New sequestrate fungi from Guyana: Jimtrappea guyanensis gen. sp. nov., Castellanea pakaraimophila gen. sp. nov., and Costatisporus cyanescens gen. sp. nov. (Boletaceae, Boletales)

Abstract

Jimtrappea guyanensis gen. sp. nov., Castellanea pakaraimophila gen. sp. nov., and Costatisporus cyanescens gen. sp. nov. are described as new to science. These sequestrate, hypogeous fungi were collected in Guyana under closed canopy tropical forests in association with ectomycorrhizal (ECM) host tree genera Dicymbe (Fabaceae subfam. Caesalpinioideae), Aldina (Fabaceae subfam. Papilionoideae), and Pakaraimaea (Dipterocarpaceae). Molecular data place these fungi in Boletaceae (Boletales, Agaricomycetes, Basidiomycota) and inform their relationships to other known epigeous and sequestrate taxa within that family. Macro- and micromorphological characters, habitat, and multi-locus DNA sequence data are provided for each new taxon. Unique morphological features and a molecular phylogenetic analysis of 185 taxa across the order Boletales justify the recognition of the three new genera.

Introduction

Gasteroid fungi comprise a diverse, artificial assemblage of fungi within Agaricomycetes (Basidiomycota) that are functionally united in their enclosed hymenial development and lack of ballistospory. This informal group includes such charismatic macrofungi as puffballs, earthstars, false earthstars, earthballs, bird’s nest and cannonball fungi, stinkhorns, and false truffles (Ingold 1965, Miller & Miller 1988). These fungi had once been treated as a cohesive taxonomic unit (e.g. class Gasteromycetes) with the assumption that ballistospory was rarely lost in basidiomycete evolutionary history (e.g. Coker & Couch 1928) or that the sequestrate state was ancestral, predating the evolution of ballistospory (e.g. Singer 1971). Other authors regarded sequestrate basidiomycetes as a polyphyletic assemblage based on morphological and developmental evidence (e.g. Reijnders 1963, 2000, Heim 1971, Moore 1998).

Application of molecular techniques in mycology has since corroborated the latter view by discovering new sequestrate taxa in numerous family and genus-level lineages in Agaricomycetes, demonstrating that the sequestrate basidioma form has independently evolved multiple times (e.g. Bruns et al. 1989, Mueller & Pine 1994, Hibbett et al. 1997, Miller et al. 2000, Peintner et al. 2001, Miller & Aime 2001, Binder et al. 2006, Lebel & Tonkin 2007, Henkel et al. 2010, Gube & Dorfelt 2012, Lebel & Syme 2012, Ge & Smith 2013). Some sequestrate fungi resulted from recent, isolated evolutionary events that led to one or a few sequestrate species within a clade of non-sequestrate relatives (e.g. Kretzer & Bruns 1997, Martin et al. 2004, Giachini et al. 2006, Smith et al. 2006, Henkel et al. 2010) whereas other sequestrate clades of earlier origin have speciated and radiated across the globe (e.g. Grubisha et al. 2002, Binder & Hibbett 2006, Hosaka et al. 2006, Lebel et al. 2015). Understanding of the multiple origins and taxonomic affinities of sequestrate fungi has provided insight into the evolutionary forces that drastically alter basidioma form, function, and basidiospore dispersal (Thiers 1984, Kretzer & Bruns 1997, Reijnders 2000, Trappe & Claridge 2005, Albee-Scott 2007).

Knowledge of the diversity and distributions of sequestrate fungi has progressively advanced for some regions of the world (e.g. Bougher & Lebel 2001, Montecchi & Sarasini 2001, Trappe et al. 2009), but tropical sequestrate fungi remain especially poorly known. While some epigeous sequestrate fungi have recently been documented from the Brazilian Amazon (e.g. Cabral et al. 2014), Mueller et al. (2007) estimated that ~30 species of hypogeous sequestrate taxa are 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 diverse assemblage of sequestrate fungi in remote, primary tropical rain forests dominated by ectomycorrhizal (ECM) species of Dicymbe (Fabaceae subfam. Caesalpinioideae), Aldina (Fabaceae subfam. Papilionoideae), and Pakaraimaea (Dipterocarpaceae). These include epigeous and hypogeous taxa from diverse basidiomycetes and ascomycetes, including ECM-forming genera such as Hysterangium (Hysterangiales), Scleroderma and Tremellogaster (Boletales), and Pseudotulostoma and Elaphomyces (Eurotiales), as well as non-ECM genera such as Protubera (Hysterangiales), Guyanagaster (Agaricales), and Geastrum (Geastrales) (Miller et al. 2001, Henkel et al. 2010, 2012, Castellano et al. 2012, Henkel unpubl.).

Within Boletaceae (Boletales), numerous genera of sequestrate fungi have been recognized from various world regions, including: the widely distributed North Temperate Chamonixia and Octaviania; Australasian Rossbeevera; South-East Asian Durianella, Spongiforma, and Rhodactina; tropical African Mackintoshia; and Australian Soliocassus and Royoungia (Binder & Bresinski 2002, Desjardin et al. 2008, 2009, Lebel et al. 2012, Orihara et al. 2012a, b, Moreau et al. 2013, Trappe et al. 2013). However, despite a high diversity of non-sequestrate, epigeous Boletaceae species in certain regions of the lowland Neotropics (e.g. Singer et al. 1983, Henkel et al. 2012, 2015) there are very few reports of sequestrate Boletaceae from the region (Mueller et al. 2007, Tedersoo & Smith 2013). Here we rectify this situation by describing three new monotypic genera of sequestrate Boletaceae from the Pakaraima Mountains of Guyana. These fungi were collected from closed-canopy, wet rainforests dominated by ECM trees, an infrequent habitat type in the lowland Neotropics (Henkel 2003, Degagne et al. 2009, Smith et al. 2013). Molecular data from the ITS and 28S rDNA, RPB1, and RPB2 loci, along with morphological features, indicate that these Guyanese sequestrate fungi are members of Boletaceae but are evolutionarily distinct from all other described genera and species within the family.

