Two new African siblings of Pulveroboletusravenelii (Boletaceae)

Abstract This paper sorts out the taxonomy of species affiliated with Pulveroboletusravenelii in the Guineo-soudanian and Zambezian woodlands of Africa. Morphological and genetic characters of African Pulveroboletus collections were studied and compared to those of North American and Asian species. A phylogenetic analysis showed that the African specimens form a subclade, sister to the Asian and American taxa. Although clamp connections have previously never been reported from Pulveroboletus, all specimens of the African subclade show very small clamp connections. Two new African species, Pulveroboletusafricanussp. nov. and P.sokponianussp. nov., are described and illustrated. Comments concerning morphology and identification, as well as distribution and ecology, are given for both species.


Introduction
Boletes belonging to Pulveroboletus Murrill are morphologically characterised by boletoid basidiomata with a pulverulent arachnoid veil. As originally indicated by Murrill (1909), this veil or cleistoblema sensu Clémençon (2012), is most often yellowish to vivid yellow and already present at a very early stage of development. Alterations broadening the circumscription of Pulveroboletus (Singer 1945(Singer , 1947(Singer , 1986, are not followed here as they have rendered the genus morphologically heterogeneous (Watling 2008) and polyphyletic (Binder and Hibbett 2006, Wu et al. 2014, Zeng et al. 2017. In its strict sense, Pulveroboletus holds few species, all of which are similar to the type, Pulveroboletus ravenelii (Berk. & M.A. Curtis) Murrill. Based on molecular data, Raspé et al. (2016) stated that what is called Pulveroboletus ravenelii outside North America belongs to a complex of different taxa. The name P. ravenelii has been used erroneously for lookalikes in Asia, Australia (Watling 2001, Horak 2011 and also Africa (Thoen and Ducousso 1989, Watling and Turnbull 1992, De Kesel et al. 2002, Yorou and De Kesel 2011, Vanié-Léabo et al. 2017, Kamou et al. 2017. So far, Raspé et al. (2016) and Zeng et al. (2017) have resolved part of the Asian complex around P. ravenelii, which now counts ten species. In a similar way, this paper aims to resolve and clarify the identity of some of the African, non-viscid, Pulveroboletus that have been kept under "Pulveroboletus aff. ravenelii".

Sampling, microscopy and morphology
Specimens were obtained from our own fieldwork or from herbarium specimens at our disposal. Protocols for field collecting, macroscopic description, drying and preservation follow Eyi Ndong et al. (2011). Codes and names of colours are according to the Methuen Handbook of Colour (Kornerup and Wanscher 1978). Microscopic structures were revived and examined in 5% potassium hydroxide (KOH) or in 10% ammonia with Congo Red. Measurements and drawings of microscopic structures were undertaken using an Olympus (BX51) compound microscope equipped with digital camera and drawing tube. Dimensions of microscopic structures are presented in the following format: (a-)b-c-d(-e), in which c represents the average, b = c − 1.96 * SD and d = c + 1.96 * SD and a and e are extreme values. Q is the length/width ratio based on at least 50 spores and is presented in the same format as spore dimensions (Eyi Ndong et al. 2011). Unless otherwise stated, herbarium specimens are deposited in BR. Duplicates from material from Togo are deposited in TOGO (Université de Lomé, Togo). Herbarium specimens from S. Badou (Benin) are deposited in UNIPAR (University of Parakou, Benin Republic). Abbreviations of herbaria follow Thiers (continuously updated). MycoBank (CBS-KNAW Fungal Biodiversity Centre, continuously updated) numbers are provided for the new species.

Alignment and phylogeny inference
Sequences of Pulveroboletus species, including the type species P. ravenelii, along with sequences of various genera of the Pulveroboletus group (Wu et al. 2014) and three Leccinoideae species as outgroup were generated or retrieved from GenBank (Table 1). The sequences were assembled in GENEIOUS Pro v. 6.0.6 (Biomatters). All sequences were aligned using MAFFT (Katoh and Standley 2013) on the server accessed at http://mafft.cbrc.jp/alignment/server/ and introns in rpb2 and tef1 were identified based on the amino acid sequence of previously published DNA sequences. Maximum Likelihood (ML) phylogenetic tree inference was performed using RAxML (Stamatakis 2006) on the CIPRES web server (RAxML-HPC2 on XSEDE; Miller et al. 2009). The phylogenetic tree was inferred by a single analysis with four partitions (one for the exons of each gene and a fourth for the introns of rpb2 and tef1), using the GTRCAT model with 25 categories. Statistical support of nodes was obtained with 1,000 rapid bootstrap replicates.

