Coccoloba-associated xerocomoid boletes (Boletaceae) from the Caribbean and Mexico: Tropicoboletus ruborculus gen. et comb. nov., revision of Xerocomus coccolobae, phylogenetic assessment of Singerocomus guadelupae comb. nov., and type studies of Xerocomus caeruleonigrescens, X. cuneipes, and X. pseudoboletinus var. pini-caribaeae

Only two Coccoloba-associated xerocomoid boletes with smooth basidiospores are currently known from the Dominican Republic, namely Boletus ruborculus and Xerocomus coccolobae. A multilocus phylogenetic analysis of four gene markers (ITS, LSU, RPB2, TEF1) reveals that B. ruborculus forms an autonomous clade in the Boletaceae corresponding to a novel genus, which is introduced here as Tropicoboletus gen. nov., whereas X. coccolobae is confirmed as a member of Xerocomus s. str. Tropicoboletus is sister to subfamily Xerocomoideae in the combined RPB2/TEF1 Boletaceae-wide analysis. Accurate morphological descriptions of the two species based on well-annotated samples are provided, accompanied by color photographs of fresh specimens in habitat and line drawings of their main anatomical features. The holotype collections of B. ruborculus and X. coccolobae were successfully sequenced and re-examined anatomically. The distribution range of Tropicoboletus ruborculus comb. nov. is extended from the original locality in Puerto Rico to the Dominican Republic and Mexico where its presence is reported for the first time. Similarly, the Dominican collections of X. coccolobae represent the first documented occurrence of this species for the Island of Hispaniola. Based on molecular and morphological evidence, we conclude that the Belizean species Xerocomus olivaceus is conspecific with X. coccolobae and is therefore reduced into synonymy. In addition, the holotypes of Xerocomus caeruleonigrescens, Xerocomus cuneipes, and Xerocomus pseudoboletinus var. pini-caribaeae were microscopically re-studied, although their exact taxonomic placement remains unresolved in the absence of any phylogenetic inference. Molecular investigation of a paratype of Boletus guadelupae resulted in a conspecificity with the recently described Singerocomus atlanticus from Brazil, extending the biogeographic coverage of Singerocomus to the Caribbean. Accordingly, the new combination Singerocomus guadelupae is proposed and S. atlanticus is synonymized. Finally, a putative novel Xerocomus s. str. species is discovered from the Dominican Republic but not formally described for the time being due to the paucity of material available.

It appears clear, however, that additional monophyletic clades are expected to be uncovered as soon as supplementary molecular investigation of poorly known or critical boletoid and xerocomoid taxa will become available in the near future, especially from remote and underexplored areas of the pantropical belt.
Although it has not been as thoroughly investigated as North America and Europe, Mesoamerica has been the object of a large number of mycological contributions published in the past decades which have enhanced our understanding of the neotropical boletes diversity and biogeography with a special emphasis on boletoid or xerocomoid taxa, underpinning the health and functioning of different ecosystems in Mexico, mainland Central America, northern South America, and the Caribbean (e.g., Singer and Fiard 1977;Pegler 1983;Singer et al. 1983;Singer 1988;Halling 1989Halling , 1992Halling , 1997Gonzáles-Velázquez and Valenzuela 1993;Gómez 1997;Halling and Mueller 1999, 2002García-Jiménez 1999, 2013Miller et al. 2000;Minter et al. 2001;Halling and Mata 2004;Flores Arzù and Simonini 2000;Franco-Molano et al. 2000;García-Jiménez 2013;García-Jiménez et al. 2013;Ortiz-Santana et al. 2007;Courtecuisse and Welti 2013;de la Fuente et al. 2018;Flores Arzù 2020). However, most of the neotropical bolete species described to date have been defined based solely on morphological, ecological, or biochemical taxonomic criteria and are in urgent need of phylogenetic reconsideration. Moreover, identification efforts are becoming rather more difficult because several Central American species show a wider distribution throughout the neotropics than previously assumed.
Two xerocomoid smooth-spored ECM bolete species associated with Coccoloba (Polygonaceae), namely Boletus ruborculus T.J. Baroni (Miller et al. 2000) and Xerocomus coccolobae Pegler (Pegler 1983), were originally described from the Greater and Lesser Antilles of the Caribbean, respectively. In order to consolidate the taxonomic concept of these two neotropical species, we carefully studied several collections for each species. Furthermore, the DNA of the original material of both species was sequenced for the first time and the holotype specimens re-examined anatomically. In the light of the obtained outcomes, Tropicoboletus is described as a new genus to science to accommodate B. ruborculus, whereas the generic affiliation of X. coccolobae in Xerocomus s. str. is corroborated by its phylogenetic placement. Furthermore, type specimens of additional neotropical species, including Xerocomus pseudoboletinus var. pini-caribaeae Singer, Xerocomus cuneipes Pegler, Xerocomus caeruleonigrescens Pegler, and Boletus guadelupae Singer & Fiard were anatomically re-studied, and the latter species was also phylogenetically investigated.

