Neoboletusantillanus sp. nov. (Boletaceae), first report of a red-pored bolete from the Dominican Republic and insights on the genus Neoboletus

Abstract Neoboletusantillanussp. nov. appears to be the only red-pored bolete known from the Dominican Republic to date. It is reported as a novel species to science based on collections gathered in a neotropical lowland mixed broadleaved woodland. A detailed morphological description, color images of fresh basidiomes in habitat and line drawings of the main anatomical features are provided and relationships with phylogenetically and phenotypically similar taxa are discussed. Three genomic regions (nrITS, nrLSU/28S and rpb2) have been sequenced in order to reinforce the recognition of the new species and to elucidate its taxonomic affiliation within Neoboletus.


Introduction
In recent times the intensive use of molecular tools applied to the investigation of the systematics of boletoid mushrooms and related groups (order Boletales) has dramatically revolutionized traditional classifications based on morphological traits, facilitating the research process and leading to the establishment of a novel scientific approach with unexpected taxonomic implications (Bruns and Palmer 1989, Binder 1999, Binder and Bresinsky 2002, Binder et al. 2005, Binder and Hibbett 2006, Nuhn et al. 2013.
Neoboletus antillanus is described herein as a new species to science using morphological and three-loci (nrITS, nrLSU/28S and rpb2) molecular data, based on multiple collections from a lowland mixed woodland consisting of a number of different neotropical broadleaved trees, in purported ECM association with the widespread, natively sand-growing littoral seagrape, Coccoloba uvifera (L.) L. (Polygonaceae), a small woody plant naturally distributed throughout the Caribbean basin (Séne et al. 2015(Séne et al. , 2018. This notable species appears to be the first and sole red-pored bolete recorded in the Dominican Republic so far and one of the very few ECM members of the Boletaceae to be found in local lowland deciduous forested ecosystem.
The present paper is one in a series of intended contributions devoted to the study of neotropical Boletales, aiming to provide new insights into the taxonomy, phylogenetic relationships, plant and substrate associations, ecological importance, conservation and biogeographic patterns of the bolete communities occurring in the Dominican Republic, with continued biodiversity investigations of underexplored areas.

Collection site and sampling
Specimens examined were collected in a hilly forest near the cemetery of Sousa, in Puerto Plata Province, Dominican Republic, and are deposited in the Herbarium of Jardín Botánico Nacional of Santo Domingo, Dr. Rafael Ma. Moscoso (JBSD) (acronym from Thiers 2019), while "ANGE" and ''MG'' refer to the personal herbarium of Claudio Angelini and Matteo Gelardi, respectively. Herbarium numbers are cited for all collections from which morphological features were examined. Author citations follow the Index Fungorum, Authors of Fungal Names (www.indexfungorum.org/authorsoffungalnames.htm).

Morphological studies
Macroscopic descriptions and ecological information, such as habitat notations, time of fruiting and associated plant communities accompanied the detailed field notes of the fresh basidiomata. Color terms in capital letters (e.g. Myrtle Green, pl. VIII) are from Ridgway (1912). Photographs of collections were taken in the natural habitat using a Nikon Coolpix 8400 digital camera. Microscopic anatomical features were observed and recorded from revived dried material; sections were rehydrated either in water, 5% potassium hydroxide (KOH) or in anionic solution saturated with Congo red. All anatomical structures were observed and 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.W2)/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. 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.

Sequence alignment, data set assembly and phylogenetic analyses
The sequences obtained in this study were checked and assembled using Geneious v. R 11.1.4 (Kearse et al. 2012) and compared to those available in GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) by using the BLASTN algorithm (Altschul et al. 1990). A general combined Maximum likelihood tree including all the Boletaceae sequences present in GenBank and UNITE (http://unite.ut.ee/) databases was generated to detect the phylogenetic position of our collections in the major clades of Boletaceae as circumscribed by Wu et al. (2014) (tree not shown). Consequently, phylogenetic analyses were restricted to the major clade including Neoboletus sequences (Pulveroboletus group, Fig. 1).
Our datasets consist of sequences of Neoboletus and other sequences with greatest similarity available in GenBank selected based on BLASTN search and previous molecular studies including Neoboletus collections (Wu et al. , 2016aSmith et al. 2015;Urban and Klofac 2015).
Sequences were aligned with MAFFT v. 7.017 (Katoh et al. 2002) and then manually adjusted using Geneious v. R 11.1.4 (Kearse et al. 2012). Two phylogenetic analyses were performed: the first phylogenetic analysis, based on a combined nrLSU/ rpb1/rpb2 dataset, was focused on the intergeneric position of the new species in the Pulveroboletus group of the Boletaceae, as delimited by Wu et al. (2014). According to the results by Wu et al. (2014), Zangia erythrocephala was chosen as outgroup taxon for the three-loci combined dataset. The second phylogenetic analysis based only on a nrITS sequence dataset was restricted to the taxa closely related to the new species (genus Neoboletus). Costatisporus cyanescens was used as outgroup taxon for this dataset following Smith et al. (2015).
The GTRGAMMA model of sequence evolution was selected for both analyses. The two phylogenetic analyses were inferred with three partitions: nrLSU(28S)/rpb1/ rpb2 and ITS1/5.8S/ITS2, respectively. The datasets were analyzed using Bayesian inference (BI) and Maximum likelihood (ML) criteria. The BI was performed with Mr-Bayes v.3.2 (Ronquist et al. 2012) with four incrementally heated simultaneous Monte Carlo Markov Chains (MCMC) run for 10 million generations, under the selected evolutionary model. Trees were sampled every 1000 generations, resulting in overall  sampling of 10001 trees; the first 2500 trees were discarded as "burn-in" (25 %). For the remaining trees, a majority rule consensus tree showing all compatible partitions was computed to obtain estimates for Bayesian Posterior Probabilities (BPP). The ML was performed with RAxML v.7.2.8. (Stamatakis 2006) and a total of 1000 bootstrap replicates (Felsenstein 1985) were computed to assess the relative robustness of the

