Phylogenetic overview of Aureoboletus (Boletaceae, Boletales), with descriptions of six new species from China

Abstract In this study, species relationships of the genus Aureoboletus were studied, based on both morphological characteristics and a four-gene (nrLSU, tef1-a, rpb1 and rpb2) phylogenetic inference. Thirty-five species of the genus have been revealed worldwide, forming eight major clades in the phylogenetic tree, of which twenty-four species have been found in China, including six new species: A. glutinosus, A. griseorufescens, A. raphanaceus, A. sinobadius, A. solus, A. velutipes and a new combination A. miniatoaurantiacus (Bi & Loh) Ming Zhang, N.K. Zeng & T.H. Li proposed here. A key to 24 known Chinese species has been provided.


Introduction
Aureoboletus Pouzar was circumscribed in 1957, based on the type species A. gentilis (Quél.) Pouzar (Pouzar 1957). It was characterised by its slimy basidiomata, glabrous to subglabrous pileus and golden yellow hymenophore unchanging when dry (Quélet 1884;Saccardo 1888;Pouzar 1957). To date, 35 species have been described worldwide, 15 of which were originally described in China (Patouillard 1895;Shi and Liu 2013;Zhang et al. 2014Zhang et al. , 2015aZhang et al. , b, 2017Zeng et al. 2015;Wu et al. 2016;Fang et al. 2019). Aureoboletus species can be found in tropical, subtropical and temperate regions of different continents, but most known species appear to exist in Asia and North America. Interestingly, they are strongly implicated as symbionts with an array of ectotrophic plants of the Fagaceae and Pinaceae families (Pouzar 1957;Yang et al. 2003;Klofac 2010;Shi and Liu 2013;Halling et al. 2015;Zeng et al. 2015;Wu et al. 2016;Zhang et al. 2017).
The establishment and acceptance of the genus Aureoboletus has a long history. Xerocomus section Auripori Singer (1942) was established to accommodate Aureoboletuslike taxa. Later, Auripori species were transferred to the genus Pulveroboletus Murrill (Singer 1947). For a long time, the genus Aureoboletus was not accepted as an independent genus by some mycologists (Smith and Thiers 1971;Corner 1972;Singer 1986;Both 1993;Šutara 2005) and species with viscid basidiomata and vivid yellow hymenophores were variously placed in genera Boletellus Murrill, Boletus L., Pulveroboletus and Xerocomus Quél. (Singer 1942(Singer , 1947(Singer , 1986Smith and Thiers 1971;Corner 1972;Both 1993;; However, Aureoboletus was accepted as an independent genus by other mycologists and the features of the genus were redefined (Watling 1965;Watling 2008;Hongo 1973;Zang 1993;Šutara 2008;Klofac 2010). A world-wide survey of the genus, based on morphological characteristics, was conducted and a key was designed to aid in the identification of 11 global Aureoboletus species (Klofac 2010).
Numerous Aureoboletus specimens have been recently obtained in China, increasing the species diversity of Aureoboletus. In this study, the species richness and phylogenetic relationships were re-evaluated, based on detailed morphological observations and a four-gene phylogenetic inference. The aims were to 1) evaluate the phylogenetic relationships within the genus; 2) redefine the characteristics of Aureoboletus; 3) elucidate the species diversity of Aureoboletus in China; 4) describe the newly discovered species.