Collection site and sampling
Specimens examined were collected in Sosúa, Puerto Plata Province, Dominican Republic and El Morro, Viejo San Juan, Puerto Rico. Dominican Republic samples are deposited at the Herbarium of Jardín Botánico Nacional of Santo Domingo, Dr. Rafael Ma. Moscoso, Dominican Republic (JBSD). The holotype collection of Xerocomus pseudoboletinus var. pini-caribaeae and a mixed type (holotype/paratype) of Boletus guadelupae examined in the present study are deposited at the Field Museum of Natural History, Chicago (F), the holotype of Boletus ruborculus is deposited at the New York Botanical Garden (NYBG), while the holotypes of Xerocomus coccolobae, X. cuneipes, and X. caeruleonigrescens and paratype specimens of B. guadelupae are all deposited at the Fungarium of the Royal Botanic Gardens Kew (K-M) (acronyms from Thiers 2022). "ANGE," "MG," "komille," and "de la Fuente" refer to the personal herbarium of Claudio Angelini, Matteo Gelardi, Kurt O. Miller, and J.I. de la Fuente, respectively. Fungarium numbers, unless otherwise stated, are cited for all collections from which morphological features were examined. Author citations follow the Index Fungorum, Authors of Fungal Names (www. index fungo rum. org/ autho rsoff ungal names. htm). Geographic distribution of some studied species have also been checked on MyCoPortal (https:// mycop ortal. org).

Morphological studies
Macroscopic descriptions, macrochemical reactions (30% NH 4 OH, 30% KOH), and ecological information, such as habitat notations, time of fruiting, and associated plant communities, accompanied the detailed field notes of the fresh basidiomes. In the field, latitude, longitude, and elevation were determined with a global positioning system (GPS) receiver. Color terms in capital letters (e.g., White, Plate LIII) are from Ridgway (1912). Photographs of collections were taken in the natural habitat using a Nikon Coolpix 8400 camera. Microscopic anatomical features were observed and recorded from revived dried material; sections were rehydrated either in water, 5% potassium hydroxide (KOH), or in ammoniacal Congo red. All anatomical structures were measured from preparations in anionic Congo red. Colors and pigments were described after examination in water and 5% KOH. Measurements were made at 1000 × using a calibrated ocular micrometer (Nikon Eclipse E200 optical light microscope). Basidiospores were measured directly from the hymenophore of mature basidiomes, dimensions are given as (minimum) average ± standard deviation (maximum), Q = length/width ratio with the extreme values in parentheses, Qm = average quotient (length/width ratio) ± standard deviation and average spore volume was approximated as a rotation ellipsoid [V = (π.L.W 2 )/6 ± standard deviation]. The notation [n/m/p] indicates that measurements were made on "n" randomly selected basidiospores from "m" basidiomes of "p" collections. The width of each basidium was measured at the widest part, and the length was measured from the apex (sterigmata excluded) to the basal septum. Radial and/or vertical sections of the pileipellis were taken midway between the center and margin of the pileus. Sections of the stipitipellis were taken from the middle part along the longitudinal axis of the stipe. Metachromatic, cyanophilic, and iodine reactions were tested by staining the basidiospores in Brilliant Cresyl blue, Cotton blue, and Melzer's reagent, respectively. Line drawings of microstructures were traced in free hand based on digital photomicrographs of rehydrated material.

DNA extraction, PCR amplification, and DNA sequencing
Genomic DNA for all samples, except for the K-M, NYBG and F specimens, was isolated from 25 mg of dried voucher specimens. DNA extraction and PCR amplification were performed as described by Alvarado et al. (2012). The universal primer pairs ITS1F/ITS4 (White et al. 1990;Gardes and Bruns 1993) and LR0R/LR5 (Vilgalys and Hester 1990;Cubeta et al. 1991) were used for the amplification of the internal transcribed spacer (ITS) and the nuclear large ribosomal subunit (LSU) regions of the nrDNA, respectively. The 6-7 region of the RPB2 gene (RNA polymerase II second largest subunit) was amplified using the primer pairs brpb2-6F2/brpb2-7R2 (Matheny et al. 2002(Matheny et al. , 2007. Primers EF1-983F andEF1-1567R (Rehner andBuckley 2005) were used for amplification of the translation elongation factor 1-α (TEF1) gene. The PCR products were purified with the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI) following manufacturer's instructions and positive reactions sequenced forward and reverse by MACROGEN Inc. (Seoul, Republic of Korea).
For the K-M, genomic DNA was extracted following an enzymatic digestion and glass-fiber filtration protocol (Dentinger et al. 2010) and for NYBG and F specimens using the NucleoSpin™ Plant II kit. Amplification of the ITS region was performed following standard conditions and using several primer combinations: ITS1F with ITS4B/  ITS4/ITS2, ITS3 with ITS4B/ITS4, and ITS8F with ITS6R (White et al. 1990;Gardes and Bruns 1993;Dentinger et al. 2010). Successful amplicons were purified using ExoSAP-IT (USB) and sequenced bidrirectionally using BigDye3.1 in a ABI3730 DNA analyzer (Applied Biosystems).
