Cantharellaceae of Guyana II: New species of Craterellus, new South American distribution records for Cantharellus guyanensis and Craterellus excelsus, and a key to the Neotropical taxa

Craterellus olivaceoluteus sp. nov. and Craterellus cinereofimbriatus sp. nov. are described as new to science. These fungi were collected from Guyana in association with ectomycorrhizal host trees in the genera Dicymbe (Fabaceae subfam. Caesalpinioideae) and Pakaraimaea (Dipterocarpaceae). Cantharellus guyanensis Mont., originally described from French Guiana, is redescribed from recent collections from Guyana, with additional range extensions for the species provided based on material examined from French Guiana, Venezuela, and north central, northeastern and southern Brazil, circumscribing nearly the entire Guiana Shield region and beyond. A new distribution record from French Guiana is provided for Craterellus excelsus T.W. Henkel & Aime. Macromorphological, micromorphological and habitat data are provided for the new species and C. guyanensis as well as DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU); additional sequence data is provided for C. guyanensis and C. excelsus specimens collected outside Guyana. The relationships of these taxa within the Cantharellaceae were evaluated with phylogenetic analyses of ITS and LSU sequence data. This work brings the total number of Cantharellaceae species known from Guyana to eight. A key to the Cantharellus and Craterellus species known from the lowland Neotropics and extralimital montane Central and South America is provided.


INTRODUCTION
summarized current knowledge of the ectomycorrhizal (ECM) fungal genera Cantharellus Adans. ex Fr. and Craterellus Pers. (Cantharellaceae, Cantharellales, Agaricomycetes, Basidiomycota) in the Neotropics. Six species originally described in Cantharellus were recorded from lowland Neotropical forests; two of them have been transferred to Craterellus (Wilson et al. 2012, Yomyart et al. 2012); two additional Cantharellus species recently were described from Brazil (Wartchow et al. 2012a, Pinheiro andWartchow 2013). Two species of Cantharellus are known from montane Colombian or Costa Rican Quercus forests (Petersen andMueller 1992, Eyssartier et al. 2003), and one extralimital species from southern South American Nothofagus forests (Petersen and Mueller 1992). For Craterellus, five species are known from tropical lowland South America when including the new species and combinations described in Wilson et al. (2012). Three additional Craterellus species are known from montane Colombian or Costa Rican Quercus forests (Wu and Mueller 1995). As currently known, species diversity of these two widely distributed ECM genera is low for the Neotropics, given that . 350 names have been proposed worldwide among the two genera.
Nearly half of the known Neotropical Cantharellaceae diversity resides in the central Guiana Shield region of northeastern South America, where species of Craterellus, and to a lesser extent Cantharellus, are well represented in primary rainforests dominated by ECM canopy trees of the genus Dicymbe (Fabaceae subfam. Caesalpinioideae), Aldina (Fabaceae subfam. Papilionoideae) and Pakaraimaea (Dipterocarpaceae) (Smith et al. , 2013). The synopsis of regional taxa provided by Wilson et al. (2012) Guyana. Additional range extensions and putative host associations are provided for C. guyanensis based on material examined from French Guiana, Venezuela and Brazil. Macromorphological, micromorphological and habitat data are provided for C. olivaceoluteus, C. cinereofimbriatus and C. guyanensis, and DNA sequence data from the nuclear ribosomal regions of the internal transcribed spacer (ITS) and 28S large subunit (LSU) are provided for each; additional sequence data is provided for C. guyanensis and C. excelsus specimens collected outside Guyana. The relationships of these taxa within the Cantharellaceae were assessed with phylogenetic analyses of ITS and LSU sequence data.
A key to the Cantharellus and Craterellus taxa known from the lowland Neotropics and extralimital montane Central and South America is provided.

