Reappraisal of the Genus Exsudoporus (Boletaceae) Worldwide Based on Multi-Gene Phylogeny, Morphology and Biogeography, and Insights on Amoenoboletus

The boletoid genera Butyriboletus and Exsudoporus have recently been suggested by some researchers to constitute a single genus, and Exsudoporus was merged into Butyriboletus as a later synonym. However, no convincing arguments have yet provided significant evidence for this congeneric placement. In this study, we analyze material from Exsudoporus species and closely related taxa to assess taxonomic and phylogenetic boundaries between these genera and to clarify species delimitation within Exsudoporus. Outcomes from a multilocus phylogenetic analysis (ITS, nrLSU, tef1-α and rpb2) clearly resolve Exsudoporus as a monophyletic, homogenous and independent genus that is sister to Butyriboletus. An accurate morphological description, comprehensive sampling, type studies, line drawings and a historical overview on the nomenclatural issues of the type species E. permagnificus are provided. Furthermore, this species is documented for the first time from Israel in association with Quercus calliprinos. The previously described North American species Exsudoporus frostii and E. floridanus are molecularly confirmed as representatives of Exsudoporus, and E. floridanus is epitypified. The eastern Asian species Leccinum rubrum is assigned here to Exsudoporus based on molecular evidence, and a new combination is proposed. Sequence data from the original material of the Japanese Boletus kermesinus were generated, and its conspecificity with L. rubrum is inferred as formerly presumed based on morphology. Four additional cryptic species from North and Central America previously misdetermined as either B. frostii or B. floridanus are phylogenetically placed but remain undescribed due to the paucity of available material. Boletus weberi (syn. B. pseudofrostii) and Xerocomus cf. mcrobbii cluster outside of Exsudoporus and are herein assigned to the recently described genus Amoenoboletus. Biogeographic distribution patterns are elucidated, and a dichotomous key to all known species of Exsudoporus worldwide is presented.

For ITS and nrLSU, PCR was carried out under the following cycling parameters: initial denaturation: 95 • C for 5 min, followed by 35 cycles: 95 • C for 30 s, 55 (or 53) • C for 30 s, 72 • C for 1 min, and final extension at 72 • C for 7 min. For tef1-α and rpb2, PCR conditions were as follows: initial denaturation at 95 • C for 5 min, followed by 40 cycles: 95 • C for 30 s, 55 • C for 45 s, 72 • C for 45 s, and final extension at 72 • C for 7 min.
Sequences were manually edited and assembled using Sequencher 4.1.4 program (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences generated for this study were submitted to GenBank and their accession numbers are cited in Table 1.
Three alignments were generated for combined ITS-nrLSU (algorithm Q-INS-i), tef1-α (algorithm E-INS-i) and rpb2 (algorithm E-INS-i) datasets with MAFFT 7 [54]. Data for each locus were manually adjusted and combined in MEGA 6.06 [55]. Sites with 99% gaps (59 positions in total) were removed using trimAl v.1.2 program [56]. Phylogenetic reconstructions were performed using the maximum likelihood (ML) and Bayesian inference (BI) methods of analysis. In both ML and BI analyses, the general time reversible model with rates that vary over sites according to gamma (GTR+G) was employed.
BI was performed with MrBayes 3.2.6 software [59], under the described model. The BI analysis was performed with two parallel searches and four chains, with three million generations and a sampling frequency of every 100th generation. Tracer 1.6.0 [60] was used to evaluate the quality of a sample from the posterior and the continuous parameters, using effective sample size (ESS). To remove the pre-stationary posterior probability distribution, a burn-in of 25% was applied.
Only bootstrap support (BS) from 50% and posterior probability (PP) values exceeding 0.7 are reported in the resulting tree ( Figure 1). A clade was considered strongly supported if it received bootstrap support (BS) greater than 70% and/or posterior probability (PP) equal to or greater than 0.95. Branch lengths were estimated as mean values over the sampled trees. The final tree was edited in Inkscape v.0.92. Figure 1. ML phylogenetic tree of Exsudoporus, Amoenoboletus and allied genera generated from a multilocus (ITS + nrLSU + tef1-α + rpb2) dataset. PP values ≥ 0.7 and BS support values ≥ 50% are shown at the nodes. Thickened branches indicate PP ≥ 0.95 and BS support ≥ 70%. Newly sequenced collections are indicated in bold; type specimens are indicated with an asterisk (*). Two-letter country codes (ISO 3166-1 alpha-2) reflecting origin of specimens are given.

