Cacaoporus, a new Boletaceae genus, with two new species from Thailand

Abstract We introduce a new genus, Cacaoporus, characterised by chocolate brown to dark brown basidiomata and hymenophore, tubes not separable from the pileus context, white to off-white basal mycelium, reddening when bruised, amygdaliform to ovoid spores and dark brown spore deposit. Phylogenetic analyses of a four-gene dataset (atp6, tef1, rpb2 and cox3) with a wide selection of Boletaceae showed that the new genus is monophyletic and sister to the genera Cupreoboletus and Cyanoboletus in the Pulveroboletus group. Two new species in the genus, C.pallidicarneus and C.tenebrosus are described from northern Thailand. Full descriptions and illustrations of the new genus and species are presented. The phylogeny also confirmed the reciprocal monophyly of Neoboletus and Sutorius, which further support the separation of these two genera.


Introduction
In the last decade or so, since molecular techniques and phylogenetic analyses have been used in taxonomy and systematics of the Boletaceae, many new species and genera have been described worldwide (e.g. Halling et al. 2012Halling et al. , 2016Zeng et al. 2012;Arora and Frank 2014;Gelardi et al. 2014Gelardi et al. , 2015, Zhao et al. 2014bWu et al. 2015Wu et al. , 2016Zhu et al. 2015). In Thailand, although the Boletaceae have been studied for a long time, only a few new Boletaceae species and a new genus have recently been described (Desjardin et al. 2009;Neves et al. 2012;Halling et al. 2014;Raspé et al. 2016;Vadthanarat et al. 2018). At the same time, many new species and genera have been described from southern and south-western China, an area with a climate and forests similar to Thailand (e.g. Li et al. 2011;Wu et al. 2015Wu et al. , 2016Zhu et al. 2015). Similarly, a high number of new species and possibly new genera are expected to occur in Thailand (Hyde et al. 2018) During our survey on the diversity of boletes in Thailand, several collections of brown to chocolate to dark brown boletes were obtained. Some collections bearing resemblance to Sutorius Halling, Nuhn & N.A. Fechner species, which typically have brown or reddish to purplish-brown basidiomata with reddish to purplish-brown hymenophore, reddish-brown spore deposit and narrowly ellipsoid to ellipsoid basidiospores (Halling et al. 2012). However, our chocolate brown bolete collections also showed differences, in particular in having a darker hymenophore, as well as in some microscopic characters like spore shape. We therefore performed a family-wide phylogeny, which showed that those brown to chocolate to dark brown boletes belong in a generic lineage, different from Sutorius. Consequently, we introduce the new Boletaceae genus Cacaoporus and describe two new species, C. pallidicarneus and C. tenebrosus, with full descriptions and illustrations.

Specimens collecting
Fresh basidiomata were collected in Chiang Mai Province, northern Thailand during the rainy season in 2013 to 2018. The specimens were photographed in situ, wrapped in aluminium foil and taken to the laboratory. After description of macroscopic characters, the specimens were dried in an electric drier at 45-50 °C. Examined specimens were deposited in the herbaria CMUB, MFLU, BKF and BR (listed in Index Herbariorum; Thiers, continuously updated).

Morphological studies
Macroscopic descriptions were made, based on detailed field notes and photos of fresh basidiomata. Colour codes were taken from Kornerup and Wanscher (1978). Macrochemical reactions (colour reactions) of pileus, pileus context, stipe, stipe context and hy-menophore were determined using 10% aqueous potassium hydroxide (KOH) and 28-30% ammonium hydroxide (NH 4 OH). Microscopic structures were observed from dried specimens, using 5% KOH, NH 4 OH, Melzer's reagent or stained with 1% ammoniacal Congo red. A minimum of 50 basidiospores, 20 basidia and 20 cystidia were randomly measured at 1000× with a calibrated ocular micrometer using an Olympus CX51 compound microscope. The notation '[m/n/p]' represents the number of basidiospores "m" measured from "n" basidiomata of "p" collections. Dimensions of microscopic structures are presented in the following format: (a-)b-c-d(-e), in which "c" represents the average, "b" the 5 th percentile, "d" the 95 th percentile, "a" the minimum and "e" the maximum. Q, the length/width ratio, is presented in the same format. A section of the pileus surface was radially and perpendicularly cut to the surface at a point halfway between the centre and margin of the pileus. Sections of stipitipellis were taken from halfway up the stipe and longitudinally cut, perpendicularly to the surface (Hosen et al. 2013;Li et al. 2011). All microscopic features were drawn by free hand using an Olympus Camera Lucida model U−DA fitted to the microscope cited above. For scanning electron microscopy (SEM), a spore print was mounted on to an SEM stub with double-sided tape. The samples were coated with gold, then examined and photographed with a JEOL JSM-5910 LV SEM.

