Sarcodon in the Neotropics: New Species from Belize, Colombia

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SARCODON IN THE NEOTROPICS: NEW SPECIES FROM BELIZE, COLOMBIA,

GUYANA, AND PUERTO RICO

Arthur Charles Grupe II

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science Biology

Committee Membership

Dr. Terry Henkel, Committee Chair

Dr. Erik Jules, Committee Member

Dr. Matthew Smith, Committee Member

Dr. Michael Mesler, Committee Member, Graduate Coordinator

July 2015

ABSTRACT

SARCODON IN THE NEOTROPICS: NEW SPECIES FROM COLOMBIA, BELIZE,

GUYANA, AND PUERTO RICO

Arthur Charles Grupe II

Eight species of the ectomycorrhizal (ECM) genus Sarcodon (Bankeraceae,

Thelephorales, Basidiomycota) are described as new to science. Sarcodon quercophilus, and Sarcodon umbilicatus are described from Quercus (Fagaceae) cloud forests within the Maya Mountains of Belize. Sarcodon pakaraimensis is described from forests dominated by the ECM trees Pakaraimaea dipterocarpacea (Dipterocarpaceae) and

Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae) in the Pakaraima Mountains of

Guyana. Sarcodon portoricensis is described from lower montane wet forest within the

El Yunque National Forest of Puerto Rico. Sarcodon colombiensis, Sarcodon rufogriseus, Sarcodon pallidogriseus, and Sarcodon bairdii are described from the

Colombian Amazon in forests dominated by Pseudomonotes tropenbosii

(Dipterocarpaceae) and “white sand” forests dominated by species of Dicymbe (Fabaceae subfam. Caesalpinioideae) and Aldina (Fabaceae subfam. Papilionoideae). The discovery of these eight new species is significant given that the majority of the approximately 87 previously described Sarcodon species are north temperate or boreal in distribution and frequently associate with coniferous host plants; these constitute the most recent records ii

for Sarcodon from the greater Neotropics. Each of the new species is morphologically consistent with accepted diagnostic characters for Sarcodon: pileate-stipitate stature, a dentate hymenophore, determinate basidiomatal development, fleshy, non-zonate context, and brown, tuberculate basidiospores. DNA (ITS) sequence analysis corroborated the generic placement of S. pakaraimensis, S. portoricensis, S. quercophilus, S. umbilicatus,

S. colombiensis, S. rufogriseus, S. pallidogriseus, and Sarcodon bairdii, and along with morphological differences, supported their recognition as distinct species.

Macromorphological, micromorphological, habitat, and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species.

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ACKNOWLEDGEMENTS

The author thanks the following for financially supporting this project: the George

Verback Scholarship from the Humboldt Bay Mycological Association, Sonoma County

Mycological Society Graduate Student Scholarship, CSUPERB Travel Scholarship, and the Greg Jennings Memorial Scholarship.

A special thank you goes out to all the people over my time at Humboldt State

University that contributed to my personal and professional development. Some of these people are: J. Uehling, D. Husbands, N. Najarian, I. Zacher, M. DeSiervo, A. Sahara, E.

Wu, K. Ludwig, S. Brandt, S. McGuire, E. DeStigter, S. Faulkner, J. Jackson, E. Alvey,

P. Lei, J. Mola, Y. Wenzel, L. McCrigler, M. Reed, A. Baker, L. Weaver, C. Satter, G.

Shelton, K. Matsunaga, E. Grupe, S. Gowan, and M. Friedman.

Field assistance in Guyana was provided by C. Andrew, P. Joseph, F. Edmond, and L. Edmond. Research permits that were granted by the Guyana Environmental

Protection Agency, and Ministry of Amerindian Affairs.

I would also like to acknowledge the contributions of my committee members.

Dr. Erik Jules for giving me my first research opportunities, and letting me borrow his prized Grateful Dead tapes. Dr. Michael Mesler for his encouragement in my development as a teacher and researcher, and for informing me that I would not be alone in “Pollinator Hell”. Dr. Matthew Smith for always being willing to help me with molecular data and problem solving. Dr. Terry Henkel for always pushing me to better myself in my academic career, showing me what it takes to execute a successful iv

international field expedition, how to spell “Pakaraimaea”, and those times we performed at the Black Alice show.

Most of all I would like to thank my family: my sister Alison Cardenas, my parents Ann Weber, Dan Weber, and Arthur S. Grupe for their unwavering support and belief in me, no matter what.

TABLE OF CONTENTS

ABSTRACT...... ii

ACKNOWLEDGEMENTS...... iv

LIST OF TABLES...... vii

LIST OF FIGURES ...... viii

INTRODUCTION ...... 1

CHAPTER 1 SARCODON IN THE NEOTROPICS I: NEW SPECIES FROM GUYANA, PUERTO RICO, AND BELIZE...... 3

Introduction...... 4

Materials and Methods ...... 6

Results...... 9

Taxonomy ...... 12

CHAPTER 2 Sarcodon in the Neotropics II. A NEW SPECIES AND DISTRIBUTION RECORD FROM COLOMBIA...... 45

Introduction...... 46

Materials and Methods ...... 48

Results...... 49

Taxonomy ...... 52

LITERATURE CITED ...... 75

LIST OF TABLES

Table I. Diagnostic morphological characters of new Sarcodon species from Guyana, Puerto Rico, Belize, and Colombia………………………………………………………19

vii

LIST OF FIGURES

Figure 1. Most parsimonious phylogram (384 steps; mid-point rooted) based on internal transcribed spacer (ITS) ribosomal DNA sequences depicting phylogenetic relationships of Neotropical Sarcodon species. Support values above the nodes are maximum likelihood bootstrap support values whereas values below the nodes are maximum parsimony support values. Nodes with bootstrap support values less than 75 are not shown. Sequences for all Sarcodon species found outside of the Neotropics were obtained from GenBank with numbers shown next to species epithets...... 11

Figure 2. Basidiomata of Sarcodon pakaraimensis (HOLOTYPE; Henkel 9513). A. Developmental series. B. Basidioma showing the black auto-oxidation and staining reaction on the pileus. C. Longitudinal section showing pink staining of the exposed pileus trama, and hollow stipe with internal squamules. Bar = 10 mm...... 16

Figure 3. Basidia and basidiospores of Sarcodon pakaraimensis (HOLOTYPE; Henkel 9513). Bar = 10 µm...... 17

Figure 4. Scanning electron micrographs of basidiospores of new Neotropical Sarcodon species (×4000). A. Sarcodon pakaraimensis (HOLOTYPE; Henkel 9513). B. Sarcodon portoricensis (HOLOTYPE; Baroni 8776). C. Sarcodon quercophilus (HOLOTYPE; CFMR-BZ-3833). D. Sarcodon umbilicatus (HOLOTYPE; Baroni 10201). ×4000

...... 21

Figure 5. Basidiomata of Sarcodon portoricensis (HOLOTYPE; Baroni 8776). Bar = 10 mm…………………………………………………………………………………….....25

Figure 6. Basidia and basidiospores of Sarcodon portoricensis (HOLOTYPE; Baroni 8776). Bar = 10 µm……...... 26

Figure 7. Basidiomata of Sarcodon quercophilus (HOLOTYPE; CFMR-BZ-3833). A. Dorsal view. B. Ventral view, showing pale hymenophore. Bars = 10 mm...... 32

Figure 8. Basidia, basidiospores, and stipitipellis terminal cells of Sarcodon quercophilus (HOLOTYPE; CFMR-BZ-3833). Bar = 10 µm...... 33

Figure 9. Basidiomata of Sarcodon umbilicatus (HOLOTYPE; Baroni 10201). Bar = 10 mm...... 39

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Figure 10. Basidia and basidiospores of Sarcodon umbilicatus (HOLOTYPE; Baroni 10201). Bar = 10 µm...... 40

Figure 11. Most parsimonious phylogram (384 steps; mid-point rooted) based on internal transcribed spacer (ITS) ribosomal DNA sequences depicting phylogenetic relationships of Sarcodon colombiensis, Sarcodon rufogriseus, Sarcodon pallidogriseus, and Sarcodon bairdii from Colombia. Support values above the nodes are maximum likelihood bootstrap support values. Nodes with bootstrap support values less than 35 are not shown. Sequences for all taxa, Sarcodon species found outside of the Neotropics, and previously described Neotropical species, were obtained from GenBank with numbers shown next to species epithets…………………………………………………………………………...51

Figure 12. Basidia (A., B.), basidiospores (C.), and terminal cells of the pileus trama (D., E.) of Sarcodon colombiensis (HOLOTYPE; Vasco 2084). Bar = 10 µm………………55

Figure 13. Basidiomata of Sarcodon rufogriseus (HOLOTYPE; Vasco 1989). Bar = 10 mm……………………………………………………………………………………….60

Figure 14. Basidia (A.), basidiospores (B.), and terminal cells of the pileus (C., D.) and stipe trama (E.) of Sarcodon rufogriseus (HOLOTYPE; Vasco 1989). Bar = 10 µm….61

Figure 15. Basidiomata of Sarcodon pallidogriseus (HOLOTYPE; Vasco 989). Bar = 10 mm. ……………………………………………………………………………………..66

Figure 16. Basidium (A.), basidiospores (B.), and basidium with basidiospore (C.) of Sarcodon pallidogriseus (HOLOTYPE; Vasco 989). Bar = 10 µm……………………67

Figure 17. Basidiomata of Sarcodon bairdii from Colombia (Vasco 990). Bar = 10 mm………………………………………………………………………………………72

Figure 18. Basidia and basidiospores of Sarcodon bairdii (HOLOTYPE; Vasco 990). Bar = 10 µm…………………………………………………………………………………73

INTRODUCTION

Fungi perform vital ecosystem services, such as saprotrophs aiding in biomass turnover, nutrient cycling and soil creation, and plant pathogens of all types that can function as ecosystem engineers by affecting the spatiotemporal composition and structure of plant communities. For instance, root pathogens such as Armillaria spp. create landscape level disturbances that influence forest succession (Hawkins and Henkel 2011). Some fungi also function symbiotically as mycorrhizae with almost all land plants, facilitating nutrient acquisition in their host plants.

Mycorrhizal fungi aid their plant hosts by greatly increasing the surface area of the host plant’s roots. This is achieved via the interface between host fine roots and the fungal hyphae, and the extension of those hyphae outward into the soil environment. The mycorrhizae extract water and mineral nutrients from the soil, which they transfer to their host plant in exchange for carbohydrates (Smith and Read 2008). There is also evidence that ectomycorrhizal (ECM) fungi have the ability to inhibit root pathogens or mitigate environmental stress on their host plants (e.g. Bandou et al. 2006).

Despite the numerous roles of fungi in ecosystems, fungal diversity is poorly understood. This is one reason why more effort is needed to document the biodiversity of fungi, particularly tropical fungi, as there has been traditionally a temperate bias towards researching these organisms. For example, a conservative estimate of ectomycorrhizal

(ECM) fungal diversity from the lowland Neotropics was previously placed at between

150-200 described species, though repeated sampling of a single study site in Guyana has

2 yielded 172 species of ECM fungi, most which are new to science, greatly increasing the known diversity of this fungal guild from the Neotropics (Henkel et al. 2012).

Efforts over the past decade to document fungal diversity in the Caribbean and the

Guiana Shield region of northeastern South America have documented a plethora of previously undescribed ECM fungal taxa, and led to the description of > 80 species and two genera new to science. This work has expanded traditionally held morphological concepts of otherwise well-studied taxonomic groups, what they can do ecologically, and discovery of taxa previously thought to occur entirely in north temperate/boreal regions, implying a much broader geographic distribution and ECM host tree range (e.g., Grupe et al. 2015, Henkel et al. 2006, Miller et al. 2001, Miller et al. 2002, Uehling et al. 2012).