Materials and Methods

Collections

Collections were made during the May–July rainy seasons of 2009, 2012, and 2015 from forests of the Upper Potaro River Basin, within a 15 km radius of a permanent base camp at 5°18’04.8” N 59°54’40.4” W, 710 m a.s.l. The collection sites were dominated by ECM Dicymbe corymbosa or co-dominated by ECM D. corymbosa, D. altsonii, and Aldina insignis (Smith et al. 2011, Henkel et al. 2012). Additional Guyana collections were made during Dec.–Jan. of 2010–2011 and June of 2012 from the Upper Mazaruni River Basin within a six km radius of a base camp at 5°26’21.3” N and 60°04’43.1” W, at 800 m a.s.l. Forests at this site were co-dominated by ECM Pakaraimaea dipterocarpacea and D. jenmanii (Smith et al. 2013).

Descriptions of macromorphological features were made from fresh material in the field. Colours were described subjectively and coded according to Kornerup & Wanscher (1978), with colour plates noted in parentheses. Fresh collections were dried using silica gel. Preserved specimens were later examined and imaged using an Olympus B×51 microscope with light and phase contrast optics. Rehydrated fungal tissues were mounted in H₂O, 3% potassium hydroxide (KOH), and Melzer’s solution. For basidiospores, basidia, hyphal features, and other structures in at least 20 individual structures were measured for each specimen examined. Length/width Q values for basidiospores are reported as Qr (range of Q values over “n” basidiospores measured) and Qm (mean of Q values ± SD). Scanning electron micrographs (SEM) of basidiospores were obtained with a FEI Quanta 250 scanning electron microscope using 20 kV. Type and additional specimens were deposited in the following herbaria: BRG, University of Guyana; HSU, Humboldt State University; PUL, Kriebel Herbarium, Purdue University; and NY, New York Botanical Garden.

DNA extraction, PCR amplification, and sequencing

DNA extractions were performed on basidioma tissue from types and additional specimens using the modified CTAB method (Gardes & Bruns 1993) or a Plant DNAeasy mini kit (QIAGEN, Valencia, CA). PCR and DNA sequencing of the nuc rDNA region encompassing the ITS 1 and 2, along with the 5.8S rDNA (ITS), nuc 28S rDNA D1–D2 domains (28S), the gene for RNA polymerase II largest subunit (RPB1) and second largest subunit (RPB2) followed the protocols and used the primers of Dentinger et al. (2010), Smith et al. (2011), and Wu et al. (2014). Newly generated sequences were edited in Sequencher v. 5.1 (Gene Codes, Ann Arbor, MI) and deposited in GenBank (Table 1).

Table 1 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 taxa described here are in bold. Unavailable sequences for individual taxa are indicated by —.

Fig. 1

Maximum likelihood (ML) phylogram (-ln = 57979.037507) based on RPB1, RPB2, and 28S ribosomal DNA sequences depicting phylogenetic relationships of the Boletaceae and new sequestrate Guyanese taxa (in red bold). ML bootstrap support values greater than 70 are shown above the nodes. Other sequestrate taxa are indicated in bold with solid black dots preceding their binomials. Previously identified clades with multiple species from the same higher taxon are collapsed into triangles for visual simplification as is a clade of outgroup taxa from several non-Boletaceae lineages of Boletales.

Taxa used, sequence alignment, and phylogenetic analysis

ITS ribosomal DNA sequences from each new species were initially subjected to BLASTn queries against GenBank in order to explore their putative phylogenetic relationships. In order to further assess their phylogenetic affinities, we used Maximum Likelihood (ML) of a concatenated dataset based on 28S, RPB1, and RPB2 sequences of diverse Boletaceae with additional Boletales taxa as outgroups. The analysis included original sequence data and additional sequences of 185 taxa from GenBank for representative species from infrafamilial clades across the family Boletaceae based on recent phylogenetic studies (e.g. Nuhn et al. 2013, Wu et al. 2014, 2015, Henkel et al. 2015). The type species and/or key representative taxa were included for as many epigeous, non-sequestrate Boletaceae genera or undescribed genus-level clades as possible (sensu Wu et al. 2014), contingent on their 28S, RPB1, and RPB2 sequences being available in GenBank. Representatives from numerous sequestrate Boletaceae taxa were also included, even if sequences were available only for one gene region. Sequences of 28S, RPB1, and RPB2 were compiled in separate nucleotide alignments of 1131 base pairs (28S), 978 base pairs (RPB1), and 903 base pairs (RPB2) using MEGA5 software (Tamura et al. 2011) and aligned with the aid of MAFFT v. 7 (Katoh & Standley 2013). The Gblocks software package (Talavera & Castresana 2007) was used to exclude ambiguous portions of the alignment, producing a final aligned dataset of 729 base pairs for 28S, 709 base pairs for RPB1, and 693 base pairs for RPB2. Boletinellus meruloides, Gyrodon lividus, Gyroporus castaneus, Phlebopus portentosus, Paragyrodon sphaerosporus, Suillus aff. granulatus, and S. aff. luteus served as Boletales outgroup taxa for the phylogenetic analysis. Maximum likelihood analysis was performed on the concatenated 28S+RPB1+ RPB2 dataset, with inclusion of the taxa in which one or more of those loci were missing, with RAxML on the CIPRES Science Gateway (https://doi.org/www.phylo.org, Stamatakis 2006, Stamatakis et al. 2008). For this analysis the three codon positions were partitioned and evaluated separately and the GTRGAMMA setting was used to determine the best ML tree and for rapid bootstrapping with 1000 replicates. Note that for the new taxon Costatisporus cyanescens, specimens Henkel 9067 and Henkel 9061 had identical ITS rDNA sequences and morphology. For the phylogenetic analysis we used 28S rDNA from Henkel 9067 and for RBP1 and RBP2 from Henkel 9061 but treated them as a single terminal taxon. For the new taxon Castellanea pakaraimophila only ITS and 28S sequences were successfully obtained. For this taxon, only 28S was included in the phylogenetic analysis.