Morphological studies
Photographs and records of basidiomata were obtained in the field. Specimens were dried in an electric drier and finally deposited in the Fungarium of Guangdong Institute of Microbiology (GDGM) or the Fungal Herbarium of Hainan Medical University (FHMU), Haikou City, Hainan Province, China. Descriptions of macro-morphological characters and habitats were obtained with photographs and field notes. Colours were described in general terms with serial numbers, for example, reddishbrown (9D8-9E8), following Kornerup and Wanscher (1978). Micro-morphological features were observed from dried materials after sectioning and mounting in 5% potassium hydroxide (KOH) solution and 1% Congo Red or Melzer's reagent under a light microscope (Olympus BX51, Tokyo, Japan). For basidiospore descriptions, an abbreviation [n/m/p] denotes n spores measured from m basidiomata of p collections; a notation (a-)b-c(-d) describes basidiospore dimensions, where the range b-c represented 90% or more of the measured values and 'a' and 'd' were the extreme values; Q referred to the length/width ratio of an individual basidiospore and Qm referred to the average Q value of all basidiospores ± sample standard deviation. All line-drawings of microstructures were made, based on rehydrated materials.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from the voucher specimens using the Sangon Fungus Genomic DNA Extraction kit (Sangon Biotech Co. Ltd., Shanghai, China), according to the manufacturer's instructions. Primer pairs LR0R/LR5 or LR0R/LR7 (Vilgalys and Hester 1990), EF1-B-F1/EF1-B-R, RPB1-B-F/RPB1-B-R and RPB2-B-F1/ RPB2-B-R (Wu et al. 2014) were used for the amplification of the large subunit nuclear ribosomal RNA (nrLSU) region, the translation elongation factor 1-alpha subunit (tef1-a), the largest subunit of RNA polymerase II (rpb1) and the second largest subunit of RNA polymerase II (rpb2), respectively. Polymerase Chain Reaction was performed in a total volume of 25 μl containing 1 μl template DNA, 9.5 μl distilled water, 1 μl of each primer and 12.5 μl PCR mix [DreamTaqtm Green PCR Master Mix (2×), Fermentas]. Amplification reactions were performed in a Tprofessional Standard thermocycler (Biometra, Göttingen, Germany) under the following conditions: at 95 °C for 4 min, then 35 cycles of denaturation at 95 °C for 60 s, annealing at 53 °C (LSU) /55 °C (tef1-a, rpb1 and rpb2) for 60 s and extension at 72 °C for 80 s, with a final extension at 72 °C for 8 min. The PCR products were electrophoresed on 1% agarose gels with known standard DNA markers and sequences were performed on an ABI Prism 3730 Genetic Analyzer (PE Applied Biosystems, Foster, CA, USA) at Beijing Genomic Institute (BGI) using the same primers. The raw sequences were assembled with SeqMan implemented in Lasergene v7.1 (DNASTAR Inc., USA). The assembled sequences of the specimens were submitted to GenBank.

Phylogenetic analyses
Newly generated sequences and related sequences downloaded from GenBank were used to reconstruct phylogenetic trees. Detailed information of samples, including species name, voucher, locality, GenBank accession numbers and references, are given in Table 1. Four sequence datasets (nrLSU, tef1-a, rpb1 and rpb2) were separately aligned with MAFFT v6.853 using the E-INS-i strategy (Katoh et al. 2002) and examined in Bioedit v7.0.9 (Hall 1999). The four datasets were analysed independently using the Maximum Likelihood (ML) method to detect the topologies of the four genes. Since no significant incongruence was detected (BS > 70%), the four single-gene alignments were concatenated using Phyutility 2.2 (Smith and Dunn 2008). Missing fragments of some gene markers of several specimens were coded as missing data, intron regions of protein-coding genes were retained in the final analyses and the ambiguously aligned regions were detected and excluded with Gblocks (Castresana 2000).
The combined final dataset was analysed using RAxML v7.2.6 (Stamatakis 2006) and MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) for Maximum Likelihood (ML) and Bayesian Inference (BI), respectively. For both BI and ML analyses, the substitution model, suitable for each gene partition, was determined using the Akaike Information Criterion (AIC), complemented in MrModeltest v2.3 (Nylander 2004). All parameters in the ML analysis were kept as defaults except for choosing GTR-GAMMAI as the model and statistical supports were obtained using rapid non-parametric bootstrapping with 1000 replicates; BI analysis using 4 chains were conducted by setting generations to 80 million and stoprul command with the value of stopval set to 0.01, trees were sampled every 100 generations, the first 25% generations were discarded as burn-ins and posterior probabilities (PP) were then calculated from the posterior distribution of the retained Bayesian trees. Phylloporus imbricatus N.K. Zeng, Zhu L. Yang & L.P. Tang and Xerocomus subtomentosus (L.) Quél. were selected as outgroups, based on Wu et al. (2016) and Zhang et al. (2017).