Molecular Phylogenetic Analysis
The aligned multigene matrix contained a total of 96 samples and 3302 aligned bases with gaps (Supplementary File S1). The ML and BI analyses generated almost identical tree topologies with minimal variation in statistical support values; thus, an ML tree with both BS and PP values was selected for the purposes of display ( Figure 1).

Taxonomy
Exsudoporus Vizzini, Simonini and Gelardi 2014, emend. Biketova and Gelardi. MYCOBANK MB 550708. Diagnosis: Basidiome stipitate-pileate with tubular hymenophore, epigeal, evelate; pileus convex to applanate, bright blood red, crimson-red, purplish-red, reddish-pink or reddish-brown, opaque to shiny, dry to subviscid with moist weather, glabrous to subpruinose or subtomentose; hymenophore poroid, adnate or slightly depressed around stipe apex; tubes yellow to olivaceous-brown; pores pinkish-red, reddish-orange, blood red to dark red, rarely yellowish-orange or yellow, often beaded with golden yellow or amber yellow droplets when young and fresh; stipe central, solid, yellowish to concolorous with the pileus, conspicuously reticulate with elongated, red meshes or deeply reticulatealveolate or can be covered with scaly patches; context pale yellow to bright yellow; tissues quickly turning dark blue or more rarely light blue or even unchanging when injured or exposed, then fading blackish; taste mild to acidic; spore print olive-brown; basidiospores smooth, subfusiform to ellipsoid or ellipsoid-fusoid; pleuro-, cheilo-and caulocystidia present; pileipellis an interwoven (ixo)trichoderm tending to a cutis; hymenophoral trama bilateral-divergent of the "Boletus-type"; clamp connections absent; stipe context varies from inamyloid to amyloid or dextrinoid; ontogenetic development gymnocarpic. Basidiomes small to medium. Pileus (2.0) 3.2-9.5 (10.0) cm broad, at first hemispherical then persistently convex and finally broadly pulvinate-flattened to slightly depressed, regularly to sometimes unevenly shaped by shallow depressions, moderately fleshy, firm at the beginning but progressively softer with age, flabby in old basidiomes; margin generally obtuse, steady to faintly wavy-lobed especially in young specimens, initially involute then curved downwards and finally completely plane or even uplifted, extending beyond the tubes up to 2 mm in primordia; surface matt, dry but slightly greasy and polished with moist weather, very finely pubescent in the early stage of development but later smooth and glabrous, not cracked; cuticle somewhat variable in color, ranging from pale pinkish, pinkish-violet to pinkish-red (Shrimp Pink, Geranium Pink, Rose Doree, pl. I; Pale Amaranth Pink, Rose Pink, pl. XII; Rose-Purple, pl. XVI) in young specimens due to the presence of a whitish pubescence which soon tends to dissolve revealing the true color below, blood red to dark carmine red, coral red or garnet red to reddish-purple (Spinel Pink, Spinel Red, pl. XXVI; Spectrum Red, Scarlet-Red, Carmine, pl. I) at maturity, often with scattered orange shades (Orange Chrome, pl. II; Scarlet, pl. I) especially towards the margin, gradually fading with age and becoming copper red to ochraceous red or even ochraceous brown (Madder Brown, Brick Red, pl. XIII; Buckthorn Brown, pl. XV) in senescence or if exposed to direct sunlight; slowly but pronouncedly turning bluish-black (Berlin Blue, pl. VIII) on handling or when injured, particularly in young and fresh basidiomes, then fading dull brownish-red (Indian Lake, Dahlia Carmine, pl. XXVI; Bordeaux, pl. XII); subcuticular layer reddish-purple (Amparo Purple, pl. XI; Lobella Violet, pl. XXXVII). Tubes at first thin then increasingly broader and shorter than the thickness of the pileus context (up to 1.8 cm long), adnate-subdecurrent at first but soon adnexed, finally depressed around the stipe apex and shortly decurrent with a tooth, pale yellow (Barita Yellow, pl. IV) at first, yellowish-olive (Yellowish Citrine, pl. XVI) to brownish-olive (Saccardo Olive, pl. XVI; Light Brownish Olive, pl. XXX; Dark Olive-Buff, pl. XL) in old fruiting bodies, bluing (Blanc's Blue, Dusky Greenish Blue, pl. XX) when cut. Pores initially forming a flat surface, later convex to ascendant, at first very small then gradually wider (up to 2 mm in diam.), simple, roundish