Four different datasets were assembled to elucidate the phylogenetic placement of the targeting taxa, viz. new genus Tropicoboletus and the species Xerocomus coccolobae and Boletus guadelupae. The first dataset focused on family Boletaceae and was generated from combined RPB2 and TEF1 sequences (Table 1). K80 + I + G and TPM1uf + I + G models were chosen for RPB2 and TEF1 alignments, respectively. The second and third datasets focused on the genus Xerocomus s. str. (LSU sequences, GTR + I + G model),     and on X. coccolobae and allied species (ITS sequences, TrN + I + G model) (Tables 2 and 3). The fourth dataset focused on Boletus guadelupae and allied species in Singerocomus based on ITS sequences (Table 4), using TPM3uf + G as the best fit model. For each molecular marker (LSU, RPB2, and TEF1), three extra single-locus alignments focused on Boletaceae were generated by retrieving all available sequences from Gen-Bank (Suppl. Mat. Figures 2-4). GTR + I + G, TIM1 + I + G and TrN + I + G models were chosen for LSU, RPB2, and TEF1, respectively. The lack of sequences from different loci for the same vouchers prevented us from generating a multilocus analysis.
Phylogenetic analyses were performed using maximum likelihood (ML) with RAxML-NG v. 1.0.1 (Kozlov et al.

2019) and
Bayesian Inference (BI) with MrBayes v. 3.2.7a (Ronquist et al. 2012) in the CIPRES science gateway (Miller et al. 2010). ML analyses were performed with 1000 bootstrap replicates (Felsenstein 1985), under the selected evolutionary models to obtain estimates for maximum likelihood bootstrap values (MLB). BI analyses were performed with one cold and three incrementally heated simultaneous Monte Carlo Markov chains (MCMC) run for 10 M generations, under the selected evolutionary models for each unlinked partition. Two simultaneous runs were performed independently. Trees were sampled every 1000 generations, resulting in sampling of 10,001 trees per single run with the first 2500 trees (25%) discarded as burn-in. For the remaining trees of the two independent runs, a majority rule consensus tree showing all compatible partitions
Both Bayesian and ML analyses produced the same topologies. Therefore, only the ML trees with both MLB and BPP values are shown (Figs. 1, 2, 3, and 4). The combined RPB2/TEF1 Boletaceae-wide data matrix comprises 285 sequences and is 2067 bp long ( Fig. 1, Suppl. Mat. Figure 1 and Table 1). The LSU data matrix of the genus Xerocomus comprises a total of 48 sequences and 865 characters ( Fig. 2 and Table 2). The ITS data matrix of X. coccolobae and allied species comprises 22 sequences and 833 characters ( Fig. 3 and Table 3). The ITS data matrix of B. guadelupae and allied species (Singerocomus) comprises 29 sequences and 870 characters ( Fig. 4 and Table 4).
In the combined RPB2/TEF1 Boletaceae-wide analysis ( Fig. 1, Suppl. Mat. Figure 1), the subfamilies recognized in recent studies (e.g., Wu et al. 2014;Gelardi et al. 2015;Henkel et al. 2016;Vadthanarat et al. 2019Vadthanarat et al. , 2022Badou et al. 2022) were also recovered. Xerocomus coccolobae and X. aff. coccolobae are nested in the genus Xerocomus s. str. (typified with X. subtomentosus). Sequences of X. olivaceus from Belize and USA (Florida) (including the holotype NR_175148) cluster with X. coccolobae in the same terminal clade (Figs. 2 and 3). The ITS P%I value of the "coccolobae clade" is = 99.7. Collections of B. ruborculus from Puerto Rico, the Dominican Republic and Mexico form a strongly supported clade which is sister to subfamily Xerocomoideae in the combined RPB2/TEF1 Boletaceae-wide analysis (with only MLB support) and in the single-locus TEF1 Boletaceae-wide analysis Basidiomes small to medium-small. Pileus (1.5-) 1.9-7.0 (-8.2) cm broad, at first hemispherical then persistently convex and finally broadly pulvinate-flattened, sometimes slightly depressed at center, regularly to unevenly shaped by shallow depressions, moderately fleshy, firm at the beginning but progressively softer with age, flabby in old basidiomes; margin even to faintly wavy-lobed, initially slightly involute Pulveroboletus spp.
