder M (2008) Durianella, a new (NSF) DEB-0918591 to T.W.H., NSF DEB-1354802 to M.E.S., NSF gasteroid genus of boletes from Malaysia. Mycologia 100: 956– DEB-0732968 to M.C.A., and the National Geographic Society’s 961. Committee for Research and Exploration to T.W.H. Additional funding Desjardin DE, Binder M, Roekring S, Flegel T (2009) Spongiforma, a for M.E.S. and K.O. was provided by the University of Florida’s new genus of gasteroid boletes from Thailand. Fungal Diversity Institute for Food and Agricultural Sciences (IFAS). Dillon Husbands 37: 1–8. functioned as Guyanese local counterpart and assisted with field Dodge CW (1931) Alpova, a new genus of Rhizopogonaceae, with collecting, descriptions, and specimen processing. Additional further notes on Leucogaster and Arcangeliella. Annals of the field assistance in Guyana was provided by Mei Lin Chin, Jessie Missouri Botanical Garden 18: 457–464. Uehling, Christopher Andrew, Valentino Joseph, Peter Joseph, Dodge CW, Zeller SM (1934) Hymenogaster and related genera. Francino Edmund, and Luciano Edmund. Jim Trappe provided useful Annals of the Missouri Botanical Garden 21: 625–708. discussions prior to description. Two reviewers provided valuable Edgar R (2004) MUSCLE: multiple sequence alignment with high comments on an earlier version of the manuscript. Research permits accuracy and high throughput. Nucleic Acids Research 32: were granted by the Guyana Environmental Protection Agency. 1792–1797. This paper is number 210 in the Smithsonian Institution’s Biological Fulgenzi TD, Henkel TW, Halling RE (2007) Tylopilus orsonianus sp. Diversity of the Guiana Shield Program publication series. nov. and Tylopilus eximius from Guyana. Mycologia 99: 622–627. 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