Recent efforts by the author in Guyana and colleagues working elsewhere in the

Neotropics suggested that the ECM tooth fungus genus Sarcodon (Bankeraceae,

Thelephorales, Basidiomycota), while previously thought to be predominantly Nearctic in distribution and associated with conifers, may be more diverse and widely distributed in the Neotropics than previously thought. This observation was based on collections of undescribed Sarcodon species from the northern Caribbean, Central America, and eastern and western Guiana Shield, in apparent mycorrhizal association with a diverse array of broadleaf hosts in the Dipterocarpaceae, Fabaceae, and Fagaceae. The work presented here encapsulates the taxonomic work to date on these Sarcodon specimens.

CHAPTER 1

SARCODON IN THE NEOTROPICS I: NEW SPECIES FROM GUYANA, PUERTO

RICO, AND BELIZE

Published in Mycologia DOI:10.3852/14-185, February 2015.

Abstract: Four species of the ectomycorrhizal (ECM) genus Sarcodon

(Bankeraceae, Thelephorales, Basidiomycota) are described as new to science. Sarcodon pakaraimensis sp. nov. is described from forests dominated by the ECM trees

Pakaraimaea dipterocarpacea (Dipterocarpaceae) and Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae) in the Pakaraima Mountains of Guyana. Sarcodon portoricensis sp. nov. is described from lower montane wet forest within the El Yunque

National Forest of Puerto Rico. Sarcodon quercophilus sp. nov. and Sarcodon umbilicatus sp. nov. are described from Quercus (Fagaceae) cloud forests within the

Maya Mountains of Belize. The discovery of these four new species is significant given that the majority of the approximately 87 described Sarcodon species are north temperate or boreal in distribution and frequently associate with coniferous host plants; these constitute the most recent records for Sarcodon from the greater Neotropics. Each of the new species is morphologically consistent with accepted diagnostic characters for

Sarcodon: pileate-stipitate stature, a dentate hymenophore, determinate basidiomatal development, fleshy, non-zonate context, and brown, tuberculate basidiospores. DNA

(ITS) sequence analysis corroborated the generic placement of S. pakaraimensis, S. portoricensis, S. quercophilus, and S. umbilicatus and, along with morphological

4 differences, supported their recognition as distinct species. Macromorphological, micromorphological, habitat, and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species. A key to

Neotropical Sarcodon species and similar extralimital taxa is provided.

Key words: Bankeraceae, Caribbean, Central America, ectomycorrhizal fungi,

Guiana Shield, Thelephorales, tooth fungi

Introduction

Sarcodon Quél. ex P. Karst. (Bankeraceae, Thelephorales) is an ectomycorrhizal

(ECM) basidiomycete genus characterized by stipitate, pileate basidiomata with determinate development, fleshy, non-zonate context, dentate hymenophore, variably tuberculate, brown basidiospores, and the presence or absence of clamp connections

(Banker 1906, Maas Geesteranus 1971). Approximately 87 species of Sarcodon have been described worldwide, mostly from high latitude forest environments (Coker and

Beers 1951, Maas Geesteranus 1971, McNabb 1971, Harrison 1973, Baird 1986, Baird and Khan 1986, Harrison and Grund 1987, Agerer 1991, Stalpers 1993, Visser 1995,

Shiryaev 2008, Mleczko et al. 2011, Baird et al. 2013, Vizzini et al. 2013). All known species of Sarcodon fruit on the soil in spatial association with ECM plants and available evidence suggests that Thelephorales species, including those of Sarcodon, are ECM

(Stalpers 1993, Tedersoo and Smith 2013). The ECM status of Sarcodon has been confirmed for Sarcodon imbricatus (L.) P. Karst. on Picea abies (L.) H. Karst. in

Germany (Agerer 1991), Sarcodon scabrosus (Fr.) P. Karst. on Pinus banksiana Lamb.

(Visser 1995) in Canada, Sarcodon leucopus (Pers.) Maas Geest. & Nannf. with other

Pinaceae hosts in Poland (Mleczko et al. 2011), and Sarcodon atroviridis (Morgan)

Banker on Abies pindrow (Royle ex D. Don) Royle in Pakistan (Niazi 2008). While largely Holarctic in distribution, some Sarcodon species are known from the Paleotropics

(Maas Geesteranus 1964, 1967, 1971, 1974a, Watling and Lee 1995), but records from the Neotropics are scarce (Baker and Dale 1951, Snell and Dick 1958, Maas Geesteranus

1974b, Singer et al. 1983). Here we provide new reports for the genus in the Neotropics from the Guiana Shield, northern Caribbean, and Central America. Sarcodon pakaraimensis sp. nov. is described from Guyana, in forests dominated by the endemic

ECM trees Pakaraimaea dipterocarpaceae Maguire & P.S. Ashton (Dipterocarpaceae) and Dicymbe jenmanii Sandw. (Fabaceae subfam. Caesalpinioideae). Sarcodon portoricensis sp. nov. is described from a lower montane wet forest of the El Yunque

National Forest of Puerto Rico. Sarcodon quercophilus sp. nov. and Sarcodon umbilicatus sp. nov. are described from ECM Quercus-dominated (Fagaceae) cloud forests of the Maya Mountains of Belize.

All of the new species are morphologically similar but distinguishable from each other, and similar to a group of previously described species defined by their non-scaly pilei, overall somber basidioma colors, and KOH-soluble bluish green pigments (Maas

Geesteranus 1971), in particular Sarcodon atroviridis (Morgan) Banker from temperate

North America, Europe, and East Asia, Sarcodon thwaitesii (Berk. & Br.) Maas G. from the Asian tropics, and Sarcodon bambusinus (Baker & Dale) Maas G. from the

Neotropics (Berkeley and Broome 1873, Morgan 1895, Baker and Dale 1951, Maas

Geesteranus 1964, 1974b, 1975). Variation in key morphological characteristics corroborated by molecular phylogenetic analysis warrant the erection of the four new species, and indicate their close relationship within the genus. Macromorphological, micromorphological, habitat, and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) are provided for each of the new species. A key to

Neotropical Sarcodon species and similar extralimital taxa is provided.

Materials and Methods

Guyana collections were made during the Dec-Jan rainy season of 2010−2011 from the Upper Mazaruni River Basin in the Pakaraima Mountains, in the vicinity of a base camp located at 5° 26' 21.3" N; 60° 04' 43.1" W; elevation ~800 m. This area is located ~15 km west of Mt. Ayanganna (2200 m), and characterized by a mixture of open savannas and closed canopy forests on white sand soils (Smith et al. 2013). Collections were made in forests co-dominated by the ECM trees P. dipterocarpacea and D. jenmanii. Puerto Rico collections were made during Jun-Jul 1998 in a lower montane wet forest along the Pico El Toro Trail in the El Yunque National Forest (previously known as the Caribbean National Forest) in the general locality of 18° 17’ N; 65° 51’ W; elevation 650−710 m. Belize collections were made in Aug 2004 and Aug 2007 from

Doyle’s Delight Mountain within the Maya Mountains near 16° 29’ N; 89° 2’ W; elevation ~1000 m, from cloud forests dominated by species of Quercus.

Macroscopic features of basidiomata were described fresh in the field. Colors were described subjectively and coded according to Kornerup and Wanscher (1978), with color plates noted in parentheses, or as capitalized Ridgway colors as reproduced by Smithe

(1975). Collections of fresh basidiomata were dried in the field using silica desiccant beads or were warm air dried using food dehydrators or a forced air drying oven.

Micromorphological features of dried specimens were examined with an Olympus BX51 microscope with light and phase contrast optics. Fungal tissue was mounted in H2O, 3% potassium hydroxide (KOH), or Melzer's solution. At least 20 individual basidiospores, basidia, and other structures were measured per collection; for basidiospores, measurements were taken from spore deposits when possible, with dimensional measurements including ornamentation. Range and mean quotients of basidiospore length divided by width (Q) were calculated. Outlying measurements observed in less than 5% of the measured population, are indicated in parentheses. Line drawings were made using tracing paper and modified with Photoshop CS5 (Adobe, San Jose, California).

Specimens were deposited in the following herbaria (Holmgren et al. 1990): BRG –

University of Guyana; HSU – Humboldt State University; CORT – State University of

New York at Cortland; NY– New York Botanical Garden; BRH – Forest Department

Herbarium, Ministry of Natural Resources, Local Government, and the Environment of

Belize; UPRRP– University of Puerto Rico at Rio Piedras.

DNA extraction, amplification, sequencing, and phylogenetic analyses.

DNA extraction, polymerase chain reactions (PCR), cloning and sequencing protocols followed Gardes and Bruns (1993), with the following modifications. DNA was extracted from dried basidiomata by placing tissue samples into 1.5 mL tubes with 0.1 mm glass beads and 400 µL of CTAB heated to 60 oC. Tissues were ground with a plastic micropestle, then incubated overnight at 57o C with 700 rpm shaking on an Eppendorf

Thermomixer R (Eppendorf International, http://www.eppendorf.com). This resulted in very dark samples, presumably from the copious pigments present in the basidiomata.

The tubes were centrifuged at 8,000 rpm to pelletize debris, and the supernatant was transferred to a new tube and brought to 500 µL with CTAB. 800 µL of chloroform was added and the tubes inverted for 1 minute prior to centrifuging at full speed for ten minutes. The supernatant was removed and extracted in 800 µL of chloroform. DNA was precipitated with two volumes of 100% isopropanol and 1/10 volume ammonium acetate at -20 oC overnight. DNA was pelleted by centrifugation at full speed for ten minutes.

The DNA pellet was washed in 70% ethanol and resuspended in 1X TE buffer. PCR reactions were performed using 1 µL of DNA diluted 1:10, 0.4 µM primer and 12.5 µl of

EcononTaq Plus 2x Mastermix as per manufacturers recommendations (Lucigen

Corporation, Middleton, WI). Cycling temperatures were as follows: initial denaturation at 94 oC for 10 minutes; cycle 35 times at 94 oC for 30 seconds, 54 oC for 30 seconds, and

72 oC for 30 seconds.

PCR products were sent to Sequetech (Mountain View, CA, http://www.sequetech.com/) for sequencing. Sequences were processed and assembled using CodonCode Aligner v.3.5.7 (CodonCode Corporation, Dedham, MA, http://www.codoncode.com/). For the new sequences, assembled sequence contigs of ITS were used in preliminary blastN queries of the GenBank (http://www.ncbi.nlm.nih.gov/) database to confirm generic affinities and indicate the most similar described and sequenced species.

Manual alignment for ITS sequences of all species included 767 characters, of which 511 nucleotide characters were included in the analysis, while 256 were excluded because they constituted gaps or were in areas that were too variable to align. The alignment is available on TreeBase at: http://purl.org/phylo/treebase/phylows/study/TB2:S16770. Maximum Parsimony (MP) analysis was completed with default settings in PAUP* 4.0 (Swofford 2003). A

Maximum Likelihood (ML) search was run in GARLI (Zwickl 2006) with the GTR+I+G model. Support for phylogenetic relationships was assessed based on 500 bootstrap replicates in PAUP (for MP) and Garli (for ML).