Chalciporus spp. Buchwaldoboletus lignicola and seven Chalciporus species (subfamily Chalciporoideae) were used as the outgroup taxa. All taxa belonging to subfamilies Austroboletoideae, Boletoideae, Chalciporoideae, Leccinoideae, and Zangioideae were collapsed into subfamily clades. All generic clades in the "Pulveroboletus group" that were highly supported were also collapsed. In the subfamily Xerocomoideae clade, Xerocomus s. str. was not collapsed to highlight the position of X. coccolobae and X. aff. coccolobae. Newly generated sequences are indicated in bold in diam.), simple, firstly labyrinthine to roundish becoming prominently angular at maturity, stretched and radially arranged towards the stipe, concolorous with the tubes and unchangeable or irregularly bluing (Paris Blue, Patent Blue, Pl. VIII) on bruising or when injured, sometimes with scattered rusty brown (Ferruginous, Pl. XIV; Mikado Brown, Pl. XXIX) stains at the orifice in aged specimens. Stipe (3.0-) 3.5-5.5 (-7.8) × 0.4-1.6 (-2.0) cm, as long as or slightly longer or shorter than the pileus diameter at maturity, central to slightly off-center, solid, firm, dry, straight or curved to occasionally sinuous, cylindrical, subcylindrical to gradually and faintly enlarged or attenuated from apex down to the base, usually ending with a short taproot at the very base; surface finely to coarsely granulose throughout, with granules more densely arranged in the upper half, devoid of reticulum or ribs, evelate; entirely ornamented by orange-brown, pale brown, brownish olive to chestnut brown granules (Carnelian Red, Vinaceous-Rufous, Cinnamon-Rufous, Hazel, Pl. XIV; Cacao Brown, Pl. XXVIII; Buffy Olive, Pl. XXX; Fawn Color, Wood Brown, Pl. XL; Light Cinnamon-Drab, Cinnamon-Drab, Pl. XLVI) on a whitish background (White, Pl. LIII) and unfrequently with a narrow purplish brown band (Pansy Purple, Pl. XII) in the upper fourth, unchangeable when pressed; basal mycelium whitish (White, Pl. LIII). Context firm and tough when young, later soft textured and eventually flabby in the pileus (up to 2.2 cm thick in the central zone, up to 1.4 cm thick halfway to margin and gradually becoming thinner towards the edge), a little more fibrous in the stipe, at first cream yellowish throughout (Baryta Yellow, Martius Yellow, Picric Yellow, Pl. IV), later very pale yellowish to whitish (Pale Viridine Yellow, Light Green-Yellow, Green-Yellow, Greenish Yellow, Pl. V; White, Pl. LIII) in the pileus and in the connection zone with the stipe, whitish in the stipe (White, Pl. LIII), rarely with scattered pinkish, purplish pink, or pinkish vinaceous spots ( base; unchangeable to slowly and faintly turning pale blue (Beryl Blue, Pallid Methyl Blue, Pale Methyl Blue, Light Methyl Blue, Pl. VIII) in the pileus context and in the connection zone with the stipe when exposed to air, unchangeable elsewhere; brownish (Auburn, Pl. II; Umber Brown, Pl. III) where eroded by maggots, cream yellowish where eaten by slugs (Light Green-Yellow, Green-Yellow, Greenish Yellow, Pl. V); subhymenophoral layer cream yellowish to pale yellowish (Baryta Yellow, Martius Yellow, Picric Yellow, Pl. IV; Light Green-Yellow, Green-Yellow, Greenish Yellow, Pl. V); exsiccate pale ochraceous on the context, brownish elsewhere (Clay Color, Tawny-Olive, Saccardo's Umber, Pl. XXIX). Odor indistinct. Taste mild. Spore print not obtained. Macrochemical spot-test reactions: 30% KOH: bright orange to vinaceous red on pileus surface, pale orange to reddish orange on context and hymenophore; 30% NH 4 OH: vinaceous red on pileus surface, none elsewhere.
Edibility unknown. Ecology and phenology: solitary to gregarious, growing on limestone among litter in a seasonally dry and moist anthropogenic lowland mixed stand under a large array of neotropical broadleaved trees including Coccoloba diversifolia (Polygonaceae), which represent its potential ECM host plant. See Parra et al. (2018) for further details on lowland vegetation in the Dominican Republic. Apparently localized in the Dominican Republic. November to December.
Known distribution: Reported to date from both the Lesser and Greater Antilles of the Caribbean (Cuba, Dominican Republic, British Virgin Islands, Martinique), south-eastern USA (Florida), and Mexico but likely widespread in Mesoamerica. Its occurrence in Brazil appears to be unlikely (see discussion below).
With the only exception of the length of hymenial cystidia which appear to be decidedly longer in the Dominican material of X. coccolobae when compared with either the protologue or the type revision, the entire overlapping of the remnant morphological, ecological and biogeographic traits of the Dominican Republic collections with the original material described from Martinique by J.P. Fiard (Pegler 1983) coupled with the phylogenetic outcomes allow us to undoubtedly attribute them to the same species. Moreover, the ITS sequence generated from the type material of X. coccolobae perfectly match those obtained from the Dominican material, thus confirming their conspecificity. Apart from a nil macrochemical reaction on external surfaces with NH 4 OH, there is no other sound morphological or ecological difference, nor molecular evidence for considering Xerocomus olivaceus B. Ortiz & T.J. Baroni (Ortiz-Santana et al. 2007) a different species with respect to X. coccolobae. The ITS sequence of the holotype material of X. olivaceus clearly nested within the terminal clade of X. coccolobae and therefore we merge the Belizean bolete into X. coccolobae as a later heterotypic synonym.