to barely angular and radially stretched at maturity, at first more or less evenly yellow (Lemon Chrome, pl. IV) but soon orange pinkish to blood red (Vinaceous, Deep Vinaceous, pl. XXVII; Eosine Pink, Spectrum Red, Scarlet-Red, pl. I), sometimes yellowish-orange (Flame Scarlet, Orange Chrome, pl. II) towards the margin and finally rusty red (Carmine, pl. I), quickly and intensely turning dark blue (Blanc's Blue, Dusky Greenish Blue, pl. XX) on bruising or when injured and finally fading to blackish (Black, pl. LIII); the hymenophore of young and fresh specimens exude abundant golden yellow (Light Orange-Yellow, pl. III), markedly salty-tasting droplets which tend to disappear with age. Stipe (4.0) 4.6-11.0 (12.0) × (0.6) 0.8-3.1 (4.0) cm, slightly longer than or as long as the pileus diameter at maturity, central to slightly off-center, solid, firm, dry, straight or faintly curved, cylindrical to fusiform, rarely progressively attenuated downwards, usually swollen in the middle part and tapering towards both the apex and the base, never clavate, ending with a long pointed taproot at the very base, frequently deeply rooting; surface showing a fine to pronounced reticulum at least in the upper half or over the upper three fourths, sometimes extending down to the base, smooth and glabrous elsewhere, evelate; lemon yellow or bright yellow to occasionally pinkish (Barita Yellow, pl. III; Light Orange-Yellow, pl. II) in the upper third or in the upper half in young specimens, orange yellowish to pinkish (Capucine Yellow, Orange, pl. II; Ochraceous-Salmon, pl. XV) elsewhere, reddish-purple to purplish-brown (Aster Purple, Dahlia Purple, Pansy Purple, pl. XII) in the lower portion, entirely blood red to carmine red or garnet red (Spinel Pink, Spinel Red, pl. XXVI; Spectrum Red, Scarlet-Red, Carmine, pl. I) in mature specimens; reticulum consisting of fine to well-defined, narrow and longitudinally stretched polygonal meshes, concolorous to the ground in early stages of development but soon blood red to carmine red (Spectrum Red, Scarlet-Red, Carmine, pl. I) lengthwise; bruising dark blue (Berlin Blue, pl. VIII) when pressed then fading sordid reddish-purple to blackish (Claret Brown, pl. I; Black, pl. LIII); basal mycelium cream yellowish (Barita Yellow, pl. IV), rhizomorphs brownish (Sayal Brown, pl. XXIX). Context firm and tough when young, later soft textured and eventually flabby in the pileus (up to 3.2 cm thick in the central zone and gradually becoming thinner towards the edge), a little more fibrous in the stipe, evenly watery yellow (Citron Yellow, pl. XVI) throughout with darker tones towards the base, with a thin reddish-purple line (Amparo Purple, pl. XI; Amaranth Purple, pl. XII; Lobella Violet, pl. XXXVII) beneath the cuticle and sometimes with reddish-purple scattered spots or shades in the stipe; turning extensively blue (Yale Blue, Vanderpoel's Blue, Blanc's Blue, Dusky Greenish Blue, pl. XX; Grayish Violaceous Blue, pl. XXII) when exposed to air with the only exception of the stipe base in young specimens, which remains practically unchangeable, finally fading dull yellowish (Aniline Yellow, Pyrite Yellow, pl. IV) to dirty yellowish-orange (Yellow Ocher, pl. XV) or occasionally reddish (Etruscan Red, pl. XXVII); reddish-purple (Pomegranate Purple, pl. XII) where eroded by maggots and bright yellow (Strontian Yellow, pl. XVI) to reddish (Etruscan Red, pl. XXVII) in places eaten by slugs; subhymenophoral layer bright yellow (Strontian Yellow, pl. XVI); exsiccate pinkish-red, wine red to dark reddishpurple (Spinel Pink, Spinel Red, pl. XXVI; Spectrum Red, Scarlet-Red, Carmine, pl. I) on pileus and stipe, brownish (Cinnamon-Brown, pl. XV) on hymenophore, beige to dull ochraceous brown on context (Ivory Yellow, pl. XXX; Clay Color, pl. XXIX). Odor intensely fruity, agreeable. Taste mild then with a weakly acidic aftertaste. Spore print olive-brown. Macrochemical spot-test reactions: 25% NH 3 : Pileus cuticle fades ochraceous, no reaction elsewhere (but bluing oxidation on tissues disappears); 30% KOH: staining dark wine red on hymenophore, bright orange to wine red elsewhere; FeSO 4 : blackish on hymenophore, no response or pale olivaceous on context, brownish-olive on pileus and stipe. Ontogenetic development gymnocarpic.