Results

blastN queries for ITS sequences of the four new species reported here produced best matches to species of Sarcodon but none exceeded 95% similarity. The MP search found only one most parsimonious tree with a score of 384 steps. The ML tree, with a likelihood score of -ln 2448.83248, had an identical topology to that of the MP tree (data

10 not shown). These analyses demonstrated that the four new Neotropical Sarcodon species described here are phylogenetically distinct from each other and are nested within a well- supported clade that also includes the north temperate species S. atroviridis (Figure 1;

100% ML and MP support). A midpoint rooted tree is shown and no outgroup was used for this analysis because ITS sequences from other genera of Thelephorales were too distant to be reliably aligned in the ITS region with species of Sarcodon. Therefore, our analysis necessarily focused on the placement of the Neotropical species and could not resolve other phylogenetic relationships within the genus nor address the position of

Sarcodon within the Bankeraceae or Thelephorales. Among the four new species, S. quercophilus and S. portoricensis had the closest affinity with S. atroviridis, with the three species occurring in a well-supported subclade (Figure 1; 96% ML, 99% MP support). Sarcodon umbilicatus and S. pakaraimensis lie in a sister relationship to the rest of the taxa of this clade in a well-supported subclade (Figure 1; 89% ML, 86% MP support). The remaining north temperate Sarcodon species included in the analysis were resolved outside of the S. atroviridis clade

(Figure 1).

Taxonomy

Sarcodon pakaraimensis A. Grupe & T.W. Henkel, sp. nov. Figures 2, 3, 4

Mycobank MB 809566

Diagnosis: Sarcodon pakaraimensis differs from other species of Sarcodon in its combination of pinkish gray, smooth to pitted, black-staining pileus, hollow stipe with internal squamules at maturity, pink staining of exposed trama, basidiospores that are 5−7

µm long, and its unique ITS sequence.

Typification: GUYANA: REGION 7 CUYUNI-MAZARUNI: Pakaraima

Mountains, Upper Mazaruni River Basin, within 0.5 km of base camp at 5° 26' 21.3" N;

60° 04' 43.1" W; elevation ~800 m; vicinity of base camp, 22 Dec 2010, Henkel 9513

(holotype BRG; isotypes NY 02329648; HSU). GenBank accession: ITS KC155390.

Pileus initially conic, broadly conic to plano-convex with age, 21−74 mm broad,

3−18 mm tall, predominantly pinkish gray (7B2−7C2) throughout initially, with irregular darker purplish spots, with age developing irregular black auto-oxidative patches, these occasionally coalescing throughout, bruising similarly; surface glabrous macroscopically with irregular fine pits, under hand lens a densely interwoven, repent mat; margin entire, slightly incurved when young, becoming irregularly wavy with age; trama cream (5A2), staining slowly to light pink (11A2−11B2) upon exposure. Hymenophore dentate, adnate; teeth crowded, 0.5−1.0 mm long at margin, 1.0−2.0 mm centrally, 2.0−3.0 mm at stipe, subacuminate, cream colored throughout in young specimens (4A3−4A4), staining dark brown (6F5) upon pressure, becoming brown-mottled with age with tips remaining

13 cream. Stipe subequal, 22−58 × 3−19 mm, tapering very slightly at the base, glabrous, grayish cream (7A2−7B2) when young, later concolorous with pileus; somewhat purplish towards apex, black at base; trama substuffed initially, later hollow with internal black erect squamules, concolorous with pileus trama, staining light pink (11A2−11B2) initially upon exposure, darkening to burgundy with time. Odor somewhat citrus-like or of conifer sap; taste mildly bitter. Macrochemical reactions: all tissues staining dark blue to black with KOH and NH4OH on dried basidiomata. Dried pileus color olive-gray (3E2−3E3) with bluish gray (23F3) spots; context yellowish gray (2C2−2C3). Basidiospores dark reddish brown (9E4−9F4) in fresh deposit, more yellowish brown (5E4) when dried;

5−7(−8) × 5−9(−10) µm including ornamentation (mean = 6.4 × 7.6 µm; n = 40), Q range

= 0.71–1.2, Q mean = 0.84, suboblate, tuberculate, light brown in H2O, pale golden- brown in KOH, inamyloid; tubercle apices mostly rounded, rarely exsculpate, also evident under SEM; hilar appendage 1−2 µm long. Basidia 27−47(−54) × 7−13 µm wide apically, 5−7 µm wide centrally, 2−3 µm at basal septum, clavate, somewhat curving subcentrally, with numerous small guttules, hyaline in H2O and KOH; basal septum with clamp connection; sterigmata (2−)3−4, curved, (4−)5−6(−7) µm long. Hymenial cystidia absent. Hymenophoral trama of parallel to slightly interwoven hyphae, diverging slightly immediately below subhymenium, in mass light gray to light orange-brown in H2O, faint tan to light blue-green in KOH; individual hyphae faint gray in H2O, faint green-blue in

KOH, 3−6 µm wide, with densely clustered small, bluish green extracellular pigment bodies scattered throughout. Pileipellis a cutis of strongly repent hyphae, in mass light

14 gray-green in H2O, dark gray-blue in KOH, suffused from suprapellis downward to

133−180 µm into subpellis with dense masses of irregularly-shaped, extracellular, granular pigment bodies, these black in H2O, bluish green in KOH and eventually dissolving and leaching into solution; individual hyphae light gray-green in H2O, faintly gray-brown to light gray in KOH, 4−6(−12) µm wide, irregularly bulging and constricting; terminal cells undifferentiated. Pileus trama in mass gray-brown to orange- brown in H2O, light gray-tan in KOH, with highly scattered, extracellular, dark bluish green to nearly black granular pigment bodies in H2O, these bluish green in KOH; individual hyphae light gray in H2O, faint gray in KOH, occasionally branching, most irregularly bulging and constricting, some cylindrical, (2−)6−11 µm wide. Stipitipellis a cutis of largely repent hyphae, grading inward to a more anticlinally interwoven subpellis, in mass gray in H2O, variably gray-blue in KOH, with scattered extracellular granular pigment bodies, these dark blue to nearly black in H2O, dark bluish green in

KOH; individual hyphae light gray in H2O, faint gray to faint gray-blue in KOH, isodiametric or frequently with irregular bulges and constrictions, 3−8(−10) µm wide; terminal cells undifferentiated. Stipe trama interwoven, in mass light gray-tan in H2O and

KOH, with frequent irregularly shaped pockets devoid of hyphae, these (20−)30−47(−91)

µm wide across longest axis; individual hyphae faint gray in H2O and KOH, isodiametric to frequently with irregular bulges and constrictions, 5−10 µm wide, slightly constricted at septa. Inner surface of hollow stipe an interwoven mass of subanticlinal hyphae continuous with the stipe trama, interspersed with fascicles of massed anticlinal hyphae

15 corresponding to inner stipe squamules; fascicle hyphae in mass gray in H2O, grayish brown in KOH. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Scattered to gregarious on humic substrata on the forest floor in mixed Pakaraimaea/Dicymbe forest; known only from the type locality in the Upper Mazaruni River Basin of Guyana.

Etymology. Pakaraimensis (-ensis Latin adj. B) = adjectival suffix indicating origin or place, referring to the type locality of the species in the Pakaraima Mountains of

Guyana.

Other specimen examined. GUYANA: REGION 7 CUYUNI-MAZARUNI:

Pakaraima Mountains, Upper Mazaruni River Basin, ~1 km SW of base camp, 29 Dec

2010, Henkel 9554 (BRG; HSU). GenBank accession: ITS KM668103.

Figure 2. Basidiomata of Sarcodon pakaraimensis (HOLOTYPE; Henkel 9513). A.

Developmental series. B. Basidioma showing the black auto-oxidation and staining reaction on the pileus. C. Longitudinal section showing pink staining of the exposed pileus trama, and hollow stipe with internal squamules. Bar = 10 mm.

Figure 3. Basidia and basidiospores of Sarcodon pakaraimensis (HOLOTYPE; Henkel

9513). Bar = 10 µm.

Commentary. Sarcodon pakaraimensis is recognized in the field by its pileate- stipitate, determinate basidioma with pinkish gray, smooth, black-staining pileus with dentate hymenophore, concolorous subequal stipe that is hollow with internal squamules when mature, and the pink staining of exposed trama. Sarcodon pakaraimensis can be differentiated from each of the other new Neotropical species described here based on key features of the pileus surface, staining reactions of the exposed trama, stipe trama, basidiospore ornamentation, and stipitipellis (Table I).

Sarcodon pakaraimensis is similar to the north temperate S. atroviridis in that both have pits or ridges on the otherwise smooth pileus that stains black when bruised

(Morgan 1895, Banker 1906, Coker and Beers 1951). Sarcodon pakaraimensis is distinguished from S. atroviridis by its pink-staining reaction of the exposed trama, lack of an olive cast to the dried basidioma, internal squamules within the hollow stipe, and shorter basidiospores (5−7 vs. 7.2−9 µm) (Morgan 1895, Banker 1906, Coker and Beers

1951, Baird et al. 2013). Additionally, ITS sequences of S. pakaraimensis are only ~86% similar to sequences of S. atroviridis from the USA and so these two species are distinct in the phylogenetic analysis (Figure 1).

Sarcodon pakaraimensis is similar to the paleotropical S. thwaitesii in basidioma color and black staining of the pileus, and the hollow stipe at maturity, but differs in its densely interwoven, matted pileipellis (vs. an erect tomentum); additionally, the interior stipe squamules and pink staining reaction of the exposed trama are lacking in S. thwaitesii (Berkeley and Broome 1873, Maas Geesteranus 1964, 1971, 1974a).

Both S. pakaraimensis and the Neotropical S. bambusinus have non-decurrent teeth, a similar pileus shape, and similar stipe lengths, but S. pakaraimensis differs from

S. bambusinus in its pileus surface of an interwoven, repent mat, pink tramal staining reaction, hollow stipe with internal squamules, and basidiospore length (5−7 vs. 6.5−9

µm) (Baker and Dale 1951, Maas Geesteranus 1974b).

Figure 4. Scanning electron micrographs of basidiospores of new Neotropical Sarcodon species (×4000). A. Sarcodon pakaraimensis (HOLOTYPE; Henkel 9513). B. Sarcodon

22 portoricensis (HOLOTYPE; Baroni 8776). C. Sarcodon quercophilus (HOLOTYPE;

CFMR-BZ-3833). D. Sarcodon umbilicatus (HOLOTYPE; Baroni 10201).

Sarcodon portoricensis A. Grupe & T.J. Baroni, sp. nov. Figures 4, 5, 6

MycoBank MB 809567

Diagnosis: Sarcodon portoricensis differs from other species of Sarcodon in its combination of grayish brown, black-staining, matted fibrillose pileus with an areolate disk at maturity, black staining trama, basidiospores that are 8−9 µm wide, and unique

ITS sequence.

Typification: PUERTO RICO: MUNICIPALITY CANOVANAS: El Yunque

National Forest; El Toro Trail, 18° 16’ 51.4”N; 65° 51’ 31.3” W, ~710 m; 5 Jun 1998,

Baroni 8776 (holotype: NY 02329649; isotype: CORT 011998). GenBank accession: ITS

KM668100.

Pileus initially conic with broad umbo, becoming planate or with an uplifted, broadly wavy margin, 12−30 mm wide, 8−12 mm tall, grayish brown (5D4), somewhat lighter near margin, staining black upon pressure, matted fibrillose throughout; disk finely areolate with age; trama 1−2 mm thick, creamy gray, blackening upon exposure.

Hymenophore dentate, adnate; teeth 1−2 mm long, light grayish brown (5C4), somewhat flattened over distal 1/3; tips bluntly acuminate, frequently bifurcate under hand lens.