Molecular phylogenetic results
For phylogenetic analyses, 304 (102 nrLSU, 59 tef1-a, 71 rpb1 and 72 rpb2) new sequences from 105 Aureoboletus collections and 171 GenBank downloaded sequences from 68 Aureoboletus samples were used as ingroups. Four sequences of P. imbricatus and X. subtomentosus, respectively, retrieved from GenBank were used as outgroups. The combined matrix of 175 samples with 3018 nucleotide sites was submitted to TreeBASE (Submission ID 25249). HKY+G, GTR+I+G, SYM+I and SYM+G were chosen as the best substitution models for nrLSU, tef1-a, rpb1 and rpb2, respectively. ML and BI analyses generated almost identical tree topologies with minimal variations in statistical support values. Thus, only a ML tree is displayed (Fig. 1).   In the multi-gene phylogenetic trees, the monophyly of Aureoboletus was statistically strongly supported (BS = 100, PP = 1); eight well supported main clades, labelled as Clade I to VIII, are shown and six well supported (BS = 100, PP = 1) new species lineages were recognised. Description. Basidiomata small to large. Pileus viscid, dry or sticky when wet, even or smooth to wrinkled, usually subtomentose, rarely glabrous, with or without veil or velar residues hanging at margin. Context white to yellowish-white, usually pinkish to reddish-brown beneath pileipellis, unchanging or changing blue or greenish or pastel red when exposed. Tubes coloured with all kinds of yellows, pale yellow, golden yellow to bright yellow, unchanging or slightly changing to blue when bruised, pores circular to angular, smaller to larger, somewhat relatively larger and shallowly depressed around the stipe, concolorous with tubes. Stipe central, cylindrical or clavate, surface glabrous to striate fibrillose, never or rare forming reticulation or Leccinum-like scabrous, dry to viscid, with white basal mycelium. Basidiospores smooth to verrucose or longitudinally striate, subfusiform, oblong ovoid to subglobose, yellowish to yellowish-brown in KOH. Hymenophoral trama boletoid, composed of subcylindrical to cylindrical hyphae, colourless. Pleurocystidia fusiform to subclavate, thin-or thickwalled, sometimes containing golden-yellow contents at first, then gradually changing to yellowish-white to hyaline in 5% KOH. Cheilocystidia present, infrequent or absent, usually similar to pleurocystidia in shape and size, if present. Pileipellis as an interwoven trichoderm, trichoderm or ixotrichoderm, consisting of erect hyphae which are occasionally branched, cylindrical to clavate, thin-to slightly thick-walled, usually less than 1 μm. Stipitipellis hymeniform, as an ixotrichoderm to intricated ixotrichoderm. Caulocystidia clavate, fusoid or ventricose-fusoid. Stipe trama composed of parallel hyphae. Clamp connections absent.
Distribution and ecology. World-wide distribution, mainly known from subtropical Asia and temperate zones of the Northern hemisphere, growing on the ground associated with Fagaceae and other broadleaf trees. Diagnosis. This species is distinguished from other Aureoboletus taxa by its smaller and glutinous basidiomata, reddish-brown to ruby pileus usually with irregular reticulation and darker folds, gelatinised veil remnants and smooth basidiospores 10-13.5 × 4.5-5 μm in size.
Ecology and distribution. Solitary or scattered on ground with humus and debris, usually growing amongst the mosses (Fissidens sp. and Pottiaceae sp.) under Fagaceae, mixed with other broadleaf trees, alt. 300-500 m; May to July, known from Guangdong and Anhui Province.  Notes. Phylogenetic analyses showed that A. glutinosus is closely related to A. marroninus, A. tenuis, A. thibetanus and A. viscidipes; however, the independent phylogenetic position and different morphological characters can distinguish A. glutinosus from these similar species. Aureoboletus marroninus differs in having a more wrinkled and darker (violet brown or maroon) pileus, white context and smaller basidiospores 8.5-10 × 4-4.5 μm (Zhang et al. 2014). Aureoboletus tenuis has relatively larger basidiomata (pileus up to 3.5 cm broad) usually lacking well-developed veil remnants on pileus margin, smaller basidiospores 11-12 × 4-5 μm and ixotrichodermial stipitipellis composed of terminal hyphae with swollen tips (Zhang et al. 2014). Aureoboletus thibetanus is readily separated by its more robust basidiomata (pileus up to 5 cm broad), white ridged reticulation on pileus surface, white stipe and yellowish granular encrustation on cystidia and only known from the temperate zone in southwest China (Patouillard 1895; Yang et al.2003;Klofac 2010). Aureoboletus viscidipes differs in having a brownish to brown pileus tinged with yellowish-white, a longer (up to 4 cm long) and nearly white stipe and a thick layer of a reflective pale-yellow substance on the surface of cheilocystidia and pleurocystidia (Wu et al. 2016). Diagnosis. This taxon can be distinguished from other Aureoboletus species by its brownish-orange to ruby pileus colour, white to yellowish-white context changing to greyish-red or greyish-rose when exposed, light yellow tubes and comparatively small basidiospores 9-10.5 × 4.5-5 μm.