Molecular analysis
The combined nrLSU/rpb1/rpb2 data matrix ( Etymology. the specific epithet antillanus (Latin) refers to the occurrence of the species in the Antilles islands of the Caribbean. Original diagnosis. Basidiomes stipitate-pileate with tubular hymenophore characterized by medium-small size, pinkish red to reddish pileus surface, orange-red pores, reddish orange to purple-red punctuations on a yellow stipe surface, golden yellow strigosity at the stipe base, yellow context, tissues bruising dark blue when injured or exposed, ellipsoid-fusiform, smooth basidiospores, ixocutis pileipellis consisting of gelatinized, repent filamentous hyphae and occurrence in neotropical lowland mixed broadleaved forests in putative ECM association with host species (Coccoloba uvifera) other than Fagaceae and Pinaceae.
Basidiomes medium-small (Fig. 3). Ontogenetic development gymnocarpic. Pileus (3.0) 3.5-7.5 (8.0) cm broad, at first hemispherical then persistenly convex and finally broadly pulvinate-flattened, sometimes slightly depressed at centre, regularly to hardly unevenly shaped, moderately fleshy, firm at the beginning but progressively softer with age, flabby in old basidiomes; margin steady to faintly wavy-lobed, initially involute then curved downwards, extending beyond the tubes up to 1 mm; surface matt, dry but slightly greasy with moist weather, very finely tomentose, not cracked; cuticle somewhat variable in color, ranging from wine red, dark red or reddish pink to pastel pink (Pomegranate Purple, pl. XII; Spinel Red, pl. XXVI; Pinkish Vinaceous, pl. XXVII; Carmine, Eosine Pink, Geranium Pink, Rose Doree, pl. I; Alizarine Pink, Jasper Pink, Old Rose, pl. XIII), gradually fading with age and becoming pinkish cream to pale ochraceous pink (Flesh Pink, pl. XIII; Pale Ochraceous-Salmon, Pale Ochraceous-Buff, Light Buff, Light Ochraceous-Buff, Warm Buff, pl. XV) with olive-brown to brownish shades (Dresden Brown, pl. XV; Olive Lake, pl. XVI; Light Yellowish Olive, Buffy Olive, pl. XXX) tending to progressively spread from the center towards the peripheral zone; slowly bluing (Methy Green, Sea Green, Prussian Green, pl. XIX; Motmot Blue, Capri Blue, pl. XX) on handling or when injured; subcuticular layer cream-yellowish (Citrine Yellow, pl. XVI). Tubes at first thin then increasingly broader and as long as or slightly longer than the thickness of the pileus context at maturity (up to 1.0 cm long), adnate but soon deeply depressed around the stipe apex, occasionally subdecurrent, bright yellow (Lemon Chrome, pl. IV) to olive-yellow (Yellowish Citrine, pl. XVI), turning blue (Prussian Green, Duck Green, Invisible Green, pl. XIX) when cut and eventually fading to drab yellowish (Aniline Yellow, Pyrite Yellow, pl. IV). Pores initially forming a concave then flat surface, at first small then gradually wider (up to 1 mm in diam.), simple, roundish to barely angular at maturity, at first bright orangered to orange (Scarlet Red, Scarlet, pl. I) although concolorous with the tubes (Lemon Chrome, pl. IV) towards the margin, soon becoming yellowish orange (Flame Scarlet, Orange Chrome, pl. II) and finally yellowish olive (Yellowish Citrine, pl. XVI) with very pale orange hues (Orange, pl. III), quickly and intensely turning blue (Prussian Green, Duck Green, Invisible Green, pl. XIX) on bruising or when injured. Stipe (3.5) 4.0-9.0 (9.5) × (1.0) 1.5-2.0 (2.5) cm, longer than or as long as the pileus diameter at maturity, central to slightly off-center, solid, firm, dry, straight or curved, at first ventricose-fusiform, later cylindrical but either sligthly swollen towards the base to decidedly clavate or tapering downwards, not to barely rooting, evelate; surface at the apex or in the upper third usually smooth to occasionally very faintly reticulate due to the sub-decurrence of the hymenophore in some specimens and bright yellow (Lemon Chrome, pl. IV) to lemon yellow (Strontian Yellow, pl. XVI), elsewhere showing a fine, purple-red, dark red to orange-red (Indian Lake, pl. XXVI; Amaranth Purple, pl. XII; Carmine, Scarlet Red, pl. I) punctuation (Fig. 2d) partly hiding the bright yellow (Lemon Chrome, pl. IV) ground color; the base is typically wrapped by a conspicuous golden yellow to brownish yellow strigosity (Fig. 2d) (Raw Sienna, pl. III; Yellow Ocher, pl. XV); bruising greenish blue (Light Blue Green, Blue Green, Forest Green, pl. XVII) throughout when pressed; basal mycelium golden yellow (Raw Sienna, pl. III; Yellow Ocher, pl. XV). Context firm when young, later soft textured and eventually flabby in the pileus (up to 1.0 cm thick in the central zone), a little more fibrous in the stipe, lemon yellow (Strontian Yellow, pl. XVI) throughout, usually with purplebrown (Indian Lake, pl. XXVI; Amaranth Purple, pl. XII) spots in the stipe, especially at the extreme base; turning blue (Methy Green, Sea Green, Prussian Green, pl. XIX; Motmot Blue, Capri Blue, pl. XX) more or less evenly when exposed to air and finally fading to drab yellowish (Aniline Yellow, Pyrite Yellow, pl. IV); subhymenophoral layer lemon yellow (Strontian Yellow, pl. XVI). Odour and taste not distinctive. Spore-print not obtained but likely olive-brown.
Ecology. solitary to gregarious, growing on limestone among litter in a seasonally dry and moist anthropised lowland mixed stand under a large array of neotropical broadleaved trees, including Coccoloba uvifera, which represents its probable ECM host tree. See Parra et al. (2018) for further details on vegetation.
Known distribution. Presently only known from the type locality in the Dominican Republic (Greater Antilles, Caribbean).