Stipe equal, 22−48 × 5−7 mm, rounded at extreme base, matted fibrillose over lower 3/4, more glabrous at apex, grayish brown (~5D3), somewhat lighter near apex and base, staining black upon pressure, solid; trama grayish brown, staining black slowly upon

23 exposure. Odor none; taste bitter from dried specimen. Macrochemical reactions: all tissues dark blue to black with KOH and NH4OH on dried basidiomata. Dried pileus color olive gray (3E2−3E3), with bluish gray spots (21F3−21F4); context light brown

(6D4−6E4). Basidiospores 7−9 × 8−9 µm with ornamentation included (mean = 8.7 × 8.6

µm; n = 20), Q range = 0.77–1.13, Q mean = 1.0, subglobose to globose, strongly tuberculate, faint tan in H2O and KOH, inamyloid; tubercle apices mostly exsculpate with rounded corners, occasionally flat or rounded, also evident under SEM; hilar appendage

1−2 µm long. Basidia (30−)33−44(−48) × 8−13 µm apically, (5−)7−12 µm wide centrally, 2−4 µm at basal septum, clavate, curving centrally, with globular contents, faint gray in H2O and KOH; basal septum with clamp connection; sterigmata 4, (5−)6−7(−8)

µm long. Hymenial cystidia absent. Hymenophoral trama parallel, in mass light gray-tan to green-blue in H2O, light red-brown in KOH; individual hyphae light gray in H2O and

KOH; extracellular, granular dark blue-green pigment bodies numerous, these partially soluble in KOH; cells 3−5(−6) µm wide. Pileipellis a cutis of largely repent hyphae, in mass light gray to green-blue in H2O, dark gray-blue in KOH, with abundant irregularly- shaped dark gray-blue extracellular pigment bodies in H2O, these only partially soluble in

KOH; individual hyphae faint gray-green in H2O, faint gray to gray-blue in KOH, commonly with irregular bulges and constrictions, occasionally cylindrical; terminal cells undifferentiated, (5−)7−12 µm wide. Pileus trama in mass light tan to brown-orange in

H2O, light green-blue in KOH; individual hyphae faint tan-gray in H2O, faint gray in

KOH, cylindrical, rarely with irregular bulges and constrictions, (3−)5−10(−12) µm wide.

Stipitipellis a cutis of largely repent hyphae, in mass light gray-green in H2O, dark gray- blue in KOH; individual hyphae light gray in H2O, light gray-blue in KOH; terminal cells

2−3 µm wide, subclavate to subcapitate, with inflated tips 3−4 µm wide. Stipe trama in mass orange-brown in H2O, light orange-brown to light green-blue in KOH; individual hyphae faint tannish orange in H2O, faint tan to faint green-blue in KOH, (4−)6−7(−8)

µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Scattered on clay soils of forest floor in a lower montane wet forest; known only from the type locality in the El Yunque National Forest,

Puerto Rico.

Etymology. Portoricensis (-ensis Latin adj. B) = adjectival suffix indicating origin or place, referring to the type locality of the species in Puerto Rico.

Other specimen examined. PUERTO RICO: MUNICIPALITY CANOVANAS:

El Toro Trail, 18° 17’ 0” N; 65° 51’ 15” W, 650 m; 2 Jul 1998, coll. N. Pérez, S.A.

Cantrell & J. Ramírez, CFMR-PR-5421 (UPRRP).

Figure 5. Basidiomata of Sarcodon portoricensis (HOLOTYPE; Baroni 8776). Bar = 10 mm.

Figure 6. Basidia and basidiospores of Sarcodon portoricensis (HOLOTYPE; Baroni

8776). Bar = 10 µm.

Commentary. Sarcodon portoricensis is recognized in the field by its initially conic pileus becoming plane with an uplifted margin, grayish brown, black-staining and matted fibrillose pileus surface with a finely areolate disk at maturity, concolorous stipe, black staining trama, and relatively long, grayish brown, frequently apically bifurcate teeth. Sarcodon portoricensis can be differentiated from each of the other new

Neotropical species described here based on key features of the pileus surface, staining reactions of the exposed trama, stipe trama, basidiospore ornamentation, and stipitipellis

(Table I).

Sarcodon portoricensis is similar to S. atroviridis in its patch-like blackening of the pileus, non-decurrent hymenophore, glabrous stipe, and basidiospore dimensions.

Sarcodon portoricensis is distinguished from S. atroviridis by its pileus surface that is matted fibrillose and areolate over the disk (vs. tomentose to felted and non-areolate), black tramal staining reaction (vs. lilac to bluish grey), and matted fibrillose stipe surface

(vs. glabrous to pubescent) (Morgan 1895, Banker 1906, Coker and Beers 1951, Maas

Geesteranus 1971, Baird et al. 2013). Additionally, the ITS sequence of S. portoricensis is only ~87% similar to sequences of S. atroviridis from the southeastern USA and so these two species are distinct in the phylogenetic analysis (Figure 1).

Sarcodon portoricensis is similar to S. thwaitesii in its pileus shape, areolation, and black staining, but differs in its grayish brown pileus and stipe, and wider basidiospores (8–9 vs. 5.4–7.2 µm) (Berkeley and Broome 1873, Maas Geesteranus

1964, 1971, 1974a).

Both S. portoricensis and S. bambusinus have a grayish brown stipe with similar dimensions, and similar basidiospore and basidium lengths, but S. portoricensis differs from S. bambusinus in its pileus surface that is matted fibrillose and finely areolate over the disk (vs. villose to subfurfuraceous), black staining trama (vs. slowly fuliginous to fuscous), and basidiospore width (8–9 vs. 5–7 µm) (Baker and Dale 1951, Maas

Geesteranus 1974b).

Sarcodon portoricensis resembles Sarcodon quietus Maas G. from the Congo in its solid stipe, black staining trama and exsculpate basidiospore ornamentation, but differs in it grayish brown (vs. distinctly olivaceous) basidioma color, adnate (vs. decurrent) hymenophore, and areolate (vs. squamulose) pileus disc (Maas Geesteranus

1967).

Sarcodon quercophilus A. Grupe & D.J. Lodge, sp. nov. Figures 4, 7, 8

MycoBank MB 809568

Diagnosis: Sarcodon quercophilus differs from other species of Sarcodon in its combination of felty matted, dull violet pileus with substriate margin, hollow stipe with internal squamules, non-staining trama, basidia that are 22–30 × 11–16 µm, basidiospores that are 5−7 µm long, and unique ITS sequence.

Typification: BELIZE: TOLEDO – CAYO DISTRICT: Chiquibul National Park,

Maya Mountains, Doyle’s Delight, elevation 1070 m; East Trail, 12 Aug 2004, coll.

Lodge, CFMR-BZ-3833 (holotype: NY 02329783; isotype: HSU). GenBank accession:

ITS KM668101.

Pileus convex, 18–33 mm broad, 40–74 mm tall, dull violet (15E4–15F4) throughout; surface an appressed felted mat, lighter concolorous and substriate at margin; trama tan, 4 mm thick above stipe, unchanging. Hymenophore dentate, adnate; teeth blunt-acuminate, pale yellow when young (3A2–4A2), browning slightly with age. Stipe subclavate, somewhat flattened, 40–75 × 4–8 mm, broadening over basal ¼ to 5.7–11.5 mm wide; surface a submoist, felted mat, dull violet (15E4–15F4); trama substuffed to hollow, with internal stipe squamules observable sections of the dried stipe (not noted in field), unchanging; basal mycelium lighter concolorous, matted. Odor none; taste bitter.

Macrochemical reactions: all tissues staining dark blue to black with KOH and NH4OH on dried basidiomata. Dried pileus color grayish green (1D3−1D4) over disk, with bluish gray spots (22F3−22F4); context grayish yellow (3C2−3C3). Basidiospores 5–7 × 7–9

µm including ornamentation (mean = 5.7 × 8 µm; n = 40), Q range = 0.63–0.88, mean Q

= 0.71, oblate, tuberculate, tan in H2O, lighter concolorous in KOH, inamyloid; tubercles tall, appearing flat in polar view, in side view exsculpate with two prominent, rounded or finely pointed to mammillate apices, these projecting outward, also evident under SEM; hilar appendage 1–2 µm long. Basidia 22–30(−36) × 10–16 µm apically, 5–10 µm centrally, 2–3 µm basally, clavate, occasionally curved near center, faint gray in H2O and

KOH; sterigmata 4, curved, 4–7 µm long. Hymenial cystidia absent. Hymenophoral trama of parallel, slightly interwoven hyphae, in mass gray in H2O and KOH; individual hyphae light gray in H2O and KOH, cylindrical, (2−)3–5 µm wide. Pileipellis a cutis of largely repent hyphae, in mass dark yellow-green in H2O, faint tan in KOH; individual hyphae faint gray to faint yellow-green in H2O, faint gray-blue in KOH, mostly cylindrical, infrequently with irregular bulges and constrictions, 4–7 µm wide, sometimes branching in subpellis; terminal cells undifferentiated. Pileus trama in mass light gray- green to brown-green in H2O, light gray-blue overall in KOH, blue-green where granular pigment bodies present, light brown-orange where pigment bodies absent; extracellular pigment bodies in KOH irregularly shaped, granular, blue-green, solubilizing gray-blue into solution; individual hyphae faint gray-tan in KOH, faint gray-green in H2O, cylindrical or often with irregular bulges and constrictions, frequently branching, 5–10

µm wide. Stipitipellis a cutis of strongly interwoven repent hyphae, in mass gray-green in

H2O, blue-green in KOH; extracellular pigment bodies abundant, of two types, some amber and irregularly shaped, these persistent in KOH and H2O, others irregularly shaped, small, granular, blue-green, solubilizing gray-blue into solution in KOH and staining tramal hyphae; individual hyphae faint gray-green in H2O, faint gray to gray-

31 blue in KOH, mostly cylindrical; 2–4 µm wide; terminal cells with slightly inflated apex and 2–8 irregular serpentine undulations. Stipe trama in mass light gray-green in H2O, light gray-tan in KOH; individual hyphae faint gray or tan in H2O and KOH, mostly cylindrical, infrequently with irregular bulges and constrictions, infrequently branching,

(4–)5–7(−9) µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Scattered on soil in montane cloud forest under

Quercus; known only from the type locality in the Maya Mountains of Belize.

Etymology. Quercophilus (-philus in Greek comp.) = Quercus (oak) loving; referring to occurrence of the species in Quercus-dominated forests.

Other specimen examined. BELIZE: TOLEDO – CAYO DISTRICT: North Ridge

Trail, 16° 29” N; 89° 02’ W, elevation 1100 m, 19 Aug 2004, coll. Lodge, CFMR-BZ-

3928 (NY 02329784; BRH).

Figure 7. Basidiomata of Sarcodon quercophilus (HOLOTYPE; CFMR-BZ-3833). A.

Dorsal view. B. Ventral view, showing pale hymenophore. Bars = 10 mm.

Figure 8. Basidia, basidiospores, and stipitipellis terminal cells of Sarcodon quercophilus

(HOLOTYPE; CFMR-BZ-3833). Bar = 10 µm.

Commentary. Sarcodon quercophilus is recognized in the field by its convex, felty matted pileus that is dull violet throughout with lighter concolorous, substriate outer margin, adnate hymenophore of pale yellow teeth, and concolorous, relatively long, subclavate, laterally compressed, hollow stipe with internal squamules. Sarcodon quercophilus can be differentiated from each of the other new Neotropical species described here based on key features of the pileus surface, staining reactions of the exposed trama, stipe trama, basidiospore ornamentation, and stipitipellis (Table I).