Aureoboletus griseorufescens
Etymology. "griseorufescens" refers to the greyish-red discolouration of context when exposed or bruised.
Ecology and distribution. Solitary or scattered on ground with humus and debris under Fagaceae trees, mixed with other broadleaf trees, alt. 200-400 m; June to September; currently only known from southern China.
Notes. Aureoboletus griseorufescens is somewhat similar to the recently reported species A. venustus from southern China; however, the latter taxon differs in having relatively larger (pileus up to 8 cm) and more viscous basidiomata, a reddish-orange pileus and broader basidiospores 7. 5-10.5 × 5-6 μm (Li et al. 2016). In addition, A. griseorufescens formed a separate species level branch at the base of the phylogenetic tree ( Fig. 1), indicating that it is in an independent phylogenetic position. Diagnosis. This species can be easily distinguished from other Aureoboletus taxa by its dry and yellowish-white to pinkish-white pileus covered with fibrillose to tomentose squamules, radish smell and ovoid basidiospores 7.5-9 × 5-6 μm.
Ecology and distribution. Solitary or scattered on ground with humus and debris under Fagaceae trees mixed with other broadleaf trees, alt. 300-1300 m; June to September; Currently known from Jiangxi and Hunan Province. Notes. The yellowish-white basidioma colour makes it easy to distinguish A. raphanaceus from the other species. Boletus orientialbus N.K. Zeng & Zhu L. Yang recently described from China is somewhat similar to A. raphanaceus in colour; however, B. orientialbus differs in having more robust basidiomata, smooth pileus, reticulate stipe and smaller basidiospores 7-10 × 4.5-5 μm (Zeng et al. 2013).

Aureoboletus sinobadius Ming Zhang & T.H. Li, sp. nov.
MycoBank No: 827101 Figs 2H, I, 3G, 7A-F Diagnosis. This species is distinguished from other Aureoboletus species by its pastel red to reddish-brown pileus, light yellow hymenophore unchanging when bruised, salty taste and two different shapes of basidiospores.
Etymology. "sino-" refers China, the holotype's location of the species; "badius" means the brownish-red or chestnut pileus colour.
Ecology and distribution. Solitary or gregarious on soil under broadleaf forests dominated by Castanopsis spp. and Cyclobalanopsis spp. and mixed with other broadleaf trees, alt. 300-1200 m; May to July, currently only known from Guangdong Province. Notes. Aureoboletus solus looks like A. tenuis; however, the latter differs from the former in its viscid basidiomata, ixotrichodermial stipitipellis, composed of terminal hyphae with slightly swollen tips and larger basidiospores (10-) 11-12 × 4-5 μm (Zhang et al. 2014). Phylogenetic analyses indicated that A. solus is closely related to A. nephrosporus, but A. nephrosporus differs in having larger basidiomata with a red to brownish-red pileus, ovoid to nephroid basidiospores 8-10.5 × 5-6 μm and cheilocystidia and pleurocystidia covered with a thick layer of a strongly refractive pale yellow substance (Wu et al. 2016). Diagnosis.This species can be easily distinguished from others in Aureoboletus by its dry and small basidiomata, brown orange to reddish-brown pileus, light yellow to pastel yellow stipe, covered with fibrillose to tomentose squamules and smooth basidiospores 10-13 × 4-6.5 μm. Etymology. "velutipes" refers to the stipe, covered with fibrillose to tomentose squamules.
Context 5-10 mm thick at centre, firm and tough in youth and, later, soft, white to yellowish-white, with more or less green tint at border contacting tubes, unchanging when exposed. Tube 3-10 mm deep, light orange to orange unchanging when bruised. Pores polygonal, 0.5-1.5 per mm, somewhat relatively larger and shallowly depressed around the stipe, orange to pale orange unchanging when bruised. Stipe 30-80 × 4-10 mm, central, solid, cylindrical or clavate, equal to slightly enlarged downwards, smooth to distinctly longitudinally streaks or broad reticulations, viscid in wet condition, concolorous with pileus. Stipe context concolorous with that of pileus, unchanging when exposed. Basal mycelium white to yellowish-white. Odour strong. Taste mild.