MATERIALS AND METHODS
Collections.-Collections were made during the May-Jul rainy seasons of 2000-2004, 2006-2010, 2012 and 2013 from the Upper Potaro River Basin, within a 15 km radius of a permanent base camp at 5u18904. 80N, 59u54940.40W, 710 m, from forests dominated by ECM Dicymbe corymbosa Spruce ex Benth. or co-dominated by ECM D. corymbosa, Dicymbe altsonii Sandw., and Aldina insignis (Benth.) Endl. ). Additional Guyana collections were made Dec-Jan 2010-2011 and Jun 2012 from the Upper Mazaruni River Basin within a 6 km radius of a base camp at 5u 26921.30N; 60u04943.10W, 800 m, from forests co-dominated by ECM Pakaraimaea dipterocarpacea Maguire & P.S. Ashton and Dicymbe jenmanii Sandw. (Smith et al. 2013 Macromorphological 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. Fungi were field-dried with silica gel. Micromorphological features of fresh specimens were examined with an EPOI field microscope with light optics; dried specimens were examined in the laboratory with an Olympus BX51 microscope with light and phase contrast optics. For basidiospores, basidia, hyphal features and other structures at least 20 individual structures were measured for each specimen examined for all species treated in this study. Rehydrated fungal tissue was mounted in H 2 O, 3% KOH, and Melzer's solution. Line drawings were made with tracing paper and modified with Photoshop CS5 (Adobe, San Jose, California). Specimens were deposited in these herbaria: BRG 5 University of Guyana; HSU 5 Humboldt State University; PUL 5 Purdue University; TL 5 Universite Paul Sabatier, Toulouse; JPB 5 Universidade Federal da Paraíba, João Pessoa; NY 5 New York Botanical Garden (Holmgren et al. 1990). Additional specimens of C. guyanensis collected by R. Singer (central Brazil) and A. de Meijer (southern Brazil) and Craterellus orinocensis Pat. & Gaillard by R. E. Halling (Venezuela) were examined on loan from the Tennessee Fungus Herbarium (TENN).
DNA extraction, amplification, sequencing and phylogenetic analyses.-DNA extraction, polymerase chain reactions (PCR), cloning and sequencing protocols used in this study for newly obtained specimens of C. guyanensis and C. excelsus were described in Wilson et al. (2012).
For this study, newly generated internal transcribed spacer (ITS) and/or 28S large subunit (LSU) sequences from C. guyanensis specimens from French Guiana, Venezuela and Brazil and a C. excelsus specimen from French Guiana were processed and assembled using CodonCode Aligner 3. 5.7 (CodonCode Corp.,Dedham,Massachusetts,http://www.codoncode.com/). For the new sequences, assembled nucleotide sequence contigs of ITS and LSU regions were used in preliminary BLAST queries of GenBank (www. ncbi.nlm.nih.gov/) and UNITE (unite. ut.ee/;Kõljalg et al. 2005) databases to confirm generic affinities. Along with ITS and LSU sequences from other Guyanese taxa reported in Wilson et al. (2012), additional ITS and LSU sequences of primarily north temperate Cantharellaceae species available on GenBank were assembled into datasets with high intrafamilial inclusivity for phylogenetic analyses. Specimen and GenBank information for all taxa used in this study is provided (SUPPLEMENTARY  TABLE I).
Initial alignment of datasets was performed with MUSCLE (Edgar 2004), followed by manual alignments using Mac-Clade 4.07 (Maddison and Maddison 2005). Maximum likelihood (ML) and ML bootstrapping analyses were performed with RAxML (Stamatakis 2006), which was implemented on the CIPRES web portal ) using 1000 bootstrap replicates to generate bootstrap statistics.
Bayesian analyses were performed with MrBayes 3. 1.3 (Ronquist and Huelsenbeck 2003) implemented on the CIPRES web portal. These analyses used four chains, sampling every 1000 tree for 10 000 000 generations. All other parameters were used at the default settings. In each analysis, two MCMC analyses were run, which produced two files with , 10 000 trees each. The first 1/10th of trees were removed as the burn-in. Both files were combined and a 50% majority rule tree was performed in PAUP* 4.0 (Swofford 2003) to ascertain the Bayesian posterior probabilities for each dataset.
While the well defined pileate-stipitate stature and presence of clamp connections may have led to placement of this species in Cantharellus under traditional morphological taxonomy (e.g . Corner 1966), molecular phylogenetic analyses indicated that C. olivaceoluteus resides in Craterellus (FIG. 1A, B).
The early development of conical primordia with subsequently occasionally perforate pilei in mature basidiomata of C. olivaceoluteus, in which the perforation is continuous with the hollow core of the stipe, are consistent with both the traditional concept of Craterellus (Corner 1966) and also the modern, in which absence of clamp connections was not considered universal in the genus (Dahlmann et al. 2000).
interwoven to subparallel, anticlinal hyphae, light tannish brown in mass in KOH; terminal elements of nearly equal lengths, undifferentiated and rounded at apex; individual hyphae faintly tan in KOH, cells 12.4-5.9 3 3.7-6.2 mm. Stipe trama hyphae light grayish brown in mass in KOH; individual hyphae branching occasionally, nearly hyaline in KOH; cells 44.5-61.7 3 4.9-9.9 mm. Clamp connections absent from hyphae of all tissues. Commentary: Craterellus cinereofimbriatus is recognized in the field by its light grayish brown, pileatestipitate, pliant basidiomata tending toward subinfundibuliform and generally 30-65 mm tall, smooth to finely rugulose, flesh gray hymenophore, planate to marginally downturned and undulating, umbilicate to perforate pileus with fimbriate extreme margin that is lighter gray, and regularly hollow stipe.