Sarcodon quercophilus is similar to S. atroviridis in the dark, somber colors of the fresh basidiomata and felty matted pelli. Sarcodon quercophilus can be distinguished from S. atroviridis by its substriate pileus margin (vs. smooth), grayish green to bluish gray dried pileus (vs. olivaceous), shorter basidia (22–30 vs. 31−42 µm), shorter basidiospores (5−7 vs. 7.2−9 µm), and stipitipellis terminal cells with serpentine undulations (Morgan 1895, Banker 1906, Maas Geesteranus 1971, Baird et al. 2013).

Additionally, the ITS sequence of S. quercophilus is only ~95% similar to sequences of

S. atroviridis from the southeastern USA and so these two species are distinct in our phylogenetic analysis (Figure 1).

Sarcodon quercophilus is similar to S. thwaitesii in overall basidioma colors, the long, hollow stipe, and basidium length, but differs in its appressed felty, marginally substriate pileus (vs. short erect tomentum throughout), appressed felty stipe (vs. tomentose to glabrescent), and narrower basidia (5–10 vs. 10–13 µm) (Berkeley and

Broome 1873, Maas Geesteranus 1964, 1971, 1974a).

Sarcodon quercophilus and S. bambusinus have similar basidioma shapes, dimensions and basidium lengths, but S. quercophilus differs from S. bambusinus in its appressed felty, marginally substriate pileus, non-staining trama, wider basidium apices

(11−16 vs. 8−11 µm), and wider basidiospores (7−9 vs. 5−7 µm) (Baker and Dale 1951,

Maas Geesteranus 1974b).

Sarcodon quercophilus resembles Sarcodon procerus Maas G. from the Congo in its basidioma colors, striate pileal margin, hollow stipe, and lack of tramal staining upon exposure, but differs in its narrower pileus (18–33 vs 40–60 mm), adnate (vs. decurrent) hymenophore, shorter, broader basidiospores (5–7 × 7–9 vs. 6.7–8 × 4.5–5.4 µm) with strongly exsculpate (vs. rounded) ornamentation (Maas Geesteranus 1967).

Sarcodon umbilicatus A. Grupe, T.J. Baroni & D.J. Lodge, sp. nov. Figures 4, 9, 10

MycoBank MB 809569

Diagnosis: Sarcodon umbilicatus differs from other species of Sarcodon in its combination of grayish brown to fuscous, rugulose, umbilicate, brown staining pileus, adnexed hymenophore, hollow stipe with internal squamules, grayish brown staining trama, basidiospores that are 6–7 × 7–8 µm, and unique ITS sequence.

Typification: BELIZE: TOLEDO – CAYO DISTRICT: Chiquibul National Park,

Maya Mountains, Doyle’s Delight, North East Trail to creek, 16° 29’ 42,0”N; 89° 02’

37.3”W, elevation 1038 m; 26 Aug 2007, Baroni 10201 (holotype: NY 02329650; isotype: CORT 011996). GenBank accession: ITS KM668102.

Pileus convex to broadly convex and umbilicate, 20–60 mm wide, 13–16 mm tall, initially grayish brown (6C2), Dark Drab in center, Natal Brown or Army Brown on margin, with age to dark brown (7F3), Sepia, margin sometimes concentrically banded; umbilicus deep, occasionally perforate and continuous with the hollow stipe; surface rugulose, matted fibrillose under hand lens, staining darker brown when handled; trama light brown (5D4), staining fuscous slowly upon exposure, 6 mm thick above stipe.

Hymenophore dentate, adnexed; teeth 3–4 mm long centrally, 2–3 mm near margin, 1 mm near stipe, acuminate, grayish orange (5B3; 5D4 in side view), Straw Yellow, becoming Drab Gray. Stipe equal, 20–55 × 4–6 mm, slightly tapered at base, concolorous with pileus, darkening to fuscous with handling, subglabrous, hollow, with minute internal squamules; basal mycelium a low grayish mat. Odor indistinct; taste bitter.

Macrochemical reactions: all tissues dark blue to black with KOH and NH4OH on dried

37 basidiomata. Dried pileus color yellowish gray to olive (3C2−3D2); context yellowish gray (2B2−2C2). Basidiospores (5–)6–7 × (6–)7–8(−9) µm including ornamentation,

(mean = 6 × 7.6 µm; n = 40), Q range = 0.63–1.0, mean Q = 0.83, oblate, tuberculate, faint brown in H2O and KOH, inamyloid; tubercles short, apices usually rounded, occasionally flattened, rarely exsculpate, also evident under SEM; hilar appendage 1–1.2

µm long. Basidia (30–)32–38(−40) × 9–10 µm apically, 5–7 µm centrally, (2–)3–4 µm basally, clavate, regularly curved centrally, faint tan in H2O, gray in KOH; sterigmata 4, barely curving, 5–6 µm long. Hymenial cystidia absent. Hymenophoral trama of parallel, slightly interwoven hyphae, diverging slightly at subhymenium, in mass light gray in

H2O, faint tan in KOH; individual hyphae light gray in H2O, faint gray in KOH, with two types of irregularly shaped extracellular pigment bodies, some which are large, amber, and non-soluble in H2O and KOH, others which are small, granular, blue-green and solubilizing gray-blue into solution in KOH; cells (2–)3–5(−6) µm wide. Pileipellis a cutis, in mass overall light brown-green in H2O, dark blue-green or faint tan in KOH depending on concentration of pigment bodies; individual hyphae faint tan in H2O, with scattered light blue-green internal granulose pigments, faint gray-blue in KOH; terminal cells undifferentiated, 4–7 µm wide. Pileus trama in mass light gray or light tan in H2O, light tan or gray-blue in KOH; individual hyphae faint gray in H2O, faint gray-blue or faint tan in KOH, frequently with irregular bulges and constrictions, occasionally cylindrical, (3–)5–7(−8) µm wide, with rare hyphal cells containing copious intracellular, evenly-dispersed pigments, these uniformly dark gray-blue in KOH, inevident in H2O.

Stipitipellis a cutis of largely repent, interwoven hyphae, difficult to separate, in mass

38 light gray-green in H2O, light grayish to greenish blue in KOH; individual hyphae faint gray in H2O, faint green-gray in KOH, mostly cylindrical with undifferentiated terminal cells, 2–3(−5) µm wide, interspersed with scattered hyphae with subclavate to subcapitate terminal cells, these (2–)3–5 µm wide. Stipe trama in mass light brown-orange in H2O, lighter in KOH, with pockets devoid of hyphae, these (7–)12–114(−156) µm across longest axis; individual hyphae faint gray in H2O and KOH, irregularly bulging, occasionally cylindrical, sometimes branching, (4–)7–12 µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Scattered on soil in montane cloud forest under

Quercus; known only from the type locality in the Maya Mountains of Belize.

Etymology. Umbilicatus (Latin adj. A) = navel-like, having a small central depression; referring to the umbilicate pileus of the species.

Other specimens examined. BELIZE: TOLEDO – CAYO DISTRICT: Chiquibul

National Park, Maya Mountains, Doyle’s Delight, North East Trail to creek, 16° 29’

42,0”N; 89° 02’ 37.3”W, elevation 1131 m; 20 Aug 2007, Baroni 10113 (CORT

011997); Doyle’s Delight, North Ridge Trail, 16° 30’ 14.3”N; 89° 03’ 04.3”W, elevation

1100 m, 24 Aug 2007, coll. S. Matola, CFMR-PR-4332 (NY 02329785; HSU); elevation

1100 m, 26 Aug 2007, coll. Lodge, CFMR-BZ-4382 (NY 02329786; HSU).

Figure 9. Basidiomata of Sarcodon umbilicatus (HOLOTYPE; Baroni 10201). Bar = 10 mm.

Figure 10. Basidia and basidiospores of Sarcodon umbilicatus (HOLOTYPE; Baroni

10201). Bar = 10 µm.

Commentary. Sarcodon umbilicatus is recognized in the field by its convex to broadly convex, umbilicate pileus with a rugulose surface that is initially grayish brown to fuscous at maturity, the dark brown to fuscous staining of the various tissue layers upon pressure or exposure, and hollow stipe with internal squamules. Sarcodon umbilicatus can be differentiated from each of the other new Neotropical species described here based on key features of the pileus surface, staining reactions of the exposed trama, stipe trama, basidiospore ornamentation, and stipitipellis (Table I).

Sarcodon umbilicatus is similar to S. atroviridis in its convex to broadly convex pileus and basidiospore widths. Sarcodon umbilicatus can be distinguished from S. atroviridis by its pileus that is consistently fuscous (7F3) at maturity (vs. brownish grey

[5D3–5E3], grayish violet [15F3] or black), matted fibrillose to rugulose, umbilicate pileus (vs. tomentose to felted/glabrous and lacking an umbilicus), dark staining reactions with pressure or exposure, concolorous stipe, and shorter basidiospores (5–7 vs. 7.2–9

µm) (Morgan 1895, Banker 1906, Maas Geesteranus 1971, Baird et al. 2013).

Additionally, the ITS sequence of S. umbilicatus is only ~82% similar to sequences of S. atroviridis from the southeastern USA and so these two species are distinct in the phylogenetic analysis (Figure 1).

Sarcodon umbilicatus is similar to S. thwaitesii in its hollow mature stipe, basidium lengths, and basidiospore dimensions, but differs in its umbilicate pileus with a rugulose surface (vs. short tomentose), and longer teeth (3–5 vs. 1–3 mm) (Berkeley and

Broome 1873, Maas Geesteranus 1964, 1971, 1974a).

Sarcodon umbilicatus and S. bambusinus have initially grayish brown pilei and concolorous stipes, a grayish brown tramal staining reaction, and tooth lengths up to 5 mm, but S. umbilicatus differs from S. bambusinus in its matted fibrillose to rugulose pileus (vs. villose to subfurfuraceous), presence of an umbilicus, and wider basidiospores

(7–8 vs. 5–7 µm) (Baker and Dale 1951, Maas Geesteranus 1974b).

Key to Neotropical Sarcodon species, with selected extralimital taxa

1. Pileus pinkish gray, with scattered darker purplish spots, with age developing irregular black patches, or grayish lilac or dark purple when young………………………………………………………………………...……...…...2

1. Pileus color not as above, some shade of grayish, orangish, with reddish tints, or yellowish brown……...……………………………………………………………..…...4

2 (1). Pileus surface irregularly pitted; a densely interwoven mat under hand lens; trama slowly staining pink upon exposure; Guyana, in Pakaraimaea (Dipterocarpaceae) forests

……………………………………………...... ………...Sarcodon pakaraimensis

2. Pileus surface not as above; trama unchanging or not staining as above.…..………………………………………………………………………..….……3

3 (2). Pileus surface appressed, felted; substriate at margin; stipitipellis terminal cells with 2–8 serpentine undulations; Belize, in montane Quercus forests…………………………………………..…...... Sarcodon quercophilus

3. Pileus surface erect tomentose or velutinous, eventually areolate; stipitipellis terminal cells undifferentiated; Malaysian Archipelago and New Guinea (Dipterocarpaceae and

Fagaceae), New Zealand in Nothofagus forests..………...... …Sarcodon thwaitesii

4 (1). Pileus surface tomentose, felted or pubescent, eventually glabrous; basidioma some shade of orangish to olivaceous brown to sepia; drying olivaceous; teeth up to 16 mm long; eastern North America, Europe, East Asia……...... Sarcodon atroviridis

4. Pileus surface not as above, or not with those combinations; basidioma some shade of grayish brown, fuscous, or reddish tints; not drying olivaceous; teeth not exceeding 6 mm……………………………………………………………………………………….5

5 (4). Pileus regularly with a deep umbilicus; Belize, in montane Quercus forests………

