Paxilloboletus gen. nov., a new lamellate bolete genus from tropical Africa

This study presents Paxilloboletus gen. nov., a new lamellate bolete genus represented by two tropical African species, Paxilloboletus africanus sp. nov. and Paxilloboletus latisporus sp. nov. Although the new taxa strongly resemble Paxillus (Paxillaceae), they lack clamp connections and form a separate generic clade within the Boletaceae phylogeny. The new species are lookalikes, morphologically only separable by their spore morphology. Descriptions and illustrations of the new genus and new species are given, as well as comments on ecology, distribution, and morphological differences with other gilled Boletaceae.


Introduction
Over the years, our survey of bolete diversity in tropical Africa (here restricted to suborder Boletineae) has resulted in a collection of specimens both with poroid and with lamellate hymenophores. Among the stipitate-pileate Boletineae, poroid forms are the most common, but there are several well-separated lamellate clades dispersed among the clades with poroid hymenophores (Farid et al. 2018;Zhang and Li 2018). The genera Paxillus Fr. (Paxillaceae) and Phylloporus Quél. (Boletaceae) are the most well recognized and globally distributed Boletineae with lamellate hymenophores.
Over the years several Paxillus and Phylloporus species have been recombined into new genera (e.g., Gilbert 1931;Singer 1942;Bresinsky et al. 1999;Farid et al. 2018;Vadthanarat et al. 2019), some outside the Boletineae, and a few new species have also been described in new lamellate genera (Singer and Digilio 1952;Zhang and Li 2018), resulting in a total of six lamellate lineages in the Boletineae: Paxillus, Phylloporus, Phyllobolites Singer (Boletaceae), Phylloboletellus Singer (Boletaceae), Phylloporopsis Angelini, Farid, Gelardi, ME Sm., Costanzo & Vizzini (Boletaceae), and Erythrophylloporus Ming Zhang & TH Li (Boletaceae). However, hitherto only Paxillus and Phylloporus are known from tropical Africa, with 6 of the 38 known Paxillus species and 15 of the 86 known Phylloporus species occurring in the region (Kirk 2021;Rammeloo 1986, 1987a). Based on a combination of phylogenetic and morphological characters, we here introduce the seventh lamellate genus in the Boletineae, Paxilloboletus gen. nov. (Boletaceae), so far only known from tropical Africa. Tanzania, Togo and Zambia), during the rainy season. Specimens were photographed in situ and field notes were taken of their macroscopic characters. Sectioned specimens were dried for 24-48 h in a field drier at 40-60 °C and immediately stored afterwards in airtight bags (type Minigrip), with or without silica gel. All studied material is deposited in the herbaria of the University of Parakou (UNIPAR), the University of Uppsala (UPS), and Meise Botanic Garden (BR) (abbreviations in Index Herbariorum; Thiers, continuously updated). Macroscopic descriptions are based on available field notes, photographs, and in some cases on observations made on the exsiccata (Badou et al. 2018). Color codes and names have been taken from Methuen Handbook of Color (Kornerup and Wanscher 1978). Macrochemical reactions (bluing) of the context and hymenophore of exsiccata were determined using Melzer's reagent. Microscopic structures were examined in 10% ammonia, with or without Congo red. Observations, measurements, and drawings of microscopic structures were carried out using an Olympus BX51 or a Leica DM 2700 M light microscope equipped with a digital camera and drawing tube.
Dimensions of microscopic structures are given as a range. Basidiospore dimensions are presented in the following format: (a-) b-c-d(-e), where c is the average, b = c -1.96 * SD (5 th percentile) and d = c + 1.96 * SD (95 th percentile), a the extreme minimum and e the extreme maximum value. Q is the length/width ratio based on at least 50 spores and is presented in the same format as the spore length and width. Pellis structures were studied from a fine surface scalp, taken either halfway between the center and the margin of the cap (pileipellis), and/or halfway up the stipe (stipitipellis).
For scanning electron microscopy (SEM), a portion of hymenophore was dried in a critical point dryer (Leica EP CDP 300), mounted on a SEM stub and coated with a layer of approximately 6 nm Pl/Pd using a High-Resolution Fine Sputter Coater for FE-SEM (JFC-2300HR Coating Unit, JEOL). Scanning electron microscopy was carried out with a JEOL JSM-7100FLV Field Emission SEM (Meise Botanic Garden).