On the basis of its subcoriaceous basidioma with hollow stipe arising from an acuminate primordium with straight margins, C. cinereofimbriatus can be identified as a species in Craterellus in both the traditional and modern senses (Corner 1966, Feibelman et al. 1997, Dahlman et al. 2000, Wilson et al. 2012). These characters, along with the subinfundibuliform stature, smooth hymenophore, and lack of clamp connections make C. cinereofimbriatus morphologically consistent with Craterellus sensu lato, despite the recent discovery of several Craterellus species in Guyana, which have clamp connections and solid stipes (Wilson et al. 2012). The position of C. cinereofimbriatus within Craterellus was corroborated by phylogenetic analyses (FIG. 1A, B). Rare fruiting of C. cinereofimbriatus in Guyana's Dicymbe forests was recorded in a long-term D. corymbosa plot study of , with its basidiomata occurring in only 0.3% of 630 quadrats sampled during the May-July rainy seasons over 7 y.
Craterellus cinereofimbriatus most closely resembles the sympatric C. excelsus in its drab, light gray to gray brown colorations overall, broadly undulating pileal margin at maturity, consistently smooth hymenophore, hollow stipe, and similarly sized and shaped basidia and basidiospores (Henkel et al. 2009). Craterellus cinereofimbriatus can be distinguished for C. excelsus by its consistently shorter basidiomata (30-60 vs. 60-135 mm) that occur solitarily or in small caespitose clusters as opposed to the frequent, large caespitose clusters of C. excelsus, more narrow central pileal perforation leading to a less broadly infundibuliform, more defined pileate-stipitate stature and regularly fimbriate-crenulate vs. smooth extreme pileal margin. These subtle, but discernable, macromorphological differences between C. cinereofimbriatus and C. excelsus were corroborated by molecular phylogenetic analyses, in which these taxa resolved as sister species within Craterellus (FIG. 1A, B).
Habit, habitat and distribution: In Guyana, in small to large troops on humic mat of forest floor in Dicymbe-dominated stands or on humic deposits on lower trunks of Dicymbe corymbosa, preferentially on sandy soils; more rarely under Dicymbe or Aldina on lateritic soils; known from the Upper Potaro and Upper Mazaruni Basins of Guyana and 100 km to the east in the lowlands of the Mabura Hill region; also known from French Guiana, Venezuela, and north central, northeastern and southern Brazil in association with putative nyctaginaceous or polygonaceous ECM host plants. Commentary: Key macromorphological characters allowing field recognition of C. guyanensis include the well defined pileate-stipitate stature, the yellow-orange to orange pileus with downturned margin and rarely exceeding 40 mm diam, the orangish cream, well defined, lamelloid hymenophore, and subequal, light orange, solid stipe. In addition the preference for sandrich soils and fruiting habit in medium to large troops has been observed regularly in Guyana and also in French Guiana and Venezuela. While rare fruiting of C. guyanensis in Guyana's Dicymbe forests was recorded in a long-term D. corymbosa plot study of , with its basidiomata occurring in 0.8% of 630 quadrats sampled during the May-July rainy seasons over 7 y; these study plots were located on lateritic soils, whereas the species has been observed fruiting and collected much more frequently in Dicymbe forests on sand soils.
Micromorphologically C. guyanensis is distinguished by the ellipsoid, smooth, hyaline to yellowish basidiospores with granular-guttulate cytoplasm, relatively short (generally , 80 mm) basidia with 3-4-5-6 sterigmata, abundant clamp connections on hyphae of all tissues, and a cuticular pileipellis with broadly cylindrical terminal elements with thickened walls. Overall, material from Guyana agrees well with the type description of C. guyanensis, from French Guiana, provided by Montagne (1854) and a modern revision of the type specimen (Eyssartier 2001) except that the purplish orange tints in the developing pilei recorded in the type have never been observed in the numerous collections from Guyana or in the recent collections Roy G178 and Henkel 9732 from French Guiana. In addition, the hollow stipe recorded by Montagne is rarely seen in the Guyana material.