Key determining features of X. coccolobae include small to medium-small sized basidiomes, finely to coarsely granulose, brownish to dark brown or less frequently chestnut brown to purplish brown pileus and stipe surfaces, bright yellow olive tubular hymenophore, whitish basal mycelium, pale yellowish to whitish context usually unchanging to irregularly staining light blue in the pileus-stipe connection zone when damaged, reddish reaction with NH 4 OH on pileus cuticle, ellipsoid to broadly ellipsoid, smooth basidiospores, slender pleurocystidia up to 106 μm long, hymenial cystidia (both cheilo-and pleurocystidia) sometimes multiseptate, a palisadoderm pileipellis of cylindrical hyphae, hymenophoral trama of the "Phylloporustype" and the occurrence in lowland xero-mesophytic mixed broadleaved forests in apparent association with Coccoloba spp. (Polygonaceae) (including C. uvifera, C. diversifolia, C. spicata, C. swartzii, C. pubescens, etc.) (Pegler 1983;Ortiz-Santana et al. 2007 as "X. olivaceus"; this study). Based on our observations, the bluing oxidation of hymenophore and context in X. coccolobae is usually absent but sometimes present and quite variable in terms of range and intensity, depending on specimens age and weather conditions. It should therefore be considered a feature of low taxonomic significance. Similarly, pileus and stipe surfaces exhibit a rather considerable color variation at maturity, although always spanning in the range of brown, making the diagnostic value of these chromatic traits overestimated in the past. This is the first verified report of X. coccolobae from the Dominican Republic. Xerocomus coccolobae has so far been reported from the Caribbean (Cuba, Dominican Republic, British Virgin Islands, and Martinique) and from Mexico (Veracruz, Quintana Roo, Yucatan) (Pegler 1983;García-Jiménez 1999;Minter et al. 2001;Ortiz-Santana et al. 2007;de la Fuente et al. 2018de la Fuente et al. , 2020. According to the present outcomes, the distribution of X. coccolobae should also be extended to south-eastern USA (Florida) in association with Coccoloba uvifera. However, the exact area of occupancy of X. coccolobae is currently indefinite but based on the current known distribution and host association it is plausible to claim that its geographical range may correspond with that of the plant genus Coccoloba, which is an important constituent of the coastal mixed vegetation communities of neotropical lowland ecosystems and that most likely represents its ECM plant associate.
The possibility of confusion with any of the numerous similar Xerocomus s. str. species cannot be ruled out. A certain morphological affinity exists between X. coccolobae and other xerocomoid taxa occurring in the same geographical macro-region, such as X. hypoxanthus, X. cuneipes, and X. pseudoboletinus var. pini-caribaeae.
Basidiomes small. Pileus (2.2-) 2.8-4.5 (-4.7) cm broad, at first hemispherical then persistently convex and finally broadly pulvinate-flattened, sometimes slightly depressed at center, regularly to unevenly shaped by shallow depressions, moderately fleshy, firm at the beginning but progressively softer with age, flabby in old basidiomes; margin even to faintly wavy-lobed, initially slightly involute then curved downwards and finally completely plane or even uplifted, shortly appendiculate and extending beyond the tubes up to 1 mm; surface matt, dry, finely tomentose but later smooth and glabrous and then slightly greasy with moist weather, not cracked; somewhat variable in color, ranging from flesh-pink to purplish pink or pinkish vinaceous to vinaceous red (Hermosa Pink, La France Pink, Shrimp Pink, Pl. I; Safrano Pink, Orient Pink, Pl. II; Venetian Pink, Alizarine Pink, Acajou Red, Vandyke Red, Pl. XIII; Deep Vinaceous, Dark Vinaceous, Pl. XXVII) but progressively fading with age becoming pinkish gray, pinkish brown, brownish pink to grayish or pale grayish brown (Light Vinaceous-Fawn, Vinaceous-Fawn, Fawn Color, Army Brown, Buffy Brown, Pl. XL; Purple-Drab, Vinaceous-Drab, Pl. XLV; Light Cinnamon-Drab, Cinnamon-Drab, Light Drab, Pl. XLVI; Pale Mouse Gray, Light Mouse Gray, Olive Gray, Mouse Gray, Pl. LI; Storm Gray, Pl. LII) starting from the center, although tending to retain pinkish hues towards the peripheral zone even in senescence, always paler at margin, outer rim usually whitish (White, Pl. LIII); slowly reddening (Pomegranate Purple, Bordeaux, Pl. XII; Deep Vinaceous, Dark Vinaceous, Pl. XXVII) on