DNA extraction, amplification, and sequencing
Genomic Deoxyribonucleic Acid (DNA) was extracted from dried specimens using the DNeasy Plant Kit (Quiagen). A total of five regions were sequenced, including the nuclear ribosomal internal transcribed spacer (ITS), partial nuclear ribosomal large subunit (LSU), and fragments of three protein-coding genes: the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2, respectively) and transcription elongation factor 1-α (TEF1-α). ITS sequencing was attempted for all studied specimens, and RPB2 sequencing was subsequently attempted for the majority, with special focus on specimens from the Democratic Republic of the Congo, where higher variability in ITS was observed. The remaining three genes were sequenced for four specimens with non-identical ITS sequences. The chosen regions were amplified using primer pairs ITS1 (White et al. 1990) and LB-w (Tedersoo et al. 2008) for ITS; LR0R and LR5 (Hopple and Vilgalys 1994) for LSU; RPB1-B-F and RPB1-B-R (Wu et al. 2014) for RPB1; fRPB2-5F (Liu et al. 1999) and bRPB2-7R (Matheny 2005) for RPB2; and EF1-983F and EF1-2218R (Rehner and Buckley 2005) for TEF1-α. Thermocycling protocols were according to Wu et al. (2014). After amplification, PCR products were cleaned with ExoSAP-IT. Sequencing was performed by Macrogen Europe (Amsterdam, the Netherlands) using the same primers as for amplification, except that ITS4 was used as a reverse sequencing primer for ITS, and internal primers bRPB2-6F (Matheny 2005) and bRPB2-6R2 (Matheny et al. 2007) were added for RPB2. Reads were trimmed and assembled using Prosed (version 3.2;Filatov 2002) or Staden (Staden et al. 2000). Sites where chromatograms show clear double peaks indicative of heterozygosity or other intragenomic variation were hand-annotated with IUPAC ambiguity codes.

Alignment and phylogenetic inference
The four-gene (LSU, RPB1, RPB2 and TEF1-α) data set of Gelardi et al. (2015) was used as a base alignment to place the specimens phylogenetically. Publicly available sequences of the lamellate genera Phylloporopsis (two specimens; Farid et al. 2018), Phylloboletellus (one specimen; Binder and Hibbett 2006) and Erythrophylloporus (six specimens; Zhang and Li 2018;Vadthanarat et al. 2019;Haelewaters et al. 2020) were added to the matrix, along with the sequences of our specimens. No sequence data is available for Phyllobolites. After preliminary results indicated the placement of our specimens in subfamily Boletoideae, we also added publicly available sequences from the additional genera Afrocastellanoa, Durianella, Guyanaporus, Heliogaster, Indoporus, Mackintoshia and Nigroboletus (one specimen each), all previously placed in subfamily Boletoideae (Desjardin et al. 2008;Gelardi et al. 2015;Henkel et al. 2016;Orihara et al. 2010;Orihara and Smith 2017;Parihar et al. 2018;Smith et al. 2015), which were not present in the base alignment. Accession numbers for these added sequences are in Table S1. Taxonomic identity of specimens was updated with reference to the current annotation of the sequences on GenBank, synonymy from Species Fungorum (Kirk 2021), as well as recent literature (Chai et al. 2019;Farid et al. 2018;Gelardi et al. 2015;Gelardi 2020;Henkel et al. 2016;Orihara et al. 2016;Orihara and Smith 2017;Parihar et al. 2018;Smith et al. 2015;Zhu et al. 2015;Wu et al. 2016aWu et al. , 2016bWu et al. , 2019Zhang et al. 2019;Vadthanarat et al. 2019).
For each of the four genes (LSU, RPB1, RPB2 and TEF1-α), new sequences were aligned separately using MUSCLE in Geneious (version 10.2.3; Drummond et al. 2010) with default settings, and trimmed to match the base alignment. The new sequences were then sequentially added to the base alignment using MAFFT with the -add option (version v7.453;Katoh and Frith 2012;Katoh and Standley 2013). These separate alignments of each gene were concatenated, and a maximum likelihood phylogenetic tree was built using the online version of RAxML XSEDE2 (version 8;Stamatakis 2006) in CIPRES portal (Miller et al. 2010) with rapid bootstrapping according to the MRE_IGN stopping criterion and the GTRGAMMA substitution model.