Results

BLASTn queries and phylogenetic analysis

ITS BLASTn queries of each of the new taxa on GenBank indicated affinities with Boletaceae at the family level, but were uninformative at the genus level, with none of the searches exceeding 89% similarity with any ITS sequences in GenBank. The ML analysis of the combined 28S, RPB1, and RPB2 dataset produced a phylogram (-ln = 57979.037507) with overall topology similar to that of previously published studies (e.g. Wu et al. 2014) (Fig. 1). The new Guyanese taxa were placed within Boletaceae, but none were nested within previously described genera, including the boletoid sequestrate genera Chamonixia, Durianella, Gastroboletus, Gastroleccinum, Heliogaster, Mackintoshia, Mycoamaranthus, Octaviania, Rossbeevera, Royoungia, Solioccasus, or Spongiforma.

Taxonomy

Jimtrappea T.W. Henkel, M.E. Smith & Aime, gen. nov.

MycoBank MB812359

(Figs 2–3, 4A)

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.

Fig. 3

Microscopic features of Jimtrappea guyanensis (holotype; Henkel 9163). A. Basidiospores. B. Bisterigmate basidium, basidioles, and cystidium. C. Four-sterigmate basidium with developing basidiospores. D. Hymenium section showing basidia, basidiospores, and dextrinoid, subcylindrical cystidia (in Melzer’s). E. Dextrinoid, sublanceolate cystidium (in Melzer’s). F. Opaque, hyaline cystidium (in KOH). B–C,F = phase contrast. Bars = 10 µm.

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.

Etymology: The genus is named in honour of Dr. James “Jim” Trappe, the world’s foremost authority on sequestrate fungi.

Diagnosis: Distinguished from other Boletaceae by a combination of the following characters: Basidiomata hypogeous to partially emergent, sequestrate, subglobose to ovate. Peridium off-white, unchanging, glabrous, thin. Gleba variously pink at maturity, unchanging, moist, loculate. Columella short, pad-like, with short sterile veins. Basidiospores statismosporic, subfusiform, smooth, pinkish to reddish brown, inamyloid, pedicellate. Basidia clavate. Hymenial cystidia cylindrical, lanceolate or ventricose, hyaline in KOH, dextrinoid in Melzer’s solution. Clamp connections absent.

Type species: Jimtrappea guyanensis T.W. Henkel et al. 2015.

Jimtrappea guyanensis T.W. Henkel, M.E. Smith & Aime, sp. nov.

MycoBank MB812360

(Figs 2–3, 4A)

Etymology: Guyana and —ensis (Latin adj. B) = adjectival suffix indicating origin or place; referring to the country of known occurrence of the species.

Diagnosis: Reminiscent of a Tylopilus species, but lacking a stipe and with a loculate gleba. Peridium pale, delicate. Basidiospores pinkish to reddish brown, smooth, and cystidia strongly dextrinoid in Melzer’s solution.

Type: Guyana: Region 8 Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, ~10 km southeast of 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 corymbosa, D. altsonii, and Aldina insignis, 29 Dec. 2009, Henkel 9163 (BRG 41210 — holotype; HSU G1115, NY 02460742 — isotypes). GenBank accession numbers ITS and 28S: JN168684, LC053660; RPB1: LC053661.

Description: Basidiomata subglobose to ovate and irregularly lobed, occasionally appearing fused, (6–)11–21 mm tall, (3–)8–29 mm broad, subfirm to soft and gelatinous with age; surface off-white to pale cream (4A1–4A2, 5A2) throughout, unchanging with pressure or slightly browning, with occasional humic stains, glabrous macroscopically, under hand lens a tightly appressed hyphal mat; base subtended by delicate white hyphal cords and occasionally concolourous ectomycorrhizas. Peridium in longitudinal 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 lens of compact, folded locules that gelatinize with maturity; in longitudinal section columella a short basal structure 1–4 mm wide, off-white, gelatinous, opaque; upward-radiating sterile veins short (1–2 mm), less evident at maturity. Odour faintly fragrant, clay-like. Taste slightly bitter, astringent. Macrochemical reactions not obtained.