handling or touching or more obviously when injured; subpellis layer reddish violet (Rose Pink, Pale Amaranth Pink, Mallow Pink, Pl. XII). Tubes wide at first in side view then increasingly broader with age and as long as or slightly longer or shorter than the thickness of the pileus context (up to 0.6 cm long), adnate but soon depressed around the stipe apex and decurrent with a short tooth, pale yellow to olive yellow and finally brownish olive (Buff Yellow, Pl. IV; Greenish Yellow, Bright Green Yellow, Oil Yellow, Javel Green, pl. V; Primuline Yellow, Pl. XVI; Raw Sienna, Pl. III) at maturity, unchangeable to erratically turning very light blue (Pale Blue-Green, Tyrolite Green, Pl. VII) when cut. Pores initially forming a concave surface, later flat then slightly convex, broad at first then gradually wider wth age (up to 2 mm in diam.), simple, firstly roundish becoming prominently angular at maturity, stretched and radially arranged towards the stipe, concolorous with the tubes and unchangeable or irregularly and very slowly and faintly bluing (Pale Blue-Green, Tyrolite Green, Pl. VII) on bruising or when injured, sometimes with scattered rusty brown (Ferruginous, Pl. XIV; Mikado Brown, Pl. XXIX) stains at the orifice in aged specimens. Stipe (2.3-) 2.6-5.8 (-9.0) × 0.5-1.0 (-1.3) cm, slightly longer than or as long as the pileus diameter at maturity, central to slightly off-center, solid, firm, dry, straight or curved to occasionally sinuous, cylindrical, subcylindrical to gradually and faintly swollen or conversely attenuated from apex down to the base, usually ending with a short taproot at the very base; surface longitudinally finely fibrillose throughout, non-reticulate, evelate; whitish to pale yellowish (White, Pl. LIII; Baryta Yellow, Pinard Yellow, Pl. IV) in the upper third, whitish (White, Pl. LIII) elsewhere but irregularly streaked or mottled bright flesh-pink, pinkish vinaceous or purplish pink to purplish red (Hermosa Pink, La France Pink, Shrimp Pink, Pl. I; Safrano Pink, Orient Pink, Pl. II; Venetian Pink, Alizarine Pink, Acajou Red, Vandyke Red, Pl. XIII), with a pale yellow to yellow (Baryta Yellow, Pinard Yellow, Picric Yellow, Pale Lemon Yellow, Pl. IV) basal tomentum, unchangeable or faintly reddening (Pomegranate Purple, Bordeaux, Pl. XII; Deep Vinaceous, Dark Vinaceous, Pl. XXVII) to rarely very slowly and faintly bluing (Pale Blue-Green, Tyrolite Green, Pl. VII) when pressed; basal mycelium yellow (Pale Lemon Yellow, Lemon Yellow, Pl. IV). Context firm and tough when young, later soft textured and eventually flabby in the pileus (up to 0.9 cm thick in the central zone, up to 0.7 cm thick halfway to margin and gradually becoming thinner towards the edge), a little more fibrous in the stipe, whitish, pale cream to very pale yellowish (White, Pl. LIII; Baryta Yellow, Pl. IV) in the pileus but cream to pale yellowish (Baryta Yellow, Pinard Yellow, Pl. IV) upon the tubes and upper fourth of the stipe, pinkish violet (Rose Pink, Pale Amaranth Pink, Mallow Pink, Pl. XII) underneath the cuticle, more or less evenly bright fleshpink, purplish pink or pinkish vinaceous to vinaceous red (Hermosa Pink, La France Pink, Shrimp Pink, Strawberry Pink, Peach Red, Pl. I; Safrano Pink, Orient Pink, Grenadine Pink, Grenadine, Pl. II; Venetian Pink, Alizarine Pink, Old Rose, Pl. XIII) in the rest of the stipe but pale brownish to dirty brown (Medal Bronze, Dark Citrine, Pl. IV; Isabella Color, Light Brownish Olive, Pl. XXX) at the very base; very slowly and faintly turning pale blue (Pale Blue-Green, Pl. VII; Pale Blue, Light Cerulean Blue, Cerulean Blue, Pl. VIII) upon the tubes and more sporadically in the connection zone with the stipe when exposed to air, occasionally bluing all over the pileus context, unchangeable or nearly so elsewhere; yellowish (Pinard Yellow, Pl. IV) to dark vinaceous red (Pomegranate Purple, Bordeaux, Pl. XII; Deep Vinaceous, Dark Vinaceous, Pl. XXVII) where eroded by maggots, whitish to pale flesh-pink where eaten by slugs (Hermosa Pink, La France Pink, Pl. I; White, Pl. LIII); subhymenophoral layer pale yellowish (Baryta Yellow, Pinard Yellow, Pl. IV); exsiccate pale ochraceous on the context, brownish elsewhere (Clay Color, Tawny-Olive, Saccardo's Umber, Pl. XXIX). Odor indistinct. Taste mild to slightly sour. Spore print olive-brown. Macrochemical spot-test reactions: 30% KOH: vinaceous red on pileus surface; 30% NH 4 OH: with vapors greenish black on pileus surface.