Ecology and distribution. Scattered on soil in tropical to subtropical forests dominated by Fagaceae (Castanopsis chinensis, C. fissa, Lithocarpus spp. and Quercus spp.). Notes. Aureoboletus miniatoaurantiacus, originally described as B. miniatoaurantiacus, is a rather common species in southern China and can be easily distinguished by its bright orange-yellow basidiomata, tomentose or pulverulent pileus surface, light orange to orange hymenophore unchanging when bruised and ovoid basidiospores. Based on a re-study of the type specimen and other collections quoted by Bi et al. in 1994, we found that the type specimen is composed of two small immature basidiomata, which are in a poor condition for morphological observation, but other voucher specimens fit well with the description of A. tomentosus. Thus, the newly described species A. tomentosus is, in fact, a synonym of A. miniatoaurantiacus, this conclusion also being supported by molecular data in this study (Bi et al. 1982;Bi et al. 1994; Wu et al.

Species delimitation, species diversity and new taxa in China
In the taxonomic circumscription of the genus Aureoboletus proposed by Pouzar (1957), 35 species were identified prior to this study, of which 20 species were recorded from China (i.e. A. auriporus, A. catenarius, A. clavatus, A. duplicatoporus, A. formosus, A. longicollis, A. marroninus, A. mirabilis, A. nephrosporus, A. quercus-spinosae, A. rubellus, A. shichianus, A. tenuis, A. thibetanus, A. tomentosus, A. venustus, A. viscidipes, A. viscosus, A. yunnanensis and A. zangii). However, the report of the North American species A. auriporus was excluded from China in this study due to a misidentification, as its correct name is A. sinobadius. The previously described species, A. tomentosus, was proven to be A. miniatoaurantiacus and so, a new combination is proposed here. Six species, A. glutinosus, A. griseorufescens, A. sinobadius, A. solus, A. raphanaceus and A. velutipes, obtained from China, are newly described in this study.
The present study demonstrates that species of Aureoboletus are very diverse in China, especially in its southern areas. Common morphological characteristics and molecular data make Aureoboletus easily distinguishable from other existing genera in Boletaceae, but some variable morphological features make it difficult to recognise some species. Careful examination showed that several morphological characteristics are available to delimit these species in China. For example, the colour of the hymenophore and pattern of the pileus are important characteristics: A. glutinosus has a light yellow to olive yellow hymenophore and a coarse pileus with irregular reticulation; A. sinobadius has a vivid yellow hymenophore and a subtomentose to glabrous and viscid pileus; A. miniatoaurantiacus has a light orange to orange hymenophore and a tomentose to pulverous pileus; regarding the size of basidiomata, A. clavatus and A. yunnanensis have relatively larger basidiomata up to 10 cm in diameter, whereas A. glutinosus and A. marroninus have smaller basidiomata usually less than 2.5 cm in diameter. The colour of the pileus and the colour and odour of the context also help to identify species in the field. In contrast to macro-morphology, several micro-morphological features can also be used to discriminate species of Aureoboletus, such as the size and shape of basidiospores and the shape and inclusion of cystidia, pileipellis and stipitipellis seem to be rather constant amongst the different species.

Phylogenetic analyses supported the presence of eight clades in Aureoboletus
In the present study, all selected samples of Aureoboletus formed a well-supported monophyletic group and eight major clades are proposed here, based on morphological characteristics and phylogenetic inference.
Clade I is characterised by the presence of a viscid pileus, a vivid yellow to greyish-yellow hymenophore that is unchanging when bruised, smooth basidiospores and ixotrichodermium pileipellis. In the present study, this group contains ten species, including the type species A. gentilis and the new species A. sinobadius. This clade is a rather homogeneous group in terms of morphology, which is consistent with the definition of Aureoboletus given by Pouzar (1957). Species in this clade can be separated from each other by pileus colour and the size of basidiospores. In addition, two unsequenced species, A. flavimarginatus and A. flaviporus, should belong to this clade, based on their morphological characteristics (viscid pileus and vivid yellow hymenophore).