Alignment and phylogeny inference
The sequences were assembled in GENEIOUS Pro v. 6.0.6 (Biomatters) and introns were removed prior to alignment based on the amino acid sequence of previously published sequences. All sequences, including sequences from GenBank, were aligned using MAFFT (Katoh and Standley 2013) on the server accessed at http://mafft.cbrc. jp/alignment/server/.
Maximum Likelihood (ML) phylogenetic inference was performed using RAxML (Stamatakis 2006) on the CIPRES web portal (RAxML-HPC2 on XSEDE; Miller et al. 2009). The phylogenetic tree was inferred by a single analysis with three partitions (one for each gene), using the GTRCAT model with 25 categories, two Buchwaldoboletus and nine Chalciporus species from sub-family Chalciporoideae were used as outgroup since Chalciporoideae always appeared as sister to the remainder of the Boletaceae in recent phylogenetic analyses (e.g. Nuhn et al. 2013;Wu et al. 2014Wu et al. , 2016. Statistical support of clades was obtained with 1,000 rapid bootstrap replicates. For Bayesian Inference (BI), the best-fit model of substitution amongst those implementable in MrBayes was estimated separately for each gene using jModeltest (Darriba et al. 2012) on the CIPRES portal, based on the Bayesian Information Criterion (BIC). The selected models were HKY+I+G for atp6 and rpb2 and GTR+I+G for cox3 and tef1. Partitioned Bayesian analysis was performed with MrBayes 3.2 (Ronquist et al. 2012) on the CIPRES portal. Two runs of five chains were run for 15,000,000 generations and sampled every 500 generations. The chain temperature was decreased to 0.02 to improve convergence. At the end of the run, the average deviation of split frequencies was 0.008147.