Peridium 94–200 µm thick, single-layered, of interwoven repent hyphae, these laterally branching, uninflated to inflated, 1–7 µm wide, occasionally swollen at septa, with irregular extracellular encrustations, granulose-guttulate, hyaline in KOH and H₂O, interspersed with golden brown, opaque conductive hyphae; terminal cells cylindrocapitate, infrequently subventricose, or rarely cylindrical or with distinct angles, occasionally with short side branches, 7–81 µm long, 2–16 µm wide at apex, 2–7 µm centrally, 2–6 µm at base, with brownish yellow, densely granulose contents in KOH, and occasionally with globose, hyaline extracellular encrustations and swollen at the basal septum. Glebal trama hyaline, of tightly packed, parallel to slightly interwoven hyphae diverging toward hymenium; hyphae cylindrical, infrequently swollen at septa or branch points; cells 8–82 × 2–8 µm, hyaline in KOH, thin-walled, often heavily gelatinized and separating in mature specimens; contents not evident or sparsely guttulate-granulose; conductive hyphae frequent in subhymenial region, 2–7(–9) µm wide, golden-brown, opaque. Hymenium lining locules composed of a palisade of basidioles, basidia and cystidia arising from dichotomously branching subhymenial hyphae. Basidia abundant in younger specimens, increasingly rare to absent with age, subclavate to clavate, tapering evenly toward base, or rarely cylindrical, 23–50 µm long, 7–12 µm broad at apex, 6–9(–12) µm at centre, (3–)4–6 µm at base, thin-walled, hyaline in KOH and H₂O, unreactive in Melzer’s solution; contents not evident or opaque granulose-guttulate; guttules refractive, variablysized, solitary to numerous; sterigmata two, three, or four per basidium, straight, even, 1.5–2 × 1–2 µm. Basidiospores statismosporic, smooth, subfusiform to fusiform, occasionally amygdaloid, bilaterally symmetrical in all views, (13–)14–18(–20) × 6–8 µm (mean = 16.0 ± 1.6 × 6.9 ± 0.6 µm; Qr = 1.88–2.83(–3.25), Qm = 2.34 ± 0.25; n = 120), initially light pinkish brown, more reddish brown at maturity in KOH and H₂O, inamyloid; sterigma detaching irregularly from basidium and leaving a pedicel (0.5–)1–3.5(–5) µm long at basidiospore base; wall 0.3–0.9 µm thick, nearly smooth under light microscopy, under SEM surface of short, irregular layers. Cystidia abundant in young specimens, less frequent with maturation, arising from lower subhymenium, not projecting above hymenial palisade, cylindrical, sublanceolate, or subventricose, rarely cylindroclavate, (30–)34–72(–88) µm long, 5–9 µm broad at apex, 6–12 µm at centre, 4–6(–8) µm at base, thin-walled, faintly grey and highly refractive in KOH and H₂O, strongly dextrinoid in Melzer’s; contents initially granulose-guttulate, later a uniform, highly refractive cytoplasm, deliquescing into locules with advanced age. Clamp connections absent.

Habit, habitat, and distribution: Solitary or in small groups semi-emergent on mineral soil/humic layer interface on the forest floor, or hypogeous deeper within mineral soil, in forests on lateritic or white sand soils under Aldina insignis, Dicymbe altsonii, D. corymbosa, D. jenmanii, or Pakaraimaea dipterocarpacea; known from the Upper Potaro and Upper Mazaruni River Basins of Guyana.

Additional specimens examined: Guyana: Region 8 Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, ~1.5 km southwest of base camp at 5°18’04.8” N 59°54’40.4” W, on Cathie’s Hill, in lateritic soil under D. corymbosa, 12 June 2012, Aime 4891 (BRG 41211; PUL F2833; HSU G1118; GenBank accession number ITS: KR261060); 100 m south-east of base camp near Dicymbe plot JP5, in alluvial sand soil under D. corymbosa, 12 June 2015, Henkel 10077 (BRG 41221; HSU G1128). Region 7 Cuyuni-Mazaruni: Pakaraima Mountains, Upper Mazaruni River Basin, ~10 km west of Mt Ayanganna in vicinity of Pegaima savanna base camp at 5°26’21.3” N 60°04’43.1” W, vicinity of base camp, in white sand soils under P. dipterocarpacea and D. jenmanii, 25 Dec. 2010, Henkel 9540 (BRG 41212; HSU G1119); 27 Dec. 2010, Henkel 9555 (BRG 41213; HSU G1120); ~200 m south of base camp, in white sand soils under P. dipterocarpacea and D. jenmanii, 1 June 2012, Henkel 9661 (BRG 41214; HSU G1121); 2 km south-west of base camp in Pakaraimaea plot 2, in white sand soils under P. dipterocarpacea and D. jenmanii, 5 June 2012, Henkel 9689 (BRG 41215; HSU G1122).

Commentary: Jimtrappea guyanensis is recognized in the field by the white peridium, unchanging tissues, pink, loculate gleba, and short columella. Micromorphologically J. guyanensis is distinguished by the smooth, subfusiform, reddish brown basidiospores and prominent dextrinoid cystidia. Smooth basidiospores are relatively rare among sequestrate Boletaceae, and the dextrinoid cystidia of J. guyanensis are unprecedented among sequestrate Boletales with smooth basidiospores (e.g. Dodge 1931, Smith & Singer 1959, Pegler et al. 1989, Pacioni & Sharp 2000, Lumyong et al. 2003, Nouhra et al. 2005, Yang et al. 2006, Desjardin et al. 2008, Moreau et al. 2011, Moreau et al. 2013, Lebel et al. 2012, Orihara et al. 2012a, b, Trappe et al. 2013, Hayward et al. 2014). In the phylogenetic analysis reported here, J. guyanensis was putatively related to the South-East Asian sequestrate Durianella echinulata, albeit without bootstrap support (Fig. 1). These two species are distinct morphologically as D. echinulata is characterized by highly ornamented spores, a blue colour change upon exposure, and a rough, warted peridium (Desjardin et al. 2008). Additionally, J. guyanensis is putatively related to a cluster of Tylopilus species, including the type species of that genus (T. felleus), but without bootstrap support (Fig. 1).

Jimtrappea guyanensis is micromorphologically most similar to the tropical African monotypic sequestrate genus Mackintoshia, originally described as a member of Agaricales (Pacioni & Sharp 2000), but now known to belong to Boletaceae based on ITS and 28S rDNA data (Fig. 1; Nuhn et al. 2013, Tedersoo & Smith 2013). Mackintoshia persica is characterized by prominent cystidia, smooth basidiospores, and a putative symbiotic association with ECM Caesalpinioideae (Pacioni & Sharp 2000). The subfusiform basidiospores and dextrinoid cystidia of J. guyanensis contrast with the ellipsoid basidiospores and non-dextrinoid cystidia of M. persica (Castellano et al. 2000, Pacioni & Sharp 2000). Although there is no bootstrap support for the placement of either M. persica or J. guyanensis in the phylogenetic analysis, they were resolved in highly divergent clades and on relatively long branches, suggesting no close relationship (Fig. 1).