Oleipherous hyphae scattered although more frequently observed in the basal stipe trama, golden yellow to brownish in 5% KOH and Melzer's. Clamp connections absent in all tissues. Hyphal system monomitic.
Edibility: Edible after prolonged cooking. Ecology and phenology: Solitary or more frequently gregarious or subcaespitose to truly caespitose, sometimes with basidiomes emerging from the stipe of adjacent fruiting bodies, growing in warm Mediterranean regions preferably on slightly to decidedly acidic, clayey or sandy soil among litter associated exclusively with hardwoods in dry, warm, exposed groves of Quercus (Fagaceae), occasionally also with Castanea sativa (Fagaceae) and Cistus (Cistaceae), sometimes in mixed woods with the presence of Pinus halepensis and P. pinea (Pinaceae), Erica spp., Arbutus unedo (Ericaceae) and Eryngium sp. (Apiaceae). Summer to late fall (August to November).
Known distribution: Previously reported from southern Europe in warm countries at low altitudes, bordering the Mediterranean basin (Portugal, Spain, Italy, Slovenia, Bulgaria, Greece) east to the Asian Middle East (Cyprus, Israel) and likely extending as far north as France. It probably also occurs in Mediterranean northern Africa. Apparently widespread throughout mainland and insular Mediterranean basin but rare and localized. Northern, eastern and southern distribution limits yet to be established. Comments: The Italian mycologists C.L. Alessio and A. Angarano were the first to struggle with the placement of Exsudoporus permagnificus in the early 1980s, assigning it three misapplied species epithets [61,62]. Alessio wrote at length and repeatedly about this species based on several collections recorded in Piedmont (northwestern Italy) in 1973 [61]. Two years later, in 1975, the species was also recorded with several specimens emerging from a common base under a single oak tree near Bologna, Emilia Romagna [63]. Alessio's first contribution was devoted to this species which, following A. Marchand's suggestion, was at first believed to represent the eastern North American species Boletus frostii Roussell [61]. At the same time, Angarano misidentified the species as another North American taxon, B. flammans E.A. Dick and Snell, based on his collections from Cannigione (northern Sardinia) [62]. Alessio continued to use the name B. frostii for collections of B. permagnificus [64]. However, Alessio reconsidered the best name for these collections and instead applied the binomial Boletus siculus Inzenga [65], a species described from Sicily in the second half of the 19th century [66]. However, as already argued by the French mycologists M. Bon [61] and G. Redeuilh [67], and the Italian F. Bellù [68], the collections of Alessio [61,64,65] and Angarano [62] represented a taxon without a valid name.        Indeed, Pöder [69], working mainly on the same Sardinian specimens previously misidentified by Angarano, described this taxon as new to science under the binomial Boletus permagnificus Pöder (see also Pöder [67] for an Italian translation of Pöder's original manuscript by F. Bellù). Despite the convincing evidence of a novel species provided by the Austrian mycologist, in the subsequent years Alessio continued to reiterate his taxonomic view by considering B. permagnificus a later heterotypic synonym of B. siculus [70][71][72][73][74] and apparently did not change his opinion throughout his life. Conversely, Bellù did not concur with Alessio's interpretations and instead agreed with Pöder that E. permagnificus was indeed a new taxon [67,75]. Furthermore, as already pointed out by Lavorato [76] and M. Contu (pers. Comm.), B. siculus might represent an older name for Alessioporus ichnusanus (Alessio, Galli and Littini) Gelardi, Vizzini and Simonini-a species described by the same Alessio and co-workers in 1984 [77]-even though the exact identity of B. siculus remains unclear [78].
In more recent times, B. permagnificus was subjected to preliminary phylogenetic analysis which led to the erection of the genus Exsudoporus encompassing E. permagnificus and its closest relatives [26]. Finally, Blanco-Dios recombined nearly all European redpored boletes in Suillellus Murrill without providing any supporting phylogenetic evidence to justify this placement [79].
Exsudoporus permagnificus is an easily recognized species in the warm regions of southern Europe and the eastern Mediterranean region of western Asia, but it appears to be practically unknown elsewhere, especially outside Europe. It is easily recognizable Indeed, Pöder [69], working mainly on the same Sardinian specimens previously misidentified by Angarano, described this taxon as new to science under the binomial Boletus permagnificus Pöder (see also Pöder [67] for an Italian translation of Pöder's original manuscript by F. Bellù). Despite the convincing evidence of a novel species provided by the Austrian mycologist, in the subsequent years Alessio continued to reiterate his taxonomic view by considering B. permagnificus a later heterotypic synonym of B. siculus [70][71][72][73][74] and apparently did not change his opinion throughout his life. Conversely, Bellù did not concur with Alessio's interpretations and instead agreed with Pöder that E. permagnificus was indeed a new taxon [67,75]. Furthermore, as already pointed out by Lavorato [76] and M. Contu (pers. Comm.), B. siculus might represent an older name for Alessioporus ichnusanus (Alessio, Galli and Littini) Gelardi, Vizzini and Simonini-a species described by the same Alessio and co-workers in 1984 [77]-even though the exact identity of B. siculus remains unclear [78].