……………………...………………..………………………...... Sarcodon umbilicatus

5. Pileus without an umbilicus……………………………….…………………………6

6 (5). Pileus surface matted fibrillose throughout, with age finely areolate over disk; all tissues quickly staining black upon pressure or exposure; Puerto Rico, in lower montane wet forests……………………………………………..…………Sarcodon portoricensis

6. Pileus surface not as above; tissues staining differently, or not at all…...……………7

7 (6). Pileus surface villose or subfurfuraceous, more repent fibrillose near margin; stipe staining slowly fuliginous to fuscous with pressure or age; Trinidad, Brazil, in lowland tropical rainforests.……...………..…………...…...... ……..…Sarcodon bambusinus

7. Pileus smooth to fibrillose, with reddish tones; stipe not staining as above; Colombia

…………………………………..………………………….……Sarcodon colombiensis

CHAPTER 2

SARCODON IN THE NEOTROPICS II. FOUR NEW SPECIES FROM COLOMBIA

Submitted to Mycologia, May 2015

Abstract: This work reports on four species of the ectomycorrhizal (ECM) tooth fungus genus Sarcodon (Bankeraceae, Thelephorales, Basidiomycota) recently discovered in the Colombian Amazon. Sarcodon colombiensis sp. nov., Sarcodon rufogriseus sp. nov., Sarcodon pallidogriseus sp. nov., and Sarcodon bairdii are described as new to science. These fungi occur in forests dominated by the ECM trees

Pseudomonotes tropenbosii (Dipterocarpaceae), Dicymbe uaiparuensis (Fabaceae subfam. Caesalpinioideae) and Aldina sp. (Fabaceae subfam. Papilionoideae). These records bring the number of Sarcodon species known from the Neotropics to 10, and demonstrate that, while being less diverse in the tropics relative to temperate and boreal regions, the genus has a wider distribution and putative host range than previously thought. Each of the new species possesses the accepted diagnostic characters for the genus: pileate-stipitate stature, a dentate hymenophore, determinate basidiomata development, fleshy, non-zonate context, and brown, tuberculate basidiospores.

Molecular phylogenetic analysis corroborated the generic placement of the species, and, in combination with morphological characters, confirmed that they are new to science.

Macromorphological, micromorphological, habitat, and DNA sequence data from the nuc

46 rDNA internal transcribed spacer region (ITS) is provided for S. colombiensis, S. rufogriseus, S. pallidogriseus, and S. bairdii.

Key words: Bankeraceae, Colombia, ectomycorrhizal fungi, Guiana Shield,

Guyana, Thelephorales, tooth fungi

Introduction

Grupe et al. (2015) summarized the current knowledge of the ectomycorrhizal

(ECM) fungal genus Sarcodon Quél. ex P. Karst. (Bankeraceae, Thelephorales,

Basidiomycota) in the Neotropics and described four new species from Belize, Guyana, and Puerto Rico. These new Sarcodon species were associated with a diverse assemblage of putative ECM broadleaf host tree species in the genera Dicymbe (Fabaceae subfam.

Caesalpinioideae), Pakaraimaea (Dipterocarpaceae), and Quercus (Fagaceae), and brought the number of species known from the Neotropics to six. Neotropical species diversity for this largely Nearctic, conifer-associated genus is still low, given that >91 names that have been proposed worldwide in Sarcodon.

Recent macrofungal collecting efforts in Amazonian Colombia have revealed a diverse assemblage of putatively ECM fungi (e.g. Vasco-Palacios et al. 2014). Here we describe Sarcodon colombiensis sp. nov. and Sarcodon rufogriseus sp. nov. from

Colombian forests dominated by the ECM trees Dicymbe uaiparuensis Cowan, Aldina sp.

(Fabaceae subfam. Papilionoideae), and Pseudomonotes tropenbosii A.C. Londoño, E.

Alvarez & Forero (Dipterocarpaceae) from El Zafire, Amazonas. Sarcodon

47 pallidogriseus sp. nov. and Sarcodon bairdii sp. nov. are described from from a white sand forest dominated by D. uaiparuensis and Aldina spp. in Puerto Santandar,

Amazonas.

Each of the four new species are morphologically similar to, but distinguishable from, a group of previously described species characterized by non-scaly pilei, overall somber colors, and KOH-soluble bluish green pigments (Maas Geesteranus 1971), including Sarcodon atroviridis (Morgan) Banker from temperate North America, Europe, and East Asia, Sarcodon thwaitesii (Berk. & Br.) Maas G. from the Asian tropics, and

Sarcodon bambusinus (Baker & Dale) Maas G. from the Neotropics (Berkeley and

Broome 1873, Morgan 1895, Baker and Dale 1951, Maas Geesteranus 1964, 1974a,

1975). The recently described Neotropical species S. pakaraimensis A. Grupe & T.W.

Henkel, S. portoricensis A. Grupe & T.J. Baroni, S. quercophilus A. Grupe & D.J.

Lodge, and S. umbilicatus A. Grupe, T.J. Baroni & D.J. Lodge, also fall into this rather narrow morphological group (Grupe et al. 2015). Nonetheless, variation in key morphological characteristics corroborated by molecular phylogenetic analysis, supports the recognition of S. colombiensis, S. rufogriseus, S. pallidogriseus, and S. bairdii as unique species. Macromorphological, micromorphological, habitat, and DNA sequence data from the nuc rDNA internal transcribed spacer region (ITS) DNA sequence data from ITS region are provided for each of the new species.

Materials and Methods

Collections Collections of S. colombiensis and S. rufogriseus were made in Jan

2012 from El Zafire in Amazonas, Colombia at 4˚00`69’’S; 69º53’97’’W; elevation

~180−220 m, along a trail in a mixed ectotrophic forest dominated by P. tropenbosii, D. uaiparuensis, and Aldina sp.. Collections of S. pallidogriseus and S. bairdii were made in

May 2005 from Resguardo de Monochoa, Comunidad de Chukiki, Puerto Santandar,

Amazonas, 0°40’S; 72°31’ W; elevation ~150 m, in forests where the putative ECM hosts are D. uaiparuensis and Aldina spp.. Macroscopic features of basidiomata were described fresh in the field. Colors were described subjectively and coded according to

Kornerup and Wanscher (1978), with color plates noted in parentheses. Collections of fresh basidiomata were dried in the field using silica desiccant beads. For S. colombiensis, a bare macromorphological description is provided because a thorough field description was not documented due to assumed conspecificity with S. pallidogriseus and S. bairdii. Micromorphological features of dried specimens were examined with an Olympus BX51 microscope with light and phase contrast optics.

Fungal tissue was mounted in H2O, 3% potassium hydroxide (KOH), or Melzer's solution. At least 20 individual basidiospores, basidia, and other structures were measured per collection; for basidiospores, dimensional measurements include ornamentation. Range and mean quotients of basidiospore length divided by width (Q) were calculated. Outlying measurements observed in less than 5% of the measured

49 population, are indicated in parentheses. Specimens were deposited in the following herbaria (Holmgren et al. 1990): HUA – University of Antioquia Herbarium.

DNA extraction, amplification, sequencing, and phylogenetic analyses.

DNA extraction, polymerase chain reactions (PCR), cloning and sequencing followed the standard protocols of Gardes and Bruns (1993); details of modifications used in this study are given in Grupe et al. (2015).

Parsimony (MP) analysis was completed with default settings in PAUP* 4.0 (Swofford

2003). A Maximum Likelihood (ML) search was run in GARLI (Zwickl 2006) with the

GTR+I+G model. Support for phylogenetic relationships was assessed based on 500 bootstrap replicates in PAUP (for MP) and Garli (for ML).

Results

blastN queries for ITS sequences of S. colombiensis, S. rufogriseus, S. pallidogriseus, and S. bairdii reported here produced best matches to species of Sarcodon but none exceeded 95% similarity. The MP search found four equally parsimonious trees with a score of 384 steps. The ML tree, with a likelihood score of

-ln 2448.83248, differed from the topology of the MP tree in that the placement of S. umbilicatus was unresolved relative to whether it is most closely related to the S.

50 atroviridis clade or the S. pakaraimensis clade (data not shown). These analyses demonstrated that S. colombiensis, S. rufogriseus, S. pallidogriseus, and S. bairdii are phylogenetically distinct from other Neotropical Sarcodon spp.. A midpoint rooted tree is shown and “thelephoroid mycorrhizal root tip” was used as the outgroup. Therefore, our analysis necessarily focused on the placement of S. colombiensis, S. rufogriseus, S. pallidogriseus, and S. bairdii and could not resolve other phylogenetic relationships within the genus, nor address the position of Sarcodon within the Bankeraceae or

Thelephorales. Sarcodon colombiensis had the closest affinity with S. pakaraimensis, with the two species occurring in a well-supported subclade (Figure 11; 97% ML, 100%

MP support). Sarcodon rufogriseus occurred in a poorly supported relationship as basal to the S. atroviridis subclade (Figure 11; 36% ML). Sarcodon pallidogriseus and S. bairdii occurred in a basal clade to the S. pakaraimensis subclades (Figure 11; 59% and

88% ML respectively).

Sequences for all taxa, Sarcodon species found outside of the Neotropics, and previously described Neotropical species, were obtained from GenBank with numbers shown next to species epithets.

Taxonomy

Sarcodon colombiensis A. Grupe & A.M. Vasco-Pal., sp. nov. Figure 12

Diagnosis: Sarcodon colombiensis differs from other species of Sarcodon in its overall basidioma colors of dark gray to black, umbonate pileus, and unique ITS sequence.

Typification: COLOMBIA: AMAZONAS: El Zafire, along trail, 4˚00`69’’S;

69º53’968’’W; elevation ~180−220 m; 9 Jan 2012, Vasco 2084 (holotype: HUA 186215).

GenBank accession: ITS KP972654.

Pileus umbonate, up to 30 mm broad, hygrophanous, dark gray to nearly black, pileus staining reactions, surface features, and trama characteristics not recorded.

Hymenophore dentane, adnate, teeth up to 3 mm long, sharp, yellowish. Stipe equal, up to 50 mm long, 3-4 mm wide, smooth surface, concolorous with the pileus, hygrophanous. Odor not recorded; flavor not recorded. Macrochemical reactions: all tissues staining dark blue with KOH on dried basidiomata. Basidiospores 5−6 (−7) × (6−)

7−8 µm including ornamentation (mean = 5.95 × 7.45 µm; n=40), Q range = 0.75−0.86, mean Q = 0.80, oblate, tuberculate, light brown in H2O, light tan in KOH, inamyloid; tubercle apices variable in height, blunt-rounded, exsculpate; hilar appendage 1 µm long.