Neoboletus antillanus phylogeny and interspecific relationships
Phylogenetic analyses corroborate the proposal of the new species N. antillanus (Figs 1, 2). It forms an independent evolutive line within Neoboletus with no evident phylogenetic relationships (it is sister to the Chinese N. magnificus in the combined analysis, but without statistical support) to allied congeneric taxa. According to the same analyses, B. brunneopanoides, a Belizean red-pored bolete species phylogenetically nested in Neoboletus, clustered in the same clade with collections named B. vermiculosus/B. vermiculosoides from North America. Should future molecular work prove conspecificity among these three species, B. vermiculosus Peck would have priority.

Taxonomic circumscription of N. antillanus
The genus Neoboletus currently encompasses fewer than ten species geographically restricted to the northern hemisphere and essentially distributed in temperate and tropical regions. However, judging from morphological traits, there might be an additional number of species, up to three times as many in fact, belonging to the same genus, most of which have not yet been molecularly investigated. It is worth noting that a group of Chinese researchers after having firstly accepted Neoboletus as an independent genus (Wu et al. 2016a), have subsequently reduced it in synonymy with Sutorius Halling, Nuhn & Fechner based on a wider interpretation of the generic boundaries within the Boletaceae (Wu et al. 2016b). As previously pointed out by Gelardi (2018), we presently disagree with this broad circumscription of Sutorius since it is, judging from the original description, easily separated from Neoboletus based on the overall dark colors, different stipe ornamentation pattern, different spore print color, pores stuffed in early developmental stages like those of Boletus s. str. and Butyriboletus Arora & J.L. Frank and non-bluing tissues (Halling et al. 2012b). Enough, in our opinion, to state they are not the same thing especially because they cluster in two different (although with a low statistical support) sister clades. Moreover, molecular studies carried out by Smith et al. (2015) on false-truffle fungi from north-eastern South America (Guyana) and our nrLSU/rpb1/rpb2 analy-sis ( Fig. 1) (Fig. 1).
Neoboletus antillanus is easily identifiable among other species of the same genus based on the following set of unique morphologically informative features: 1) mediumsmall size, 2) reddish to pinkish red then pinkish cream pileus surface, 3) pores orange red to yellowish orange, 4) stipe ornamented over the lower three fourth by purple-red to reddish orange punctuations on a yellow background, 5) lowermost part of the stipe prominently strigose with golden yellow to brownish yellow hairs, 6) yellow context, 7) tissues bruising dark blue when injured, 8) ellipsoid-fusiform, smooth basidiospores, 9) ixocutis pileipellis consisting of gelatinized, repent filamentous hyphae and 10) occurrence in neotropical lowland mixed broadleaved forests. To date, N. antillanus has never been found with host species other than local autoctonous broadleaved trees and does not appear to be associated with either Pinaceae or Fagaceae (the latter plant family is not present in Dominican Republic). Moreover, such a purported ECM association of N. antillanus with the endemic C. uvifera might implicate a neotropical origin. Further suggestion supporting a symbiotic relationship between N. antillanus and C. uvifera is the co-occurrence at the same locality with Cantharellus coccolobae Buyck, P.-A. Moreau and Courtec., which is strictly associated with seagrape in tropical America (Buyck et al. 2016).