In more recent times, B. permagnificus was subjected to preliminary phylogenetic analysis which led to the erection of the genus Exsudoporus encompassing E. permagnificus and its closest relatives [26]. Finally, Blanco-Dios recombined nearly all European red-pored boletes in Suillellus Murrill without providing any supporting phylogenetic evidence to justify this placement [79].
Exsudoporus permagnificus is an easily recognized species in the warm regions of southern Europe and the eastern Mediterranean region of western Asia, but it appears to be practically unknown elsewhere, especially outside Europe. It is easily recognizable based on the following diagnostic key features, which place this species in a morphologically unique position among red-pored European boletes: small to medium-sized basidiomes, overall bright red coloration, cylindrical to fusiform stipe that is never clavate but is always covered by a well-developed raised reticulum, hymenophore exuding golden or amberyellow droplets in young and fresh specimens, yellowish context and basal mycelium, deep blue staining reaction upon bruising or injury, ellipsoid-fusiform, smooth basidiospores, ixocutis pileipellis mainly consisting of filamentous to cylindrical hyphae, subcaespitose to caespitose growth and the occurrence under broadleaved trees or shrubs in warm environments. As far as the pileipellis structure is concerned, inflated to nearly globose cells up to 18-20 µm wide are sometimes observed in the subpellis (as broad as 25 µm) [80][81][82]. Another peculiar organoleptic trait that has likely gone unnoticed but is worthy of mention is the salty taste of the droplets; this neglected feature has not been previously reported for E. permagnificus but should be examined in the remaining extraeuropean species to evaluate its taxonomic significance.
The amyloidity of tissues with Melzer's reagent is a point that deserves further discussion. Bozok et al. reported the amyloid reaction of the hyphae of the stipe context as an additional attribute of E. permagnificus [38]. The holotype material and all collections from Israel studied in the present paper also exhibited the same positive reaction of the stipe base context: moderately amyloid in AB B11-03, AB B13-165, AB B15-271 and AB B20-370, weakly amyloid in AB B11-06 and IB 19800750 and variably amyloid and dextrinoid in AB B15-254 and AB B15-274 ( Figure 5). As a matter of fact, in the original diagnosis, Pöder noted that the stipe trama was almost black with Melzer's reagent, suggesting amyloid tissues [69]. Icard and Hurtado [80], Lannoy and Estades [5], and Horak [83] also observed a positive amyloid reaction in this species. However, other authors reported a negative reaction with Melzer's reagent [14,82,[84][85][86]. As already suggested by Assyov, contrasting results might be due to different procedures followed in testing the amyloidity of fungal tissues [84]. However, we have tested the iodine reaction of the context at the stipe base according to Imler's procedure [4] on several of our Italian collections using the same techniques and surprisingly we observed dissimilar results depending on the studied samples; some of them showed a strong (GS1717) to weak (MG238, MG662, GS336) amyloid reaction, whereas some others exhibited an inamyloid reaction (MG558, MG637, GS784, GS10151), and a few others even displayed a dextrinoid reaction (MG418, GS1001, GS1275). Consequently, this macro-chemical reaction should be carefully re-evaluated, as it seems to be quite inconstant and variable.
A chromatographic analysis of the pigments in E. permagnificus was carried out by Davoli and Weber, which led to the isolation of variegatic acid, xerocomic acid and variegatorubin [87,88].
A bolete sample (LE 17906) identified by Bedenko [125] as B. permagnificus from the Belgorod Region in the Russian Federation has been morphologically re-examined by T. Yu. Svetasheva and I. V. Zmitrovich and was confirmed to be another species of Boletaceae (likely a member of either the genus Rubroboletus or Neoboletus). Unfortunately, the specimen is poorly preserved and no DNA sequences could be generated from this material, so it is not possible to identify this specimen to the species level.
Edibility: Edible after prolonged cooking although with a somewhat acidic taste [129]. Ecology and phenology: Solitary to most frequently gregarious, growing in open stands, clearings or shaded lawns on sandy soil associated with various Quercus spp. (including Q. chapmanii, Q. laurifolia and Q. virginiana) (Fagaceae) or mixed with Pinus clausa (Pinaceae) and Carya (Juglandaceae). Late spring to fall (April to December), uncommon to rare.