DNA analyses
The alignment contained sequences from 50 specimens and was 2,649 characters long (TreeBase number 23416). In the phylogram obtained ( Fig. 1), Pulveroboletus formed a highly supported clade (BS = 100%). Interestingly, the African species formed a highly supported sub-clade sister to the Asian and American species, which together formed another highly supported sub-clade.  Etymology. Epithet refers to its very wide distribution throughout tropical Africa. Description. Basidiomata medium-sized, covered by a general veil when young. Pileus 60-70 mm diam., at first broadly convex then pulvinate to plano-convex, upper layer dark brown (6E6-6F6), dry, mat, tomentose to felty, very soon cracking, becoming tomentose-scaly, bright yellow (2A4-5) in the deeper layers, predominantly yellow with age; scales appressed, slightly fibrillose, leather brown to greyish-brown (6E4-5), thicker and darker in the centre, thinner, paler and more diffused towards the margin; margin at first incurved, appendiculate with age, vivid yellow, beset with sulphur-yellow pulverulent material. Hymenophore tubulate, separable, straight to slightly sinuate, almost free around the stipe or depressed and then only slightly decurrent with a tooth; tubes up to 10 mm long, yellow to greyish-yellow (1B3), cyanescent when cut; pores regular, mostly round or slightly angular, slightly elongated around the stipe, small (14-16/mm), yellow (1A2-2A2), with age greyish-brown (5E4-6), cyanescent. Stipe cylindrical, 60-80 × 8-12 mm, central, solid, uppermost part vivid yellow (2A4-5), often with some reddish fibrils and smooth, lower part sheathed from the base up with a mat, dry, fibrillose-cottony, greyish to brownish-grey (5-6EF3-4) layer, the latter cracking transversally, forming brownish-grey to olive brown patches (4DE4-6), first exposing a greyish-white layer, then the bright yellow deeper layers; ring at first prominent, loose membranous-cottony, vivid yellow (2AB3-5), covering the pores, later tearing, leaving fibrillose to membranous material on both pileus margin and upper third of the stipe, pulverulent, becoming greenish from spores.
Macrochemical reactions: tubes brown to reddish-brown with KOH and NH 4 OH (in collections Rammeloo 5922 and De Kesel 2163).

Species delimitation
The African collections represent two separate species, Pulveroboletus africanus sp. nov. and P. sokponianus sp. nov., both macroscopically similar to Pulveroboletus ravenelii. In the latter, however, the disc becomes reddish-orange to reddish-brown at maturity and it grows in temperate conifer woods (Bessette et al. 2016), montane Quercus forests in Costa Rica (Halling and Mueller 2005) and Colombia (Franco-Molano et al. 2000) and Pine-oak forests in the Dominican Republic/Belize (Ortiz-Santana et al. 2007). The phylogenetic analysis showed that the African specimens form a well-supported subclade within Pulveroboletus, sister to the Asian and American taxa. Although clamp connections have previously been reported to be absent in Pulveroboletus (Watling 2008, Zeng et al. 2017, all specimens of the African subclade show very small clamp connections. Macroscopically, both African taxa can be distinguished based on the colour of the scales on the pileus and the stipe, being brown in P. africanus and greenish-grey or yellow in P. sokponianus. In P. africanus, the basal mycelium and context in the base of the stipe is generally yellow whereas it is whitish to whitish-yellow in P. sokponianus. While bluing of the context depends on the freshness and the maturity of the basidiomes, it seems more pronounced in P. africanus. Although cystidia have been reported to be rather constant and of little use to separate Asian taxa (Zeng et al. 2017), this seems to be true for the spores of the African taxa, but not for cystidia. In P. africanus, the cheilocystidia are hyaline and narrowly fusiform, whereas they are broadly fusiform and yellow pigmented in P. sokponianus. Further striking characters of distinction is the brownish intracellular pigmentation in the hyphae of the pileal and stipital scales, present in P. africanus but absent in P. sokponianus.
Young basidiomes of Pulveroboletus sokponianus are strongly reminiscent of the Asian P. brunneopunctatus G. Wu & Zhu L. Yang, but the latter has a viscid veil and smaller cheilocystidia. Using the key of the Chinese species (Zeng et al. 2017), Pulveroboletus africanus approaches closest to P. brunneoscabrosus Har. Takah. The latter has a viscid veil, reddish-brown scales and white to yellowish-white basal mycelium.

Distribution and ecology
Both new species are endemic to tropical Africa. Pulveroboletus africanus was found in Benin, Burundi, Guinea, DR Congo, Malawi, Mozambique, Togo, Zimbabwe and possibly also Zambia. It prefers regions with a pronounced wet/dry season alternance and occurs in a wide variety of woodlands, savannah woodlands and gallery forests across tropical Africa. It seems absent in the dense rainforests (Congolian region). The species is terricolous and most probably ectomycorrhizal (EcM), occurring in EcM dominated forests up to 1500 m elevation. It is difficult to ascertain if the species associates with Caesalpiniaceae (Berlinia, Brachystegia, Isoberlinia, Julbernardia) and/or with Uapaca (Phyllantaceae). Only Uapaca is well represented throughout its distribution range. In Eastern Africa (Zambezian region), it is also found under Brachystegia spp. and Julbernardia spp.; in West Africa (Soudano-Guinean region) under Berlinia grandiflora and Isoberlinia spp., Thoen and Ducousso (1989) mention it under Uapaca chevalieri Beille. The species may also occur in Zambia (ut Pulveroboletus aff. ravenelii, fig. 1H in Watling and Turnbull 1992).
Pulveroboletus sokponianus has so far only been found in a variety of savannah woodlands and gallery forests in the Soudano-Guinean transition zones of Benin and Togo, probably also in Ivory Coast (see fig. 3a in Léabo et al. 2017). The species is terricolous, most probably ectomycorrhizal (EcM) and most often found under Isoberlinia doka (Caesalpiniaceae). Since habitat destruction and felling of host trees is common practice in Benin, Yorou and De Kesel (2011) placed this species (mentioned as P. ravenelii) under the IUCN threat category 'vulnerable'.