Clade II is characterised by the presence of a dry (or slightly sticky when wet) pileus, a vivid yellow to olive yellow hymenophore that is unchanging when bruised, smooth basidiospores and trichodermium pileipellis. This clade includes six species, of which A. velutipes has distinctive morphological characteristics, such as a villous pileus and stipe, pale yellow to olive yellow hymenophore and swollen tips in terminal cells of the stipitipellis.
Clade III is well-characterised by the presence of a viscid pileus with well-developed yellowish to subhyaline veil remnant at the margin, greyish-yellow to olive yellow hymenophore, smooth to longitudinally costate basidiospores and ixotrichodermium pileipellis. Aureoboletus longicollis, originally described from Malaysia, is a well-defined species in this clade and is readily distinguished by its more viscid and larger basidioma, longer stipe and longitudinally costate basidiospores. A Chinese species, A. viscosus, shares similar traits with A. longicollis and the two species cannot be separated from each other in morphology. In this study, we did not have access to specimens of A. longicollis from Malaysia for morphological and phylogenetical study and it is not possible to make a taxonomic decision on whether A. viscosus is the same or a different species to A. longicollis without phylogenetic data. Thus, the name A. longicollis is temporarily used in this study and further studies with more materials are needed. The other species in this clade are characterised by smooth basidiospores and they can be distinguished from each other by their pileus colour and the size of basidiospores.
Clade IV contains the species A. auriflammeus and A. miniatoaurantiacus, which are mainly characterised by their bright orange yellow basidiomata, tomentose pileus surface and ovoid basidiospores. Species in this clade can be easily distinguished from others in this genus.
Clade V is characterised by the presence of a dry or somewhat tacky pileus, greyishyellow to vivid yellow hymenophore changing to olive yellow when mature and oblong, ovoid to nephroid basidiospores. This clade contains five species, including the two species A. solus and A. raphanaceus described above.
Clade VI is composed of four distinct species, which have all been recently added to Aureoboletus, based on phylogenetic analyses (Halling et al. 2015, Wu et al. 2014. Aureoboletus projectellus, A. mirabilis and A. russellii were originally described from North America and have a dry or coarsely tomentose pileus, distinct coarse reticulations on the stipe and larger basidiospores (up to 20 μm); however, the basidiospores of A. projectellus and A. mirabilis are smooth, while A. russellii has longitudinally costate basidiospores (Murrill 1938;Singer 1945;Smith and Thiers 1971;Pegler and Young 1981;. A. shichianus, originally described from southwest China, is a remarkable species in this clade and differs from the others by its small basidiomata, tomentose pileus, radially arranged pores, comparatively long and glabrous stipe and scabrous basidiospores with nodules. Species in this clade are quite diverse, though the coarsely reticulated stipe and ornamented basidiospores are unique in the genus.
Clade VII is currently formed by a single species, A. clavatus. The most striking characteristics are the large basidiomata with slightly viscid pileus, yellowish-white context staining yellowish-olivaceous when exposed, pale yellow to olivaceous-yellow hymenophore, subglobose basidiospores and the pileipellis composed of a turf of clavate hyphae. Besides the slightly viscid pileus, this species shares nearly none of the basic morphological traits of the genus Aureoboletus. However, phylogenetic analyses showed that it belongs to Aureoboletus and formed a separate branch.
Clade VIII is formed by a single species A. griseorufescens. Morphologically, A. griseorufescens is similar to those species in Clade I with a vivid yellow hymenophore and subviscid pileus; however, the most striking characteristic of A. griseorufescens is its white to yellowish-white context changing to greyish-red or greyish-rose when exposed. In the phylogenetic tree, A. griseorufescens formed the basal branch of Aureoboletus with highly-supported values, which showed that it might be an early divergent species from Aureoboletus.
In this study, some evolutionary patterns of morphological characteristics were also discovered. The traits of dry or viscid pileus surface and hymenophore colour appear to be relatively stable evolutionary characteristics and were wellsupported by monophyletic clades on the phylogenetic tree. The shape and surface ornamentation of basidiospores are not reliable characteristics for delimiting Aureoboletus, but are useful for species identification. Basidiospores ornamentation may have evolutionarily originated several times within Aureoboletus history. More morphological and molecular data are needed to understand this trait.