Known distribution: Reported from eastern and southeastern USA (Tennessee and the Coastal Plain of North Carolina south to Florida and west to Texas), reported as far north as Long Island (New York) (MyCoPortal); records from Costa Rica, Mexico, Belize and Guatemala putatively belong to another related species (see below).   Comments: This species has been sufficiently described and illustrated. The exact collecting date of the original material is not clear and was not given in the publication or on the packet, but authentic samples from the description were collected in north Florida during June and September. Curiously, Both indicated a different collection (paratype F2204) as the type of E. floridanus [128]. This is likely because there were some confusing annotations on the specimens at FH and other herbaria and because F2428 does not appear to be present at either FH or F. An attempt was made to sequence the ITS region from two paratypes of E. floridanus from FH (F2204 and F650), but due to the age and condition of the specimens, this was unsuccessful. We consider it necessary to designate a new specimen of E. floridanus from the vicinity of the type locality as a modern epitype. Phylogenetic analysis shows that this entity might be a species complex, and E. floridanus s. l. appears to encompass two different species. One is the true E. floridanus, which is distributed in eastern and southeastern North America, including our specimens from Florida that were collected nearby the type locality. A second, morphologically similar species is recorded here from Central America, based on collections from Belize and Costa Rica. It seems likely that other collections from Belize [138] and Guatemala [147] might have been misidentified as E. floridanus s. str. but actually belong to this second species. Records from Mexico that were identified as E. floridanus s. str. [143][144][145][146] should be examined and/or have their DNA sequenced to determine which of the two species they represent. However, since many taxa from Florida extend into Mexico, it seems likely that both species may be present in Mexico.
Similar to E. permagnificus, the amyloid reaction of the hyphae of the stipe context with Melzer's reagent varies from weakly dextrinoid (FLAS-F-61008) and dextrinoid (FLAS-F-59069) to mixed amyloid and dextrinoid (FLAS-F-59142), as well as weakly amyloid (FLAS-F-61189). Farid et al. also noticed a dextrinoid reaction in the studied specimen (Farid 499) [148].  Holotype: USA, Vermont, Brattleboro, C.C. Frost. Preserved in VT and selected by Halling as lectotype (VT3156) [149]. According to Halling, several additional specimens currently housed in VT, FH and NYBG can be considered "original material" because these are labeled in Frost's handwriting and were apparently identified by Frost [149].
Ecology: Solitary to scattered or gregarious, growing primarily in forests but sometimes also in open grassy places, in association with Quercus (Fagaceae) or in mixed stands with Fagus (Fagaceae), Tsuga (Pinaceae) and Arbutus menziesii (Ericaceae). Summer to autumn (June to October), occasional to fairly common.
Known distribution: Reported from eastern North America, Canada (Quebec), and USA (New England south to Florida and west to Wisconsin and Arizona), repeatedly recorded in Mexico, Guatemala and Costa Rica, but identity not confirmed to date based on molecular data. Southern limits yet to be established. Comments: This species has been sufficiently described and illustrated in several monographic treatments and popular field guides over the past century (see above). Similar to the case for E. floridanus (see above), there is cryptic diversity within E. frostii s. l., including at least four distinct clades. Our molecular evidence indicates that specimens from the Eastern USA are found in three (Figure 1, E. cf. frostii 1, 3 and 4) of the four clades, so it is premature to epitypify E. frostii until further studies are carried out in New England to determine which taxon is likely to represent the type. Exsudoporus cf. frostii 1 (from New Hampshire, Ohio, Michigan, Tennessee) or E. cf. frostii 4 (from Ohio, Michigan and Florida) probably represent the original species as described by J.L. Russell from Vermont and New York [130,149].
One of the clades (Figure 1, E. cf. frostii 2) in the E. frostii complex includes specimens from Mexico and Costa Rica that likely represent a new species. It is clear that records of E. frostii s. l. from Mexico [144,145,163,[171][172][173][174][175][176], Guatemala [147] and Costa Rica [163,177,178] are in need of urgent taxonomic revision because they likely represent an undescribed taxon. The occurrence of Boletus frostii in India as reported by Sharma et al. and Verma and Pandro are doubtful and likely represent a different taxon too [179,180].