Edibility unknown. Ecology and phenology: solitary to gregarious, growing on limestone among litter in a seasonally dry and moist anthropogenic lowland mixed stands under a large array of neotropical broadleaved trees including Coccoloba spp. (C. uvifera, C. diversifolia, C. pubescens, C. spicata, etc.) (Polygonaceae), which represent its putative ECM host trees. See Parra et al. (2018) for further details on lowland vegetation communities in the Dominican Republic. November and December.
Known distribution: It is known to date only from Mexico and the Greater Antilles of the Caribbean (Dominican Republic and Puerto Rico) but almost certainly also occurring in Belize and neighboring countries of mainland Central America. Apparently localized and infrequent.
Notes: Tropicoboletus is a novel genus segregated from the polyphyletic Boletus s.l. Multilocus phylogenetic analysis clearly resolved Boletus ruborculus with strong statistical support in a monophyletic lineage sister to subfamily Xerocomoideae (Fig. 1). The isolated phylogenetic placement of Tropicoboletus justifies its recognition as an independent genus.
There does not appear to be one exclusive morphological trait that could serve alone to separate Tropicoboletus from similar genera in the Boletaceae; however, a combined set of features allows a prompt circumscription of this new genus. The only known species T. ruborculus can be recognized, even in the field, with reasonable certainty as it is easily distinguished by a combination of macro-morphological characters: basidiomes with a diminutive size and xerocomoid silhouette, flesh-pink, pinkish red or vinaceous red to brownish red pileus and stipe surfaces, yellowish olive tubular hymenophore, slowly and erratically bluing tissues on exposure, vivid yellow basal mycelium, a sordid green reaction on pileus surface with ammonia vapors and the occurrence in lowland mixed broadleaved tropical woodlands in probable association with Coccoloba spp. In addition, some anatomical key features integrating macroscopic identification include ellipsoid-fusiform, smooth basidiospores, predominantly and distinctly ventricose-fusiform to ampullaceous hymenial cystidia, a trichodermal pileipellis and the hymenophoral trama of the "Phylloporus-type" (Miller et al. 2000;this study). Beside the peculiar ecosystem where this bolete resides, the distinctive and conspicuous yellow basal mycelium is the most reliable diagnostic attribute for a proper recognition of the species in the field. This clear-cut feature, however, has not been previously emphasized. In the protologue of B. ruborculus (Miller et al. 2000) nothing is said about the color of the basal mycelium, either because its importance was underestimated or because it was simply overlooked. The association with Coccoloba species is most likely but not yet confirmed by direct observation of the ectomycorrhizae. Interestingly, specimens collected in Puerto Rico under C. uvifera (including the type specimen) exhibit a brighter red pileal surface when compared to basidiomes occurring with C. diversifolia from the Dominican Republic, but they are otherwise identical from both morphological and phylogenetic aspects.
Confident identification of T. ruborculus is also reinforced in the present study by the availability of additional verified samples recently yielded in Puerto Rico and Mexico which were placed in the same clade as the Dominican vouchers. However, just a handful of collections of this rare species are presently known from the neotropics, making T. ruborculus a sparingly encountered species. Prior to the present study, T. ruborculus resulted unnoticed from Mexico and the Dominican Republic as this species was known only from the type locality in Puerto Rico (Miller et al. 2000). Presumably, it is native to Central America and most likely widespread throughout the neotropics but to what extent is the actual distribution range of T. ruborculus remains to be determined. One might hypothesize that the distribution of this species roughly overlaps with that of Coccoloba, which appears to represent its alleged strict symbiotic partner.
Despite its resemblance with several other red-colored xerocomoid boletes, macro-and micro-morphological features of T. ruborculus are reliable and compelling enough to allow a clear-cut delimitation from lookalikes such as Boletus guadelupae and Xerocomus caeruleonigrescens, which grow in the same ecosystem in alleged association with various species of Coccoloba.
More collections of T. ruborculus are surely needed for a better taxonomic understanding of the generic limits of Tropicoboletus and a more accurate knowledge of its ecology and geographic distribution patterns in Mesoamerica. Furthermore, future extensive research in undersampled ecological niches might uncover additional members of Tropicoboletus, especially from the paleotropics.

Clarification on type collections of Boletus guadelupae
There appears to be much confusion with dates and collection numbers of the type specimens of B. guadelupae. Based on data provided in the protologue (Singer and Fiard 1977), the holotype was collected in Basse Terre, Matouba, Guadelupe, 31 Jul 1975 (collection number not specified), whereas J.P. Fiard 563B (collected in Trace de Sofaia, Guadelupe, 25 Jul 1975) is designated as paratype, both collections having been deposited at the Field Museum of Natural History, Chicago (F).