To more closely examine relationships among our specimens, we assembled a two-gene ITS and RPB2 dataset including our specimens. We also performed a BLAST search of publically available ITS sequences from GenBank and Unite, and added hits with > 90% sequence identity to any of our specimens across the full ITS region. In contrast to the four-gene dataset, where only approximately 570 bp of RPB2 between conserved domains 6 and 7 were included, the two-gene dataset included the full length of our RPB2 sequences, approximately 1, 100 bp from conserved domain 5-7. As outgroups, we included publically available ITS and RPB2 sequences from three additional members of Boletoideae for which corresponding long RPB2 sequences were available: Boletus edulis, Porphyrellus porphyrosporus, and Strobilomyces strobilaceus (Table S2). LSU sequences were also used for the outgroups to extend the ITS sequences to the LB-w binding site to match the sequences generated in this study. The two genes were aligned separately with MAFFT in generalized affine (e-ins-i) mode for ITS, and global (g-ins-i) mode for RPB2. The two alignments were trimmed and then concatenated, and a maximum likelihood tree was generated as above. Trees were visualized using the ggtree package (version 2.4.1; Yu et al. 2017) in R (version 4.0.3; R Core Team 2020).

DNA analyses
We obtained ITS sequences for eleven specimens from four countries in West and Central Africa (Table 1). RPB2 sequences were obtained for a subset of eight specimens, and LSU, RPB1, and TEF1-α were obtained for a further subset of four specimens (Table 1). A BLAST search of GenBank revealed one additional ITS + LSU sequence (accession number FR731194; Tedersoo et al. 2011), from an ectomycorrhizal root tip in Madagascar, which was a 100% match to ADK-5006 from the Democratic Republic of the Congo. The ITS sequences fell into two clusters, each with greater than 99.5% sequence similarity, and with ~ 97% similarity between the two clusters. Variable sites within each cluster (max 2) were ambiguous in some of the specimens, indicating genetic mixing within clusters. RPB2 sequences had > 99.9% similarity within the same two clusters, and ~ 99% similarity between clusters. We consider this consistent clustering of the specimens using two unlinked loci to be evidence that the clusters represent two closely related but distinct species.
The topology of our four-gene ML-tree ( Fig. 1; version with all tips expanded in Fig. S1) is in general agreement with previous results. Although our phylogeny places Phylloboletellus within Cyanoboletus Gelardi, Vizzini and Simonini with 94% bootstrap support, this result is based only on LSU, and the very short branch lengths at the root of Cyanoboletus suggest that this placement may not be reliable.   Our four specimens SAB-0715, SAB-0716, ADK-5006, and ADK-5720 clustered together with 100% bootstrap support in the four-gene phylogeny. They were placed within subfamily Boletoideae, clearly separated from the other lamellate genera. Within Boletoideae, the new specimens were placed as sister to Boletus L., sensu stricto (but including "Alloboletus", "Inferiboletus", and "Obtextiporus" sensu Dentinger et al. 2010), also with 100% bootstrap support. The two-gene phylogeny (Fig. 2) separates the new specimens, along with the environmental sequence, into the same two clusters identified on the basis of sequence similarity, with 100% and 99% bootstrap support, supporting the hypothesis that these clusters represent phylogenetically distinct species. Because these specimens are not phylogenetically nested within any known genus, and their lamellate hymenium clearly distinguishes them from all closely related species, we describe them as a new genus with two species. Description Basidiomata epigeal, putrescent, pileate, stipitate, evelate, with lamellate hymenophore, medium to small sized; pileus convex to slightly depressed, tomentose, usually with persistently incurved margin; hymenophore easily separated from context of pileus, strongly decurrent, with lamellae regularly bifurcating and anastomosing, yellow, becoming yellowish brown; stipe solid, dry, tomentose, with or without ridges or reticulation in its uppermost part; basal mycelium whitish; context whitish to yellowish white throughout, unchangeable when exposed, strongly amyloid in the lamellae; taste fungal, insignificant; odor weak, insignificant; spore print yellowish brown, without olivaceous tint; basidiospores ellipsoid-fusiform, smooth under SEM; caulo-, cheilo-and pleurocystidia of similar shape, the latter much more abundant than cheilo-and caulocystidia; pileipellis a tomentum; hymenophoral trama divergent near the pileal context, subregular to regular toward the lamella edge, gelatinized; clamp connections absent. Etymology: refers to its distribution throughout tropical Africa.

Taxonomy
Habitat and distribution P. africanus has this far been collected in West Africa, specifically Benin (gallery forest with Berlinia grandiflora and Uapaca guineensis), Togo (miombo forest with Uapaca togoensis and Monotes kerstingii) and Guinea (woodland with Isoberlinia spp., Uapaca togoensis and Anthonotha crassifolia); in Central Africa, specifically the Democratic Republic of the Congo (miombo dominated by Julbernardia globiflora) and Zambia (soil and relic miombo woodland); and in East Africa in Tanzania (primary miombo forest). It is also known in Madagascar from sequence data (EcM root tip of Uapaca bojeri in littoral forest).