Specimens of C. guyanensis from French Guiana, Venezuela and Brazil examined for this study agree well with the Guyana material across the pertinent character set, with some minor variations. The French Guiana collection (Henkel 9732) varied from the Guyana material in its more subglobose basidiospores (mean Q 5 1.3 vs. 1.6). The Brazilian collection (Singer 43724) had slightly more ellipsoid basidiospores (mean Q 5 1.8 vs. 1.6) and somewhat shorter basidia (41-57 vs. 49.4-81.5 mm). The two Venezuelan collections , as well as an additional southern Brazilian specimen (de Meijer 1944), were identical to the Guyana material in all characters. Minor variations between specimens notwithstanding, the morphological character package shared by specimens examined from across the Guiana Shield argue strongly for a single regional FIG. 7 species. In addition, molecular phylogenetic analyses including C. guyanensis specimens from Guyana, French Guiana, Venezuela and Brazil confirmed their conspecificity (FIG. 1A, B). While evidence exists for broad geographic distributions of some ECM fungal species in the lowland South American tropics (Wartchow and Maia 2007, Menolli et al. 2009, Henkel et al. 2011, Uehling et al. 2012, Wartchow 2012, C. guyanensis constitutes the first case of an individual species being documented over a geographical area circumscribing nearly the entire Guiana Shield region, with a N-S distribution of , 1200 km, and E-W of , 2500 km. Consideration of the de Meijer specimen from Parana in southern Brazil would extend the known distribution of C. guyanensis a further 3000+ km southward. Cantharellus guyanensis also appears to have broad host-plant range, occurring in forests dominated by ECM Dicymbe or Aldina spp. in Guyana and in mixed, non-ectotrophic rainforests in spatial proximity to trees or lianas of ECM Coccoloba, Guapira or Neea spp. in French Guiana, Venezuela and northeastern Brazil (e. g. Béreau et al. 1997, Terborgh et al. 2006, Alves-Araú jo et al. 2008). Potential host plants for Singer's collection from north central Brazilian campina forest include ECM Aldina spp. and possibly Glycoxylon inophyllum (Mart. ex Miq.) Ducke (Sapotaceae) (Singer et al. 1983), as also noted in Singer's personal communication to RH Petersen included with the specimen housed at TENN. Until recently C. guyanensis was one of the few valid Cantharellus species known from the lowland South American tropics (Wartchow et al. 2012a, Wilson et al. 2012). Three new Brazilian species have since come to light that deserve comparison with C. guyanensis. Cantharellus aurantioconspicuus Wartchow & Buyck from the Atlantic coastal forest of Brazil is similar to C. guyanensis in its well defined pileate-stipitate habit, overall orange coloration, well developed lamellate hymenophore, and basidiospore and basidium sizes and dimensions, but differs in its more robust stature with a consistently broader pileus (25-90 mm vs. 10-35 mm) and lack of thickened walls in the pileipellis terminal cells (Wartchow et al. 2012b). Cantharellus amazonensis Wartchow from near Manaus in the central Brazilian Amazon has similarly small basidiomata (30-35 mm diam) but differs fundamentally in its bright red pileus, non-anastomosing lamellae, primarily six-sterigmate basidia (vs. 3-4-5-6) and lack of thickened walls in the pileipellis terminal cells (Wartchow et al. 2012b). Cantharellus protectus Wartchow & FGB Pinheiro, recently described from the Brazil's Atlantic coastal forest, is exceedingly similar to C. guyanensis in basidioma stature and size, predominantly yelloworange pileus and well defined lamellate-intervenose hymenophore but differs in its darker orange vs. pinkish orange to light orange stipe, consistently smaller basidiospores (5.5-7.5 3 3.5-5 mm vs. 7-9.2 3 4.5-6.2 mm), shorter basidia (36-58 vs. 49.4-81.5 mm) that are predominantly six-sterigmate, and slightly thickening walls (to 0.5 mm) in the pileipellis terminal cells (Pinheiro and Wartchow 2013 Commentary: Craterellus excelsus, previously known only from the type locality in Guyana, recently was collected in French Guiana. The French Guiana material agrees well both macro-and microscopically with C. excelsus (Henkel et al. 2009) and appears conspecific in the ITS phylogenetic analysis (FIG. 1A).
The record of C. excelsus from French Guiana is a significant range extension of , 500 km eastward for the species and likely a host extension because basidiomata were collected close to ECM Coccoloba lianas. The species was known previously only from Dicymbe-dominated forests in Guyana. KEY