Basidia (25−) 28−37 (−41) × 10−12 µm apically, 6−8 (−10) µm centrally, 2−4 µm at basal septum, clavate, occasionally with a central constriction, light tan to light gray in

H2O, faint gray in KOH; basal septum with clamp connection; sterigmata four, curved,

4−5 (−7) µm long. Hymenial cystidia absent. Hymenophoral trama subparallel to slightly

53 divergent, in mass light reddish brown to light gray in H2O, bright green-blue in KOH; individual hyphae light tan to light gray in H2O, faint gray to faint green-blue in KOH,

(3−) 5 (−10) µm wide, with copious blue-black pigment bodies. Pileipellis a cutis of repent hyphae, in mass light tan to light gray in H2O, light tan to light blue-green in

KOH, with blue-black pigment bodies scarce compared to other tissues, these eventually dissolving and leaching into solution; individual hyphae light tan to faint gray in H2O, light gray in KOH, 4−6 (−10) µm wide, infrequently branching near the basal clamp connection; terminal cells undifferentiated. Pileus trama in mass brown-orange in H2O, green-blue or light tan in KOH, pigment bodies present, of two types, some small, clustered, and blue-black, dissolving to bluish green and leaching into solution in KOH, others relatively large and copious, dark yellow-orange in H2O, more or less soluble in

KOH; individual hyphae frequently terminating in bifurcating tips of unequal lengths, others cylindrical and unbranched, (4−) 8−11 (−12) µm wide. Stipitipellis a cutis of repent hyphae, in mass gray or tan in H2O, light tan in KOH, with scattered clusters of irregularly-shaped, extracellular, granular pigment bodies, these blue-black in H2O, bluish green in KOH and eventually dissolving and leaching into solution; individual hyphae light gray to faint tan in H2O, gray in KOH, cylindrical, (2−) 3−4 (−5) µm wide; terminal cells undifferentiated. Stipe trama in mass brown-orange or faint tan in H2O, light tan to light green-blue in KOH, with scattered extracellular granular pigment bodies, these dark blue to nearly black in H2O, dark bluish green in KOH; individual hyphae light gray to faint tan in H2O, light gray to light green-blue in KOH, more or less cylindrical,

(5−) 8−11 (−15) µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Solitary to scattered, on soil in forests with D. uaiparuensis, Aldina sp., or P. tropenbosii; known only from the type locality in El

Zafire, Colombia.

Etymology. Colombiensis (-ensis Latin adj. B) = adjectival suffix indicating origin or place, referring to the type locality of the species in Colombia.

Figure 12. (A, B) Basidia, (C) basidiospores, (D, E) and terminal cells of the pileus trama of Sarcodon colombiensis (HOLOTYPE; Vasco 2084). Bar = 10 µm.

Commentary. Sarcodon colombiensis can be differentiated from each of the four

Neotropical species recently described in Grupe et al. (2015) and here based on key features of basidioma color and terminal cells of the pileus trama hyphae (Table I).

Sarcodon colombiensis and the Neotropical S. bambusinus have a similar pileus shape, non-decurrent teeth, similar tooth lengths, stipe lengths, and basidium lengths, but

S. colombiensis differs from S. bambusinus in its dark gray to black (vs. vinaceous drab, becoming fuscous or fuliginous with age) basidioma colors, yellowish teeth (vs. pallid fuliginous to ochraceous), and shorter basidiospores (5−6 vs. 6.5−9 µm) (Baker and Dale

1951, Maas Geesteranus 1974a).

Sarcodon colombiensis resembles the Paleotropical S. thwaitesii in pileus shape, stipe length, surface texture of the stipe, and basidium lengths. Sarcodon thwaitesii is distinguished from S. colombiensis by its grayish lilac to dark purple colors, white, grayish, or brown teeth (vs. yellowish), and shorter basidiospores (5−6 vs. 7.6−9.4 µm)

(Berkeley and Broome 1873, Maas Geesteranus 1964, 1971, 1974b).

Among extratropical species, S. colombiensis is most similar to the north temperate

S. atroviridis in that both have a similar stipe surface texture, and basidia sizes (Morgan

1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013). Sarcodon colombiensis can be distinguished from S. atroviridis in its umbonate pileus (vs. convex to planar), shorter teeth (< 3 vs. 1−16 mm), shorter basidiospores (5−6 vs. 8–9 µm), and bifurcate pileus trama hyphae (vs. unbranching) (Morgan 1895, Banker 1906, Coker and Beers 1951,

Baird et al. 2013). Additionally, the ITS sequence of S. colombiensis is only ~85%

57 similar to sequences of S. atroviridis from the southeastern USA and these two species are distinct in the phylogenetic analysis (Figure 11).

Sarcodon rufogriseus A. Grupe & A.M. Vasco-Pal., sp. nov. Figures 13,14

Diagnosis: Sarcodon rufogriseus differs from other species of Sarcodon in its combination of overall basidioma colors of dark gray to black with reddish tones

(7E1−7F1), umbonate pileus with a smooth to fibrillose pileus surface, unchanging pileus trama, initially reddish brown stipe trama, and unique ITS sequence.

Typification: COLOMBIA: AMAZONAS; El Zafire at 4˚00`69’’S; 69º53’968’’W; elevation ~180-220 m; along trail, Vasco 1989 (holotype: HUA 186216). GenBank accession: ITS KR698937.

Pileus conical when young, parabolic to plane with age, 12−70 mm broad, 2−3 mm tall, dark gray to black with reddish tones (7E1−7F1), hygrophanous; surface smooth to fibrillose, umbonate; margin eroded; trama not staining upon exposure. Hymenophore dentate; teeth 2−4 mm long, apices sharply conical, concolorous with pileus, delicate, easily rubbed off. Stipe subequal, 30−50 × 3−6 mm, cylindrical to slightly clavate, concolorous with pileus; surface glabrous; interior trama initially reddish brown, turning black with damage or age; base without basal mycelium. Odor not distinguishable; taste bitter. Macrochemical reactions: black with KOH on fresh basidioma. Basidiospores 5−7

× 7−9 µm including ornamentation (mean = 5.75 × 7.7 µm; n =20), Q range =

0.62−0.857, Q mean = 0.75, suboblate, tuberculate, gray-tan in H2O, tan in KOH,

58 inamyloid; tubercles prominent in polar view, less so in side view, predominantly exsculpate; hilar appendage 1−2 µm. Basidia (25−) 30−38 (−42) × (9−) 11−13 µm wide apically, 5−9 µm wide centrally, 3−4 µm at basal septum, clavate, gray in H2O and KOH; basal septum with clamp connection; sterigmata four, curved, 5−7 µm long. Hymenial cystidia absent. Hymenophoral trama parallel, predominantly gray in mass in H2O, bright blue-green in KOH; individual hyphae gray in H2O, light gray or light blue-green in

KOH, 3−5 µm wide, with copious granular dark gray-blue pigment bodies, these soluble in KOH. Pileipellis a cutis of repent hyphae, gray-blue in mass in H2O, light tan or blue- green in KOH, with copious granular dark gray-blue pigment bodies, these soluble in

KOH; individual hyphae gray to light tan in H2O, light gray in KOH, 4−6 µm wide, cylindrical; terminal cells undifferentiated. Pileus trama tan to brown-orange in mass in

H2O, tan to light tan or bright blue-green where pigment bodies have dissolved in KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light gray to light tan in H2O, gray or light blue in KOH, frequently terminating in bifurcating tips of unequal lengths, others cylindrical and unbranched, (3−) 6−10 µm wide. Stipitipellis a cutis of repent hyphae, in mass brown-orange or dark blue where pigment bodies are dense in H2O, tan or bright blue-green in KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light tan or light gray in H2O, light gray or light blue-green in KOH, cylindrical, 2−4 µm wide; terminal cells undifferentiated. Stipe trama brown-orange in mass in H2O, tan or blue-green in

KOH, copious granular dark gray-blue pigment bodies present, soluble in KOH;

59 individual hyphae color light tan in H2O, light tan or light gray in KOH, frequently terminating in bifurcated tips of unequal lengths, others cylindrical and unbranched, 4−8

(−10) µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Solitary to gregarious on soil in forests with either

D. uaiparuensis, Aldina sp., or P. tropenbosii, known only from the type locality in El

Zafire, Colombia.

Etymology. Rufo- (Latin adj. A) = adjectival prefix indicating red, and -griseus

(Latin adj. B) = adjectival suffix indicating gray, referring to the reddish gray coloration of the fresh basidiomata.

Figure 13. Basidiomata of Sarcodon rufogriseus (HOLOTYPE; Vasco 1989). Bar = 10 mm.

Figure 14. Basidia (A.), basidiospores (B.), and terminal cells of the pileus (C.,D.) and stipe trama (E.) of Sarcodon rufogriseus (HOLOTYPE; Vasco 1989). Bar = 10 µm.

Commentary. Sarcodon rufogriseus can be differentiated from each of the other

Neotropical species described in Grupe et al. (2015) and here based on its overall

62 basidiomata color, features of the pileus surface, lack of a pileus staining reaction, and the morphology of the pileus trama terminal cells (Table I).

Sarcodon rufogriseus and the Neotropical S. bambusinus have a similar pileus shape, non-decurrent teeth, and similar tooth, stipe, and basidium lengths, but S. rufogriseus differs from S. bambusinus in its basidioma colors of dark gray to black with reddish tones (vs. vinaceous drab, becoming fuscous or fuliginous with age), maximum pileus size (70 vs. 50 mm), smooth to fibrillose pileus (vs. villose to subfurfuraceous), and shorter basidiospores (5−7 vs. 6.5−9 µm) (Baker and Dale 1951, Maas Geesteranus

1974a).

Sarcodon rufogriseus resembles the Paleotropical S. thwaitesii in pileus shape, stipe length, surface texture of the stipe, and basidium lengths. Sarcodon thwaitesii is distinguished by its grayish lilac to dark purple colors, tomentose pileus, and taller basidiospores (7.6−9.4 vs. 5−7 µm) (Berkeley and Broome 1873, Maas Geesteranus

1964, 1971, 1974b).

Among extratropical species, Sarcodon rufogriseus is most similar to the north temperate

S. atroviridis in that both have a similar range of pileus sizes and shapes, stipe surface texture, and basidium sizes (Morgan 1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013). Sarcodon rufogriseus can be distinguished from S. atroviridis in its colors of dark gray to black with reddish tones (vs. brownish gray, grayish violet, or black), unchanging pileus trama (vs. lilac, later bluish gray), shorter teeth (1−5 vs. 1−16 mm), shorter basidiospores (5−7 vs. 8–9 µm), and bifurcate pileus and stipe tramal hyphae (vs. unbranching) (Morgan 1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013).

Additionally, the ITS sequence of S. rufogriseus is only ~82% similar to sequences of S. atroviridis from the southeastern USA and these two species are distinct in the phylogenetic analysis (Figure 11).

Sarcodon pallidogriseus A. Grupe & A.M. Vasco-Pal., sp. nov. Figures 15,16

Diagnosis: Sarcodon pallidogriseus is distinct from all other described Sarcodon species in its combination of pale gray colors, campanulate, fibrillose, centrally scabrous pileus, and velutinous stipe with unchanging trama.

Typification: COLOMBIA: AMAZONAS: Puerto Santandar, Vereda Puerto

Fresco, Chukiki, mixed forest, 0˚40`S, 72˚31`W, 150 m, 23 Sep 2005, Vasco 989

(holotype: HUA 186217). GenBank accession: ITS KR698939.

Pileus campanulate, 8−16 mm broad, 4 mm tall, gray (24D1, 24B1−24C1), drying to darker gray with orange or green tones, hygrophanous; surface slightly rugose, overall interwoven fibrillose, center with scales; margin entire, gray with orange tones (5C4); trama gray (24D1), soft. Hymenophore dentate, adnate; teeth 2 mm broad, smaller toward the margin, conical, acute apex, surface pale orange (6A3) to grayish orange when mature

(5B2−5B3), drying to dark brown. Stipe subequal, 30−45 mm long, 2−5 mm broad, cylindrical, fragile, pale gray (24D1), bruising black; surface fibrillose or velutinous; trama solid-spongy, not hollowing, cream-colored (2A2), unchanging; basal mycelium white. Odor: none; taste bitter. Macrochemical reactions: KOH black on all surfaces.

Basidiospores 5−6 (−7) × (6−) 7−8 µm including ornamentation (mean = 5.65 × 7.15 µm; n = 20 ), Q range = 0.71−0.85(−1.0) µm, Q mean =0.79 µm, suboblate, tuberculate,

64 greenish brown in H2O, pale golden brown in KOH, inamyloid; tubercles mostly prominent and short; apices infrequently exsculpate, frequently rounded, rarely pointed; hilar appendage 1−2 µm long. Basidia (30−) 34−41 (−44) × 10−13 (−15) µm wide apically, (3−) 5−8 µm wide centrally, 2−4 µm at basal septum, clavate, hyaline in H2O and KOH; basal septum with clamp connection; sterigmata four, curved, 6−8 µm long.