Context 3-6 mm thick in the center of the pileus, gradually thinner toward the margin, pure white throughout, Bootstrap support values > 50 are shown near nodes. Names of lamellate taxa are red, and new sequences for this study are bold. Monophyletic taxa represented by multiple specimens, and not containing mixed lamellate and non-lamellate taxa, are collapsed. Naming and coloring of subfamily-level clades within Boletaceae, as well as rooting, follow Wu et al. (2014) ◂ becoming dirty yellowish white in the stipe of older specimens, unchanging when exposed. Context strongly amyloid (Melzer's reagent), at least in lamella (especially the hymenium itself).
Lamellar trama divergent near pileus, with compact mediostratum and gelatinized lateral strata, subregular to regular toward the lamella edge, in the center composed of thin-walled, hyaline hyphae (diam. 4-5 µm) with locally roughened surface, beneath the subhymenium mixed with conspicuously inflated (diam. 8-16 µm) hyphae, constricted at the septa. Pileipellis a tomentum composed of intermixed, hyaline and pigmented hyphae, all smooth, thin-walled, with cylindrical, non-inflated end-cells of 5.2-9.8 µm diam; the pigmented pileal hyphae normally septate, filled with a yellowish to pale brownish thrombomorphic deuteroplasm, unchanged in KOH, becoming only slightly browner in Melzer's reagent. Stipitipellis in the upper part of the stipe with sparse hymenial elements, elsewhere composed of parallel hyphae supporting a collapsed tomentum, similar to the pileipellis. Clamp-connections absent in all tissues. Etymology: latisporus means "with wider spores" and refers to the fact that this taxon is morphologically separated from the typus generis by this character.
Habitat and distribution P. latisporus sp. nov. has only been collected from a single locality in Central Africa, specifically in the Democratic Republic of the Congo (Mikembo sanctuary, muhulu type of miombo with, Julbernardia globiflora, Brachystegia microphylla, Brachystegia spiciformis, Marquesia macroura and Uapaca kirkiana).
Context 5-8 mm, very thick in the center of the pileus, thinner toward the margin, pure white throughout, becoming dirty yellowish white in the stipe of older specimens, unchanging when exposed. Context of exsiccata moderately amyloid (Melzer's reagent), at least in the lamella, especially the hymenium.

Discussion
The basidiomata of Paxilloboletus resemble taxa in Paxillus Fr. (Paxillaceae). This is mainly due to the persistently incurved margin, tomentose cap, and separable, decurrent, lamellar hymenophore with yellow hymenium. However, the absence of clamp connections and the phylogenetic position outside Paxillaceae (Fig. 1)  Within the Boletaceae phylogeny, Paxilloboletus spp. take a position outside all known genera, and distant from other lamellate lineages. Neves et al. (2012) stated that the lamellate hymenophore configuration is a synapomorphy that distinguishes Phylloporus from the other genera in the family Boletaceae. The separate placement of lamellate genera of boletes within Boletaceae, including Paxilloboletus, clearly suggests homoplasy for the presence of a lamellate hymenophore. In particular, the phylogenetic position of Paxilloboletus as a lamellate sister of Boletus has the potential to set a clear boundary for both genera in a natural classification.
Within the Boletaceae, the basidiomata of Paxilloboletus spp. resemble the ones in Phylloporus, Sect. Immutabiles Heinem and Rammeloo (1987b) because of an unchanging context and smooth spores under SEM. Heinemann and Rammeloo (1987a) and later Watling and Turnbull (1993) studied a white capped Phylloporus from Zambia (collection FP335, deposited at NDO and K) and placed it in this section, under Phylloporus albocarnosus Heinem. Re-examination of the data available from FP335 shows that it is different from the type of Phylloporus albocarnosus (Goossens-Fontana 935, BR) by its very pale almost white pileus, tomentum-like pileipellis, and slightly wider spores with internal amyloid granulation (P. Heinemann notes). Re-examination of the pileipellis of the type of Phylloporus albocarnosus shows a pellis with clearly inflated to globular terminal elements (physalotrichoderm) and inamyloid lamella. Collection FP335 belongs to Paxilloboletus (P. africanus) and is not conspecific with P. albocarnosus. The latter is now only known from the type material.