Basidiospores of the Asian and Australasian genus Rossbeevera are nearly smooth except for broad longitudinal ridges that give them a slight to distinct polar angularity that is lacking in J. guyanensis (Lebel et al. 2012, Orihara et al. 2012b). Rossbeevera species also lack cystidia and have basidiomata that turn blue with exposure (Lebel et al. 2012, Orihara et al. 2012b). Additionally, Rossbeevera is phylogenetically distant from J. guyanensis (Fig. 1), resolving in a well-supported, previously recovered “leccinoid” clade with other sequestrate and non-sequestrate taxa (Nuhn et al. 2013, Wu et al. 2014). The fusoid, smooth, pedicellate basidiospores of J. guyanensis also resemble those of species of Hysterangium (Hysterangiales) which otherwise differ in having a dendroid columella, a dark greenish or brown gleba with gel-filled locules, and in lacking cystidia (Castellano et al. 1989).

Species in a few other temperate sequestrate genera of Boletales have large, fusoid, smooth basidiospores that could potentially be confused with those of J. guyanensis, but differ, in addition to lacking cystidia, in the following ways: Alpova species have gel-filled locules and are associated primarily with Alnus; Melanogaster species have a black gleba with gel-filled locules; Rhizopogon species have an olivaceous to dark brown gleba and are associated with Pinaceae; and Truncocolumella species have a greenish brown gleba, a dendroid columella, and are associated with Pinaceae hosts (Trappe et al. 2009).

Castellanea pakaraimophila T.W. Henkel & M.E. Sm., gen. nov.

MycoBank MB812361

(Figs 4A, 5–6)

Fig. 5

Basidiomata of Castellanea pakaraimophila. A. Dorsal view (left) and ventral views (middle, right) showing short stipe (holotype; Henkel 9514). B. Longitudinal section showing highly folded gleba, basally thickened peridium, and short stipe (Henkel 9670). C. Orangish brown peridium (Henkel 9670). Bars = 10 mm.

Fig. 6

Microscopic features of Castellanea pakaraimophila (holotype; Henkel 9514). A. Basidiospores. B–C. Four-sterigmate basidia with mature basidiospores. D. Glebal trama showing distinct mediostratum and strongly diverging lateral stratum. B–C = phase contrast. Bars = 10 µm.

Etymology: The genus is named in honor of Dr. Michael A. Castellano, a world authority on sequestrate fungi.

Diagnosis: Distinguished from other Boletacaeae by a combination of the following characters: Basidiomata hypogeous to partially emergent, sequestrate, ovate, with a short stipe. Peridium orange-brown, unchanging, subglabrous, thin. Gleba brown, unchanging, loculate. Columella short, pad-like, with a single sterile vein. Basidiospores statismosporic, subfusiform, smooth, yellowish brown, often dextrinoid, pedicellate. Basidia subclavate. Cystidia and clamp connections absent.

Type species: Castellanea pakaraimophila T.W. Henkel & M.E. Sm. 2015.

Castellanea pakaraimophila T.W. Henkel & M.E. Sm., sp. nov.

MycoBank MB812362

(Figs 4A, 5–6)

Etymology: Pakaraimaea and —philus (Gk.) = loving; in reference to occurrence of the species as basidiomata and mycorrhizas with Pakaraimaea dipterocarpacea.

Diagnosis: Differs from other known sequestrate taxa by the combination of the grey-orange to orange-brown peridium, brown unchanging gleba, basidiospores that are often dextrinoid and released in tetrads, and absence of cystidia.

Type: Guyana: Region 7 Cuyuni-Mazaruni: Pakaraima Mountains, Upper Mazaruni River Basin, ~10 km west of Mt Ayanganna in vicinity of Pegaima savanna base camp at 5°26’21.3” N 60°04’43.1” W, northern vicinity of base camp, in white sand soil under P. dipterocarpacea and D. jenmanii, 22 Dec. 2010, Henkel 9514 (BRG 41216 — holotype; HSU G1116, NY 02460743 — isotypes). GenBank accession number ITS and 28S: KC155381.

Description: Basidiomata irregularly flattened-ovate, 7–12 mm tall, 12–16 mm broad, subfirm, softer with age; surface light greyish orange (5A5–5B5–5B6) to orange-brown (7C8–7D8–7E8) with occasional darker humic stains, unchanging with pressure, glabrous macroscopically, under hand lens a dense repent mat of light orange hyphae, with age viscid to nearly glutinous; base subtended by a short, concolourous stipe, this 1.5 × 1.5 mm, with a single concolourous hyphal cord. Peridium in longitudinal section extremely thin over apical ¾ (< 0.25 mm), concolourous with the surface, over basal ¼ thickening to 0.75 mm and there off-white, single-layered, separable. Gleba dark brown (6E7–6F7, 7E7–7F7) throughout, unchanging with exposure, of irregularly shaped locules with interior surfaces minutely brownish hispid under hand lens; locule walls translucent-gelatinous; columella arising from the thickened basal peridium, with a single narrow gelatinous vein extending to apex. Odour slightly of iodine; taste not obtained. Macrochemical reactions not obtained.