Hymenial cystidia absent. Hymenophoral trama interwoven, faint tan or light blue-green in mass in H2O and KOH; individual hyphae light gray or pale blue-green in H2O and

KOH, 3−5 µm wide, with dense clusters of small, bluish green extracellular pigment bodies scattered throughout. Pileipellis a cutis of strongly repent hyphae, in mass light gray-green in H2O, dark gray-blue in KOH, with dense clusters of small, bluish green extracellular pigment bodies scattered throughout, these dark blue-gray in H2O, bright blue-green in KOH and eventually dissolving and leaching into solution; individual hyphae light gray-green in H2O, faintly gray or gray-brown in KOH, 3−5 µm wide, cylindrical; terminal cells undifferentiated. Pileus trama in mass gray-brown to orange- brown in H2O, light gray-tan in KOH, with highly scattered, extracellular, dark bluish green to nearly black granular pigment bodies in H2O, these bluish green in KOH; individual hyphae light gray-brown in H2O, light gray in KOH, cylindrical, (3−) 4−7 µm wide. Stipitipellis a cutis of repent hyphae, in mass in H2O orange-brown to dark blue where pigment bodies are dense and copious, in KOH light orange-brown or light blue- green where pigment bodies are dissolved; individual hyphae light gray in H2O, faint gray to faint gray-blue in KOH, cylindrical, 3−4 µm wide; terminal cells undifferentiated.

Stipe trama in mass orange-brown in H2O, light brown-orange to light blue-green where pigment bodies have dissolved in KOH; individual hyphae faint gray to faint tan in H2O, light gray in KOH, cylindrical, 5−8 (−10) µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Gregarious on sandy soil in mixed D. uaiparuensis and Aldina sp. forest; known only from the type locality of Chukiki, Puerto Santandar, in

Amazonas, Colombia.

Etymology. Pallido- (Latin adj. A) = adjectival prefix indicating pale coloration, and -griseus (Latin adj. B) = adjectival suffix indicating gray, referring to the pale gray color of the fresh basidiomata.

Figure 15. Basidiomata of Sarcodon pallidogriseus (HOLOTYPE; Vasco 989). Bar = 10 mm.

Figure 16. Basidium (A.), basidiospores (B.), and basidium with basidiospore (C.) of

Sarcodon pallidogriseus (HOLOTYPE; Vasco 989). Bar = 10 µm.

Commentary. Sarcodon pallidogriseus can be differentiated from each of the other

Neotropical species described in Grupe et al. (2015) and here based on its pale gray basidioma color, shape and surface features of the pileus, lack of a tramal staining reaction upon exposure, and surface features of stipe (Table I).

Sarcodon pallidogriseus and the Neotropical S. bambusinus have a similar pileus shape, non-decurrent teeth of equal lengths, stipe lengths, and basidium lengths, but S. pallidogriseus differs from S. bambusinus in its campanulate pileus shape (vs. orbicular, conical to plano-convex), pale gray basidioma color (vs. vinaceous drab, becoming fuscous or fuliginous with age), pileus surface of slightly rugose, fibrillose, with fibrils being interwoven and scales centrally (vs. villose to subfurfuraceous), and shorter basidiospores (5−6 vs. 6.5−9 µm) (Baker and Dale 1951, Maas Geesteranus 1974a).

Sarcodon pallidogriseus resembles the Paleotropical S. thwaitesii in its black surface staining reaction, lack of a tramal staining reaction, stipe length, and basidium lengths. However, S. thwaitesii is distinguished by its grayish lilac to dark purple colors, tomentose pileus, and longer basidiospores (7.6−9.4 vs. 5−6 µm) (Berkeley and Broome

1873, Maas Geesteranus 1964, 1971, 1974b).

Among extratropical species, Sarcodon pallidogriseus is most similar to the north temperate S. atroviridis in that both have basidiomata that exhibit some shade of gray, stipe surfaces that stain black upon pressure but a lack of a tramal staining reaction, and basidium sizes (Morgan 1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013).

Sarcodon pallidogriseus can be distinguished from S. atroviridis in the lack of a pileus trama staining reaction (vs. lilac, later bluish gray), orangish teeth (vs. white, yellowish,

69 brown), pileus surface of slightly rugose, fibrillose, with fibrils being interwoven and scales centrally (vs. tomentose to felted or glabrous), fibrillose to velutinous stipe surface

(vs. felted, but predominantly glabrous), cream colored stipe trama (vs. lilac to dark violet) and shorter basidiospores (5−6 vs. 8–9 µm) (Morgan 1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013). Additionally, the ITS sequence of S. pallidogriseus is only ~86% similar to sequences of S. atroviridis from the southeastern USA and these two species are distinct in the phylogenetic analysis (Figure 11).

Sarcodon bairdii A. Grupe & A.M. Vasco-Pal., sp. nov. Figures 17,18

Diagnosis: Sarcodon bairdii differs from all other described species of

Sarcodon in its combination of yellow-gray, convex to plane, subumbilicate pileus that has a fibrillose and centrally squamulose surface, yellow-gray, unchanging pileus trama, and brownish gray, fibrillose stipe.

Typification: COLOMBIA: AMAZONAS: Puerto Santandar, Vereda Puerto

Fresco, Chukiki, mixed forest, 0˚40`S, 72˚31`W, 150 m, 23 Sep 2005, Vasco 990

(holotype: HUA 186218). GenBank accession: ITS KR698938.

Pileus broadly convex to plane, subumbilicate in the center, 26−45 mm broad, yellow-gray (4B4−4B5), slightly hygrophanous; fibrillose surface with interspersed fibers, that is centrally squamulose; margin eroded, rimose, with olive tones (4E4−4E3); trama 2 mm wide, spongy, yellow-gray (4B2), unchanging. Hymenophore dentate, adnate, teeth 1−3 mm long, tapered, with acute apices, grayish brown (5F2), teeth shorter

70 at margin. Stipe subequal, 30−50 mm long, 3−11 mm in diameter, central, cylindrical, tapering toward the base, brittle, brownish gray (5D2) towards apex, darker towards the base (5E3−5D3); surface fibrillose with, fibers interspersed, bruising; interior trama solid to substuffed, spongy, yellow-gray (4B2), bruishing dark blue. Odor non-distinguishable; taste dry bitter taste. Macrochemical reactions: black with KOH on fresh basidioma.

Basidiospores (5−) 6−7 × 7−8 (−9) µm including ornamentation (mean = 6.2 ×7.8 µm; n

= 20), Q range = 0.66−0.875, Q mean = 0.79, oblate, tuberculate, dark gray to light tan in

H2O, light tan in KOH, inamyloid; tubercles short, commonly rounded, infrequently flat topped, rarely exsculpate; hilar appendage 1−2 µm. Basidia (28−) 33−46 × 10−12 (−14)

µm wide apically, (2−) 4−9 (−11) µm wide centrally, 2−3 µm at basal septum, clavate, light gray in H2O, light gray to light tan in KOH; basal septum with clamp connection; sterigmata four, curved, 5−7 µm long. Hymenial cystidia absent. Hymenophoral trama parallel, greenish brown in mass in H2O, orange-brown to dark gray-blue in KOH; individual hyphae light gray in H2O, light tan or light gray in KOH, 5−7 (−10) µm wide, with copious granular dark gray-blue pigment bodies, these soluble in KOH. Pileipellis a cutis of repent hyphae, dark gray-green to brown-green in mass in H2O, orange-brown or dark gray-blue in KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light gray in H2O, light gray to light tan in KOH, 6−9

µm wide, cylindrincal; terminal cells undifferentiated. Pileus trama dark gray-green to brown-green in mass in H2O, orange-brown or dark gray-blue in KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light

71 gray in H2O, light gray to light tan in KOH, cylindrical, 7−10 (−12) µm wide. Stipitipellis a cutis of repent hyphae, in mass dark orange in H2O, dark orange to darkest blue in

KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light gray in H2O, light gray to light tan in KOH, more or less cylindrical, with irregular bulges and constrictions, 4−7 µm wide; terminal cells undifferentiated. Stipe trama brownish green in mass in H2O, light tan or dull blue-green in KOH, with copious granular dark gray-blue pigment bodies, these soluble in KOH; individual hyphae light brown-green in H2O, light gray to light tan in KOH, 10−16 µm wide. Clamp connections abundant on hyphae of all tissues.

Habit, habitat and distribution. Gregarious on sandy soil in forests with D. uaiparuensis and Aldina sp., known only from the type locality of Chukiki, Puerto

Santandar, in Amazonas, Colombia.

Etymology. The species is named after Dr. Richard E. Baird, a world authority on hydnoid fungi of the Thelephorales.

Figure 17. Basidiomata of Sarcodon pakaraimensis from Colombia (Vasco 990). Bar =

10 mm.

Figure 18. Basidia and basidiospores of Sarcodon bairdii (HOLOTYPE; Vasco 990). Bar

= 10 µm.

Commentary. Sarcodon bairdii can be differentiated from each of the other

Neotropical species described in Grupe et al. (2015) and here based on overall basidioma color, shape and surface features of the pileus, lack of a pileus tramal staining reaction, and surface features of the stipe (Table I).

Sarcodon bairdii and the Neotropical S. bambusinus have a similar pileus shape, non-decurrent teeth of equal lengths, stipe lengths, and basidium lengths, but S. bairdii differs from S. bambusinus in its pileus shape being convex to plane, subumbilicate in the center (vs. orbicular, conical to plano-convex), basidioma color of yellowish gray (vs. vinaceous drab, becoming fuscous or fuliginous with age), pileus surface that is fibrillose with interspersed fibers and centrally squamulose (vs. villose to subfurfuraceous), and basidiospores that are both shorter and wider (6−7 × 7−8 vs. 6.5−9 × 5−7 µm) (Baker and

Dale 1951, Maas Geesteranus 1974a).

Sarcodon bairdii resembles the Paleotropical S. thwaitesii in the pileus trama lacking a staining reaction, and stipe lengths, and basidium lengths. Sarcodon thwaitesii is distinguished by its grayish lilac to dark purple colors, tomentose pileus, and basidiospore dimensions (7.6−9.4 × 5.4−7.2 vs. 6−7 × 7−8 µm) (Berkeley and Broome

1873, Maas Geesteranus 1964, 1971, 1974b).

Among extratropical species, Sarcodon bairdii is most similar to the north temperate S. atroviridis in that both have basidiomata that exhibit some shade of gray, stipes that bruise black, comparable basidiospore widths, and basidium sizes (Morgan

1895, Banker 1906, Coker and Beers 1951, Baird et al. 2013). Sarcodon bairdii can be distinguished from S. atroviridis in the pileus surface being fibrillose with interspersed fibers and centrally squamulose (vs. tomentose to felted or glabrous), unchanging pileus trama (vs. lilac, later bluish gray), the stipe surface being fibrillose (vs. felted to pubescent or glabrous), stipe trama that is yellow-gray before bruising a dark blue (vs. lilac to dark violet), and shorter basidiospores (6−7 vs. 7–9 µm) (Morgan 1895, Banker

1906, Coker and Beers 1951, Baird et al. 2013). Additionally, the ITS sequence of S. bairdii is only ~86% similar to sequences of S. atroviridis from the southeastern USA and these two species are distinct in the phylogenetic analysis (Figure 11).

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