Ecology: Solitary to scattered, growing in subalpine coniferous forests (1800-4200 m alt.) dominated by Abies spp. (including A. chayuensis, A. georgei, A. mariesii and A. veitchii), Pinus densata and Tsuga diversifolia (Pinaceae). Summer to autumn (July to October), uncommon. Comments: Exsudoporus ruber was originally described from Yunnan Province (southwestern China) by Zang [132] and was also examined in his additional publications [133,135]. This species was recently re-described based on fresh collections, taxonomically revisited in the light of molecular inference and subsequently transferred to the genus Butyriboletus [136]. Two combinations of B. rubrus nom. inval. and Bu. rubrus were published with a grammatical mistake. The epithet was intended as the masculine form of the neuter basionym epithet "rubrum", so the correct one should be "ruber". A more comprehensive phylogenetic analysis carried out in the present study indicates that this species is more appropriately placed in Exsudoporus. This placement is supported by the close phylogenetic relationship between E. ruber and E. frostii s. l. but is also strengthened by the evident morphological resemblance with other species of Exsudoporus. Exsudoporus ruber was incorrectly identified from Japan as B. frostii by the Mycological Society of Japan during a past investigation of the mycoflora of Mount Fuji (Y. Taneyama, personal observation). Later, it was fully characterized by Takahashi et al. under the name Boletus kermesinus Har. Takah., Taneyama and Koyama, based on collections from Nagano Prefecture [134]. There was no mention in the original diagnosis or protologue of B. kermesinus about the yellow droplets exuding from the hymenophore, since at the time of publication it could not be determined with confidence whether it was a diagnostic trait for this species. However, these droplets are clearly visible in photos of this species, including from the holotype material ( Figure 6H). Similarly, hymenophoral droplets have not been reported in the literature on E. ruber. According to the original diagnosis, E. ruber differs from B. kermesinus by the squamulose-punctate stipe surface, reddish tubes and lack of cheilocystidia [132,134]. These discrepancies, however, turned out to be unreliable [136]. Molecular phylogenetic inference confirmed that the Japanese Boletus kermesinus is conspecific with E. ruber, a heterotypic synonymy previously conjectured by Wu et al. [136] based solely on morphology.
Unfortunately, it has not been possible to examine or sequence DNA from the holotype in Amoenoboletus species, there are no data in the literature. Present studies of two specimens of A. weberi (FLAS-F-61525 and FLAS-F-68076) and one specimen of A. mcrobbii (K-M000025241) show an inamyloid reaction.
Hymenophoral droplets of Amoenoboletus spp. have not been reported in the published sources. However, pale greenish-yellow droplets are clearly visible in the picture of A. weberi 102459 on the Mushroom Observer website, and I.G. Safonov noted this feature in the description of the specimen [189].

Discussion
Over the past several years, numerous publications have treated Exsudoporus species as members of the genus Butyriboletus [18,136,[190][191][192][193]. However, our integrated morphological and molecular analyses indicate that Exsudoporus is the unequivocally resolved sister group of Butyriboletus, as previously hypothesized by Vizzini [26] and confirmed by Gelardi et al. [25], Bozok et al. [38], Loizides et al. [39] and Farid et al. [148]. Since there are distinct morphological features associated with each of the two lineages and they are unambiguously autonomous evolutionary lineages, we recognize these as two distinct genera.
Boletus subsplendidus W.F. Chiu is the most closely related taxon to Exsudoporus and forms a monotypic generic clade (BS = 100%, PP = 1.00). A branch that unites these two sister clades has weak statistical support (BS = 52%, PP = 0.89). Boletus subsplendidus was also placed in Butyriboletus by Wu et al. but with the recognition of the genus Exsudoporus it will need to be transferred to a separate genus [18]. Boletus subsplendidus is a small to medium sized bolete with reddish-brown to dark brown pileus, 2.5-6 cm diam., stipe 5-9 × 0.7-2 cm, yellow on the upper part and pinkish to reddish towards the base, with a reddish reticulum covering nearly the entire length of the stipe or at least the upper half, orange-red to yellow hymenophore, tissues turning blue when exposed or bruised and basidiospores 9-12 × 3.5-4.0 µm, Q m = 2.7 [18,194,195].
Along with Exsudoporus, Liang et al. also decided to include Butyriboletus hainanensis N.K. Zeng, Zhi Q. Liang and Dong Y. [190] in the genus Butyriboletus, despite the fact that the topology of these three well-supported clades in their phylogram is similar to the current phylogenetic reconstruction. They indicated that Bu. hainanensis is phylogenetically distinct from the described Butyriboletus taxa. There are also clear morphological features that distinguish Bu. hainanensis from the other known species in Butyriboletus. These characters include a blue-red-black color change of the hymenophore and context, a thick pileal context and a thin hymenophore (similar to that in Baorangia) and a stipe with a faint reticulation. Butyriboletus hainanensis and two unknown allied species from China constitute a well-supported generic clade (BS = 99%, PP = 1.00) and further research is needed to describe this group in more detail.