Peridium 25–190 µm thick, single-layered, of tightly interwoven, repent hyphae, yellowish brown in KOH and H₂O, becoming more parallel and hyaline toward gleba; individual hyphae 2–5 µm wide, thin-walled; terminal cells cylindrical to subcapitate, 19–36 × 2–3 µm. Glebal trama with a distinct mediostratum and lateral stratum; mediostratum hyaline in H₂O and KOH, 12.4–29.6 µm wide, of parallel, slightly interwoven hyphae; individual hyphae 2–8 µm wide; lateral stratum divergent at a right angle from mediostratum, hyaline in H₂O and KOH, grading imperceptibly into the densely interwoven subhymenium. Hymenium a palisade of tightly packed basidia and basidioles. Basidia faintly grey in H₂O and KOH, changing in shape with maturity; in developing basidiomata (e.g. Henkel 9670) subclavate, infrequently cylindro-clavate, rarely cylindrical, 36–54 µm long, 6.0–12.5 µm broad at apex, 5.0–11.5 µm at centre, 5.0–8.5 µm at base, thin-walled; sterigmata four, straight, 4–7.5 × 0.9–1.5 µm; in fully mature basidiomata (e.g. Henkel 9514) consistently clavate, 20–25 µm long, with four short (~1 µm), highly reduced sterigmata. Basidiospores statismosporic, smooth, subfusiform, bilaterally symmetrical in all views, 12–18 × 5.5–8(–10) µm (mean = 14.7 ± 1.20 x 7.1 ± 0.91 µm; Qr = (1.5–)1.9–2.7, Qm = 2.1 ± 0.25; n = 61), light yellowish brown in H₂O and KOH, often with one dextrinoid guttule, with a short pedicel ± l µm long, frequently released in tetrads. Cystidia and clamp connections absent.

Habit, habitat and distribution: Solitary or in a small group partially emergent on mineral soil/humic layer interface on the forest floor under P. dipterocarpacea, or immersed in decaying wood humus at base of dead P. dipterocapacea; known only from the type locality in the Upper Mazaruni River Basin of Guyana.

Additional specimen examined: Guyana: Region 7 Cuyuni-Mazaruni: Pakaraima Mountains, Upper Mazaruni River Basin, ~10 km west of Mt Ayanganna in vicinity of Pegaima savanna base camp at 5°26’21.3” N 60°04’43.1” W, 150 m northeast of base camp, in wood humus at base of dead P. dipterocarpacea, 3 June 2012, Henkel 9670 (BRG 41217; HSU G1123). GenBank accession number ITS: LC054831.

Commentary: Castellanea pakaraimophila is recognized in the field by the ovate basidiomata, orange-brown peridium, dark brown loculate gleba, short stipe, unchanging tissues upon exposure, and association with P. dipterocarpacea. Micromorphologically C. pakaraimophila is characterized by the smooth, yellowish brown, frequently dextrinoid basidiospores that abscise in tetrads, and well-defined mediostratum of the glebal trama. Castellanea pakaraimophila has been confirmed as an ECM symbiont of P. dipterocarpacea based on analysis of ITS rDNA sequences from ECM roots (Smith et al. 2013). In the phylogenetic analysis reported here, C. pakaraimophila is putatively related to a cluster of Tylopilus species, including the type species of the genus T. felleus, but without bootstrap support (Fig. 1).

Castellanea pakaraimophila is similar to Mackintoshia persica because both have dextrinoid or partially dextrinoid, light yellowish brown or ochraceous-yellow basidiospores (Castellano et al. 2000, Pacioni & Sharp 2000). However, C. pakaraimophila differs from M. persica in its subfusiform basidiospores released in tetrads and lack of cystidia (Pacioni & Sharp 2000). Additionally, the glebal trama in M. persica ranges from 200–330 µm wide, is gelatinous, and lacks a distinct mediostratum. The protologue description of M. persica notes that basidium morphology changes with basidioma age in a manner similar to that seen in C. pakaraimophila (Pacioni & Sharp 2000). However, with C. pakaraimophila the basidia become smaller and more angular with age, whereas the initially clavate basidia of M. persica become long-utriform to fusiform (Pacioni & Sharp 2000). The two species are also unrelated phylogenetically (Fig. 1).

Some Rossbeevera species can nominally resemble C. pakaraimophila because they have nearly smooth basidiospores and lack cystidia. However, the basidiospores of Rossbeevera are non-dextrinoid, individually abscised, slightly to distinctly longitudinally ridged, and barely angular to stellate in polar view. In contrast, the dextrinoid basidiospores of C. pakaraimophila lack angularity and are frequently abscised in tetrads. Basidiomata of Rossbeevera species also undergo a blue or blackish colour change upon bruising or exposure (Lebel et al. 2012, Orihara et al. 2012b).

Based on the basidiome colour and basidiospore shape, C. pakaraimophila bears some resemblance to species of Alpova (Paxillaceae) and Mycoamaranthus (Boletaceae). However, Alpova species have smaller basidiospores, a pseudoparenchymatous peridium, abundant clamp connections, and are usually associated with Alnus (Dodge 1931, Nouhra et al. 2005, Moreau et al. 2011, Moreau et al. 2013, Hayward et al. 2014). Species of Mycoamaranthus, though similar in peridial micromorphology to C. pakaraimophila, have finely ornamented to spinulose basidiospores, a bright yellow peridium, and are currently only known from Africa, South-East Asia, and Australasia (Castellano et al. 2000, Lumyong et al. 2003). The fusoid, smooth, pedicellate basidiospores of C. pakaraimophila also resemble those of species of Hysterangium (Hysterangiales) which otherwise differ in having a dendroid columella, a dark greenish or brown gleba, and in lacking cystidia (Castellano et al. 1989).

Species in a few other temperate sequestrate genera of Boletales have large, fusoid, smooth basidiospores that could potentially be confused with those of C. pakaraimophila, but differ, in addition to having non-dextrinoid basidiospores, in the following ways: Melanogaster species have a black gleba with gel-filled locules; Rhizopogon species have olivaceous to dark brown gleba colours and are associated with Pinaceae; and Truncocolumella species have greenish brown gleba colours, a dendroid columella, and are associated with Pinaceae (Trappe et al. 2009).

Costatisporus T.W. Henkel & M.E. Sm., gen. nov.

MycoBank MB812363

(Figs 4C, 7–8)

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.

Fig. 8

Microscopic features of Costatisporus cyanescens (holotype; Henkel 9061). A. Basidiospores. B. Three-sterigmate basidium with developing basidiospores. C. Four-sterigmate basidium. D. Peridium hyphae with ring-like external encrustations. B–D = phase contrast. Bars = 10 µm.