From taxa in Phylloporus Quél. (Boletaceae) and Phylloporopsis Angelini, A. Farid, Gelardi, M.E. Sm., Costanzo and Vizzini (Boletaceae), the basidiomata of Paxilloboletus spp. differ by a tomentum-type of pileipellis without inflated end-cells and sparse, yellowish thromboplerous hyphae, an unchanging white to yellowish-white context, and lacking olivaceous tints in the spore print. Erythrophylloporus Ming Zhang and T.H. Li (Boletaceae) differs from Paxilloboletus by its yellowish-orange to red pileus, orange to red lamellae, vivid yellow context and ovoid to broadly ellipsoid spores (Vadthanarat et al. 2019). Paxilloboletus differs from the lamellate Phylloboletellus Singer (Boletaceae) by smooth, non-winged, ellipsoid to slightly fusiform basidiospores and from the ill-known velate genus Phyllobolites Singer (Boletaceae) by its nonverrucose spores and complete lack of velum (ring) on the stipe.
All collections of Paxilloboletus africanus and P. latisporus systematically show a moderate to strong amyloid reaction in the entire hymenophore, a characteristic that is missing in all other lamellate Boletaceae. On exsiccata, regardless of their age (3-21 years), this reaction is immediate and gains intensity for about 2 min. However, after 15 min, the dark blackish-blue stain starts to fade and disappears within the following hour. Fleetingamyloid reactions in the trama have been reported from many boletes (Bozok et al. 2019), but the reaction in both Paxilloboletus is much stronger and not localized in the trama of the gills. In fact, seen under the microscope, the amyloid reaction (on a perradial section of a gill) mainly takes place in the hymenium and not, or hardly, in the lamellar trama. This is possibly due to the gelatinization of the gill trama of Paxilloboletus. In only few boletes, such as Caloboletus calopus (Pers.) Vizzini and Suillellus luridus (Schaeff.) Murrill, strong amyloid reactions have been reported (Bozok et al. 2019). In boletes the taxonomic value of this characteristic is considered either unclear, controversial or important (Watling 1971). Notwithstanding this situation, and because all of our collections respond homogeneously, we here consider this feature diagnostic for Paxilloboletus.
Due to their identical macro morphology, basidiomes of Paxilloboletus africanus and P. latisporus are difficult to set apart in the field. Microscopically, the taxa can only be separated by measuring a large number of spores, at least 50-100 and preferably from a spore print. While the average spore length is not different between the two species, the average spore width ranges from 4.1 to 4.5 µm in P. africanus and from 4.7 to 5.0 µm in P. latisporus. Although less pronounced, the average Q value also differs, namely Q = 1.8 in P. latisporus and Q = 2 in P. africanus.
Paxilloboletus is most likely ectomycorrhizal, and an ITS sequence matching P. africanus has previously been isolated from ectomycorrhizal root tips of Uapaca bojeri (Phyllanthaceae) in Madagascar (L. Tedersoo, GenBank accession number FR731194). In the absence of data directly linking the new taxa to associate host trees in continental Africa, we can only work with field observations. In almost all collections, regardless of the species, the field data indicate the proximity of Uapaca spp. (Phyllanthaceae) and a set of trees of subfamily Detarioideae of the Fabaceae. In West Africa (Guinea, Togo, Benin), these Detarioideae belong to Isoberlinia and Berlinia, while in Eastern Africa (DR Congo, Zambia and Tanzania) they belong to Brachystegia, Julbernardia and Isoberlinia. Compared to P. africanus, P. latisporus has a more restricted distribution which is potentially tied to a specific host. In fact, it was only found in mature miombo forests of DR Congo (Lubumbashi, Mikembo sanctuary) with Uapaca, Brachystegia and Julbernardia, and also large specimens of the ectomycorrhizal tree Marquesia macroura (Dipterocarpaceae). In recent years, we observe more and more that ectomycorrhizal taxa are separated based on a combination of molecular and very subtle morphological characteristics (Delgat et al. 2019;De Kesel et al. 2016;Vadthanarat et al. 2021). Sibling species typically show few and feeble morphological differences. They are thought to be the result of recent divergence, likely involving an EcM host switch, in which sporocarp morphologies haven't had the time to diverge as well. Since P. africanus and P. latisporus resemble each other so strongly, we consider them pseudocryptic species. Without molecular data (Fig. 2), these taxa would not have been separated, simply because subtle differences in spore width are -traditionally -not considered strong enough characteristics to separate taxa.