In this study, we also analyzed an additional generic lineage, Amoenoboletus, which has some morphological and genetical similarity with Exsudoporus. This group forms a strongly supported clade (BS = 100%, PP = 1.00) and includes at least six different species, but some other taxa (e.g., B. rubropictus and B. guatemalensis) should be evaluated in the future based on molecular data to see whether they are phylogenetically related or not.
In the present study we have performed the first comprehensive molecular phylogenetic reconstruction of the genus Exsudoporus with the inclusion of several new sequences of multiple genetic markers. Our analysis includes accessions across the Northern Hemisphere and has verified the separation of this group from the genus Butyriboletus. Phylogenetic results clearly support the separation of these two genera, and their independence is also reinforced by consistent morphological differences. Species of Exsudoporus exhibit several critical characters that discriminate them from Butyriboletus species, including: (1) an overall reddish color of the basidiomes, (2) red pores, (3) non-stuffed pores (although Smith and Thiers [7], quoting Coker, indicated stuffed pores for E. frostii), (4) hymenophore exuding golden-yellow droplets in fresh, young specimens, (5) stipe surface strongly and coarsely reticulate to reticulate-alveolate or with scaly patches, (6) generally stronger bluing reaction on bruising (with the exception of E. ruber) and (7) hyphae of the stipe base context usually weakly to strongly amyloid, although the iodine test may also result in a negative or even "pseudoamyloid" (dextrinoid) reaction. In sharp contrast, Butyriboletus is characterized by: (1) a different combination of colors with predominantly yellow tints, (2) yellow pores, (3) stuffed pores in early developmental stages, (4) a hymenophore that never exudes droplets, (5) a very shallowly and densely reticulate stipe surface, (6) generally weaker bluing reaction and no bluing at all in the context of the mid or lower stipe and (7) consistently inamyloid hyphae of the stipe base context [16,28,32,196,197].
Based on phylogenetic evidence, the closely related eastern North American species E. frostii and E. floridanus have been recognized as congeneric to the type species, the European E. permagnificus. Moreover, the Asian Leccinum ruber is an additional representative of the genus and accordingly is transferred to Exsudoporus based on morphological affinities and molecular evidence. There also appear to be at least four additional taxa from the USA, Mexico and Central America (Belize, Guatemala, Costa Rica) that were previously misidentified as either B. frostii or B. floridanus [138,[143][144][145][146][147]163,171,[173][174][175][176][177][178]198]. However, the taxonomic and geographic limits of these tentative new species are in need of further investigation.
SEM images of the basidiospores of E. permagnificus were previously published by Assyov and clearly show a smooth spore wall. Although we did not take SEM pictures of the basidiospores of E. frostii, E. floridanus and E. ruber, it is likely that they also possess a smooth spore wall [120].
Krisai-Greilhuber and Takahashi et al. reported an inamyloid reaction of the hyphae for E. frostii and E. ruber, respectively [131,134]. Conversely, we observed an amyloid reaction in several specimens of E. permagnificus as well as in the herbarium material of E. frostii, E. floridanus and E. ruber. A fleeting amyloid reaction was also reported previously for E. frostii by Smith and Thiers [7]. Moreover, a dextrinoid reaction was also observed in E. floridanus and E. permagnificus.
Results obtained in this study highlight a Holarctic distribution of Exsudoporus. Species in the genus are widespread across temperate to subtropical latitudes in both the Western Hemisphere (E. frostii and E. floridanus in eastern North America) and the Eastern Hemisphere (E. ruber in East Asia). The genus also extends into warm climatic regions, including two undescribed neotropical species reported from Central America and E. permagnificus, which occurs in dry and warm Mediterranean environments in Europe and the Middle East. It is worth noting that, with the exception of E. ruber, all other Exsudoporus species appear to be associated with angiosperms, especially with diverse species of oaks. Conversely, E. ruber seems to be restricted to the subalpine belt in association with coniferous trees such as Abies, Pinus and Tsuga. However, the evolutionary paleogeographic dynamics that led to the present distribution and ecological preferences of Exsudoporus species remain unknown.
Further research will be required to better assess the ecological requirements and distribution limits of the known species to formally describe cryptic species detected in this study and also to ascertain whether or not there are other overlooked species of Exsudoporus that remain undescribed from other parts of the world.