Etymology: Costatus (L. adj. A) = ribbed or ridged and — sporus (L. adj. A) = –spored; in reference to the distinctively ridged ornamentation of the basidiospores.

Diagnosis: Distinguished from other Boletacaeae by a combination of the following characters: Basidiomata hypogeous to partially emergent, sequestrate. Peridium greyish yellow, staining dark blue, glabrous to subtomentose, thin. Gleba brown, unchanging, loculate, sterile veins absent. Basidiospores statismosporic, subglobose to oblong, light brown, inamyloid, with costate ornamentation of longitudinal ridges pole to pole, these entire or discontinuous, pedicel infrequent. Basidia clavate. Cystidia and clamp connections absent.

Type species: Costatisporus caerulescens T.W. Henkel & M.E. Sm. 2015.

Costatisporus cyanescens T.W. Henkel & M.E. Sm., sp. nov.

MycoBank MB812364

(Figs 4C, 7–8)

Etymology: Cyanescens (L. adj. A) = becoming dark blue; referring to the dark blue auto-oxidation reaction of the bruised peridium.

Diagnosis: Easily differentiated from other sequestrate taxa by the off-white to greyish yellow peridium that stains dark blue, brown acolumellate gleba, strong chocolate nutty odour, and large basidiospores with costate ornamentation.

Type: Guyana: Region 8 Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, ~15 km east of Mt Ayanganna, 2.5 km southeast of base camp at 5°18’04.8” N 59°54’40.4” W, in Lance plot 1, solitary on lateritic mineral soil/humic layer interface under D. corymbosa, 19 June 2009, Henkel 9061 (BRG 41218 — holotype; HSU G1117, NY 02460744 — isotypes). GenBank accession numbers ITS: KT447439; RPB1: LC053663; RPB2: LC053664.

Description: Basidiomata subglobose to ovate and slightly lobed, 12–26 mm tall, 12–33 mm broad; surface initially off-white to light to greyish yellow (4A3–4A4, 4B3) where unstained by soil, developing increasingly deep blue (23C8– 23D8–23E8) stains slowly and progressively over 5–10 minutes where squeezed or bruised, firm, softer in areas, with small, possibly invertebrate mycophagist excavations, glabrous macroscopically, under a hand lens matted tomentose, with age gelatinizing in areas to dark brown and viscid; base subtended by one to several tan hyphal cords. Peridium in longitudinal section thin, 0.3–0.7 mm, appearing single-layered macroscopically, white initially, bluing slowly but intensely on exposure in younger specimens, separable. Gleba densely loculate with brown-hispid interior surfaces under hand lens, initially brown (7E8) to reddish brown (7F6– 7F7–7F8, 8F8) throughout, with age gelatinizing and darker brown (9F4–9F5–9F6), with advanced age violet brown (10F5) over outer 1/6 with locule structure breaking down and blue stains evident on glebal trama under hand lens, acolumellate, with a greyish, gelatinous sterile basal pad 1 × 5 mm. Odour strong, variously described as chocolate-nutty, musty, soapy, putty-like, or of mushroom buillon; taste indistinctive, fungoid. Macrochemical reactions: KOH reddish brown on peridium and gleba; NH4OH negative on all surfaces.

Peridium 150–615 –m thick, two-layered; outer layer 50–415 µm thick, dark yellow to brownish, of tightly interwoven hyphae; individual hyphae with yellow cytoplasmic pigment in H₂O and KOH, occasionally hyaline; intercalary cells 20–92.5 × 5–10 µm, thin-walled, frequently with spiraled to ring-like extracellular encrustations; inner layer 50–250 µm thick, hyaline, of loosely interwoven to parallel hyphae, these 3–8 µm wide, thin-walled. Glebal trama composed of mediostratum and lateral stratum; mediostratum hyaline in H₂O and KOH, 25–45 µm wide, often splitting at locule junctions, of parallel to loosely interwoven hyphae, these hyaline in KOH and H₂O or infrequently with pale yellow cytoplasmic pigments, 2–8 µm wide, thin-walled; lateral stratum moderately to strongly diverging, 10–49 µm thick; individual hyphae hyaline in H₂O and KOH, 4–7 µm wide, thin-walled, grading into interwoven subhymenium hyphae, these hyaline to pale yellow, 5–9 µm wide, thin-walled. Hymenium a palisade of basidia and basidioles. Basidia subclavate to clavate, hyaline to faintly grey in H₂O and KOH, 39.0–66.4 µm long, 7.5–12.2 µm broad at apex, 3–9 µm at the centre, 2.5-–5.8 µm at the base, thin-walled; sterigmata three or four, 4.5–5.5 × 1–2 µm. Basidiospores statismosporic, yellowish to light brown in H₂O and KOH, inamyloid, with complex costate ornamentation of 5–10 longitudinal, somewhat spiraled main ridges running pole to pole; ridges entire or discontinuous and occasionally bifurcating, with numerous narrow, shallow, nearly perpendicular cross-ridges, subglobose to oblong with ornamentation included, 17–25 × (10–)13–20 µm (mean = 19.9 ± 1.41 × 15.9 ± 1.96 µm; Qr = 1.00–1.80(–2.30), Qm = 1.27 ± 0.20; n = 118), ovate to subfusiform and basally acuminate with ornamentation excluded, 14–21 × 8–11.5 µm (mean = 16.8 ± 1.32 µ 10.0 ± 0.65 µm; Qr = 1.36–2.21, Qm = 1.69 ± 0.17; n = 100); pedicel infrequent, 0.5–4 × 1–2 µm. Cystidia and clamp connections absent.