In Singer et al. (1983), the collection number of the holotype from Matouba is specified as J.P. Fiard 563C. This is inconsistent with the information reported in Pegler (1983), where the holotype collection from Matouba is reported under the number J.P. Fiard 563A (with a wrong date "July 1075") and strangely enough the type is said to be housed at K-M instead of F as previously reported in Singer and Fiard (1977). This is another contradictory information which can be explained by the fact that the type was later duplicated at K-M. However, according to ICN rules, there cannot be two holotypes of the same species deposited at two different official herbaria. Therefore, we must assume that part of J.P. Fiard 563A was housed at F (labeled as "Fiard 563" and representing the holotype as reported in the protologue and based on the collection date provided by Pegler) and part at K-M (representing an isotype, but see below). The same thing probably happened to the paratype J.P. Fiard 563B, which was split between F and K-M. We have also been able to retrieve J.P. Fiard 563C (K-M000193861) at K-M (Fig. 8e), but unlike what written by Singer et al. (1983) this sample originates from Martinique and is dated 05 Jul 1976. Consequently, J.P. Fiard 563C cannot be considered the holotype and the type data reported in Singer et al. (1983) are evidently wrong. Likewise, the specimen K-M000193860 (which is referred to as J.P. Fiard 563B on 25 Jul 1975, heavily infected by Hypomyces sp.) housed at the Royal Botanic Gardens Kew as the holotype of B. guadelupae is not the holotype nor an isotype but a simple isoparatype (Fig. 8e). We are not able to address why the letter "B" after Fiard's collection number was handwritten modified as "A" in the accompanying label (Fig. 8e), but no doubt the correct number is the original J.P. Fiard 563B. Another authentic specimen K-M000193859, which was collected by Fiard on 23 Jul 1975 and not metioned in the protologue, has a wrong collection number (J.P. Fiard 563B, again with the label handwritten modified by replacing the letter "A" with the letter "B" after Fiard's collection number) (Fig. 8e), but it can be neither J.P. Fiard 563B nor J.P. Fiard 563A, because these vouchers were collected on 25 Jul 1975 and 31 Jul 1975, respectively. Accordingly, the original collection number of K-M000193859 presently remains unknown. K-M000193861 (J.P. Fiard 563C) (Fig. 8e) from Martinique is an additional authentic collection.
With regard to the type material preserved at F, which consists of three fragmented dried specimens (Fig. 8f), it is clearly visible that the upper right label (coll. Fiard 563B) refers to the paratype (although the date 25 Aug 1957 is obviously incorrect) while the bottom left label (Fiard 563, dated 31 Jul 1975) refers to the holotype. Unfortunately, it is impossible to know which of the two collections the dried specimens refer to. We are inclined to believe that the specimens refer to the holotype collection, but we have no proof to demonstrate it. One might hypothesize that J.P. Fiard 563A and J.P. Fiard 563B could have mixed into a single bag, so as to become a sole collection (this would explain the presence of two labels in the same packet). On the other hand, considering all material of B. guadelupae presently housed in the Fungarium of the Kew Gardens, we must conclude that an isotype of this species has never been deposited at K-M, since none of the available collections is dated 31 Jul 1975, unless we accept that the type at F is a mixture of the holotype and paratype specimens, in this case K-M000193860 would actually represent the isotype of B. guadelupae.
This kind of incrustation can be explained by hydrophobin (surface active protein) coating of basidiospores and has similar rodlet pattern to assembled Class I hydrophobins on aerial hyphae of Schizophyllum commune Fr. and pileus surface of Agaricus bisporus (J.E. Lange) Imbach (Wösten et al. 1993;Lugones et al. 1996). Hydrophobins of mushroom-forming fungi (Agaricomycetes) are far from being well studied . So far, Pisolithus tinctorius (Mont.) E. Fisch. (putative P. albus (Cooke & Massee) Priest), Suillus luteus (L.) Roussel, and Paxillus involutus (Batsch) Fr. are the only species of the order Boletales where hydrophobins, which are predominantly produced during ectomycorrhiza formation, were formally detected and characterized (Tagu et al. 1996;Duplessis et al. 2001;Rineau et al. 2017). However, there is no information on basidiospore coat hydrophobins in Boletales in the available literature. They likely play an important role in spore protection and germination, as it has been shown in anamorphic fungi (Cai et al. 2021).

Additional remarks on some incertae sedis genera
In the combined RPB2/TEF1 phylogeny focused on the whole family Boletaceae, we recovered a well-supported clade  (Fig. 1). All these genera are currently settled in the Boletaceae but with an uncertain phylogenetic placement (incertae sedis). The "Bothia clade" might represent an additional subfamily within the Boletaceae, although the only known synapomorphy of this grouping appears to be the cyanophily of the basidiospore wall as previously highlighted by Farid et al. (2018). On the other hand, Pseudoboletus, Gymnogaster and Baorangia do not appear to be strictly related to one another from morphological, ecological, or trophic standpoints and their phylogenetic close proximity could be only artifactual. Obviously, further investigation on morphological, ecological, and chemical features and additional molecular loci will be required to better clarify the taxonomic boundaries of these genera and their reciprocal phylogenetic relationships. Accordingly, we believe it is premature to introduce formal ranks for these two groups of genera and we therefore refrain from proposing new subfamilies for the time being.