Habit, habitat and distribution: Solitary to scattered and hypogeous to partially emergent on mineral soil/humic layer interface on the forest floor, under D. corymbosa or A. insignis; known only from the type locality and a second site ~8 km distant in the Upper Potaro River Basin of Guyana.

Additional specimens examined: Guyana: Region 8 Potaro-Siparuni: Pakaraima Mountains, Upper Potaro River Basin, ~15 km east of Mt Ayanganna, within 10 km radius of base camp at 5°18’04.8” N 59°54’40.4” W, ~8 km southeast of base camp on lateritic soil-leaf litter interface under A. insignis, 25 July 2009, Henkel 9067 (BRG 41219; HSU G1124). GenBank accession number ITS and 28S: LC053662; ~1 km southeast of base camp immersed hypogeously in decaying wood humus under D. corymbosa on lateritic soils, 11 June 2015, Henkel 10060 (BRG 41222; HSU G1125); GenBank accession number ITS: KT380011; 16 June 2015, Henkel 10100 (BRG 41223; HSU G1126); vicinity of base camp, under D. corymbosa, in Guyanagaster plot 40, 21 June 2015, Aime 5850 (BRG 41224; HSU G1127; PUL F2871).

Commentary: Costatisporus cyanescens is recognized in the field by the blue-bruising peridium with occasional mycophagist excavations, and dark brown to violet-brown, finely loculate, acolumellate gleba that gelatinizes with maturity. Micromorphologically, the basidiospore ornamentation of longitudinal main and lateral secondary ridges is distinctive. The basidiospore ornamentation is remarkably similar to that in species of the epigeous bolete genus Boletellus, which has no known sequestrate members (Singer 1986, Mayor et al. 2008, Halling et al. 2015). The similar basidiospore ornamentations of Costatisporus and Boletellus are apparently coincidental, as C. cyanescens has no close phylogenetic relationship with Boletellus, which occurs in the Xerocomoideae clade (Fig. 1; Table 1).

The basidiospores of South-East Asian sequestrate Rhodactina (Boletaceae) species are longitudinally ridged but lack the intervening secondary ridges observed in C. cyanescens, are purple in water mounts, and dextrinoid (Pegler et al. 1989, Yang et al. 2006). Although no 28S, RPB1, or RPB2 sequences were available for the genus Rhodactina, comparison of the ITS1 sequence from Rhodactina incarnata with that of C. cyanescens did not indicate a close relationship. The tropical Asian genus Durianella has sequestrate basidiomata that undergo a deep blue colour change upon exposure, but also have a well-developed columella, fibrillose exoperidial warts, and echinulate basidiospores (Desjardin et al. 2008).

Species of the sequestrate genera Rossbeevera (East Asia, Australasia) and Chamonixia (mostly North Temperate) feature longitudinal ridging of the basidiospores and often undergo a blue, green, or blackish colour change upon bruising or exposure (Smith & Singer 1959, Lebel et al. 2012, Orihara et al. 2012b). Basidiospore ornamentation in Rossbeevera lacks intervening subridges and has 4–5 short, broad longitudinal ridges which contribute to their slight to stellate polar angularity (Lebel et al. 2012, Orihara et al. 2012b), contrasting with the 5–10 spiraled, acute ridges of C. cyanescens that impart a consistently stellate polar shape. Furthermore, Rossbeevera is phylogenetically distant from C. cyanescens (Fig. 1). The ridged basidiospore ornamentation of Chamonixia species superficially resembles that of C. cyanescens, but their longitudinal ridges are straight with rounded margins and lack intervening subridges (Smith & Singer 1959).

Although the longitudinally ridged basidiospore ornamentation of C. cyanescens bears some resemblance to that seen in Gautieria (Gomphales) and Austrogautieria (Hysterangiales), no species of these genera undergo a blue colour change upon bruising or exposure (Zeller & Dodge 1918, Stewart & Trappe 1985). Additionally, Gautieria species differ from C. caerulescens in having gently rounded basidiospore ridges that terminate before the poles, a persistent dendroid columella, and globose cells in the peridium (Dodge & Zeller 1934). While Austrogautieria species are acolumellate and have apically convergent basidiospore ridges with subacute margins, only one species, the Australian A. manjimupana, overlaps with C. caerulescens in the number of basidiospore ridges (5–10); all other species of Austroguatieria have 8–14 ridges (Stewart & Trappe 1985). Austrogautieria manjipumana differs from C. cyanescens in the smaller (14–20 × 8–13 um), more ellipsoid basidiospores that lack intervening subridges (Stewart & Trappe 1985). Additionally, Gautieria and Austrogautieria, as members of the Phallomycetidae, are phylogenetically distant from Boletales (Giachini et al. 2006, Hosaka et al. 2006).

In the phylogenetic analysis presented here, C. cyanescens is well supported as sister to Sutorius (formerly the Tylopilus eximius complex), which is a genus of dark maroon to purple, epigeous bolete species occurring in the Americas, Australasia, and sympatrically with C. cyanescens in Guyana (Fulgenzi et al. 2007, Halling et al. 2012). The molecular-based analysis may reflect evolutionary reality, but Costatisporus and Sutorius are very different morphologically. Sutorius species have robust, pileate-stipitate basidiomata with exposed hymenophores that bruise cinnamon-brown, ballistosporic basidia, and smooth, fusiform basidiospores, among many other different micromorphological features (Fulgenzi et al. 2007, Halling et al. 2012). The Costatisporus-Sutorius clade indicated here is supported as sister to the recently characterized, mostly epigeous genus Neoboletus (Fig. 1) (Wu et al. 2014, 2015). While the sole sequestrate species of Neoboletus, N. thibetanus, stains blue upon exposure, it is easily distinguished from C. cyanescens by its notable stipe and bright yellow peridium. In the future it will be necessary to sequence additional loci from species within this emerging clade to shed light on the putatively strong relationships between Sutorius, Neoboletus, and C. cyanescens.