Phylogenetic analysis
A total of 325 sequences were newly generated and deposited in GenBank (Table 1). The alignment contained 1,013 sequences from four genes (186 for atp6, 358 for tef1, 326 for rpb2, 143 for cox3) from 362 voucher specimens and was 2946 characters long (TreeBase number 23886).
Diagnosis. Similar to the genus Sutorius in having brown basidiomata with brown encrustations in the flesh but differs from Sutorius in having the following combination of characters: brown to chocolate brown or greyish-brown to dark brown or blackish-brown basidiomata, without violet tinge, chocolate brown to dark brown hymenophore, tubes not separable from the pileus context, white to off-white basal mycelium which turns reddish-white to pale red when bruised, amygdaliform to ovoid with subacute apex in side view to ovoid basidiospores and dark brown spore deposit.
Description. Basidiomata stipitate-pileate with poroid hymenophore, small to medium-sized, dull, brown to greyish-brown to dark brown or blackish-brown. Pileus convex when young becoming plano-convex to slightly depressed with age, with deflexed to inflexed margin; surface even to subrugulose, minutely tomentose or slightly cracked at the centre; context soft, yellowish to greyish off-white then slightly greyish-orange to dull orange to greyish-brown when exposed to the air, patchy or marmorated with greyish-brown to dark brown, sometimes with scattered small dark brown to brownish-black encrustations, not or inconsistently reddening when cut. Hymenophore tubulate, adnate, subventricose to ventricose, slightly depressed around the stipe; tubes brown to greyish-brown to dark brown, not separable from the pileus context; pores regularly arranged, mostly roundish at first becoming slightly angular with age, sometimes irregular, elongated around the stipe, dark brown to greyishbrown at first, becoming brown to chocolate brown with age. Stipe central, terete to sometimes slightly compressed, cylindrical to sometimes slightly wider at the base; surface even, minutely tomentose, dull, dark brown to greyish-brown, basal mycelium white to off-white becoming reddish-white to pale red when touched; context solid, yellowish to orange white to yellowish-grey to pale orange to dull orange to reddishgrey, marmorated or virgated with brownish-grey to greyish-brown to dark brown, sometimes scattered with small reddish-brown to brownish-black fine encrustations, unchanged or inconsistently reddening when cut. Spore print dark brown.
Basidiospores amygdaliform to ovoid or ovoid with subacute apex in side view, thin-walled, smooth, slightly reddish to brownish hyaline in water, slightly yellowish to greenish hyaline in KOH or NH 4 OH, inamyloid. Basidia 4-spored, clavate to nar-rowly clavate without basal clamp connection. Cheilocystidia fusiform or cylindrical with obtuse apex, sometimes bent or sinuate, thin-walled, often scattered with small brownish-yellow to yellowish-brown crystals on the walls in KOH or NH 4 OH. Pleurocystidia narrowly fusiform with obtuse apex or cylindrical to narrowly subclavate, sometimes bent or sinuate, thin-walled, densely covered with small reddish-brown to brownish dark encrustations on the walls when observed in H 2 O, which are discoloured then dissolved in KOH or NH 4 OH. Pileipellis a trichoderm becoming tangled trichoderm to tomentum, composed of thin-walled hyphae; terminal cells mostly slightly sinuate cylindrical to irregular with rounded apex or clavate to elongated clavate. Stipitipellis a trichoderm to tangled trichoderm or disrupted hymeniderm, composed of loosely to moderately interwoven cylindrical hyphae anastomosing at places. Clamp connections not seen in any tissue.
Typus generis. Cacaoporus tenebrosus Distribution. Currently known from Thailand. Notes. Sutorius most closely resembles the new genus. In the field, Cacaoporus is easily distinguished from the Sutorius by the following combination of characters: chocolate brown to dark brown to blackish-brown basidiomata, which are darker than in Sutorius and never purplish-brown like in Sutorius species; chocolate brown to dark brown hymenophore, which is much darker than in Sutorius and never reddish-to purplish-brown like in Sutorius; tubes that are not separable from the pileus context but can be separated in Sutorius; off-white basal mycelium that more or less turns red when bruised, which is never the case in Sutorius. Etymology. Refers to the context, which is paler than in the other species, especially at the stipe base and in the pileus.
Diagnosis. Cacaoporus pallidicarneus is characterised by having a paler context than the other species and basidiospores that are amygdaliform or elongated amygdaliform to ovoid in side view, sometimes with subacute apex, shorter basidia and fusiform to narrowly bent fusiform to narrowly fusiform hymenophoral cystidia.
Description. Basidiomata small to medium-sized. Pileus (1.6)2.4-5.5 cm in diameter, convex when young becoming plano-convex with age; margin deflexed to inflexed, slightly exceeding (1-2 mm), surface even to subrugulose, minutely tomentose, dull, at first brown to greyish-brown to blackish-brown (8F3-4) sometimes paler (8C2) at places, becoming paler to greyish-brown (8E3-5) with age; context 4-9 mm thick half-way to the margin, soft, yellowish to greyish off-white then slightly  pale orange to greyish-orange (6A3 to 6B3) when exposed to the air, with patchy or marmorated with greyish-brown (8E3) especially when young, scattered with reddish-brown to brownish-black of fine encrustations at places, slightly reddening when cut. Stipe central, terete or sometimes slightly compressed, cylindrical with slightly wider base, (2.0)2.8-3.7 × 0.4-0.7 cm, surface even, minutely tomentose, dull, greyish-brown to dark brown (8 E/F 3-4 to 8F2), basal mycelium white to offwhite becoming pale red (7A3) when bruised; context solid, yellowish to greyish off-white then orange white to pale orange (5A2-3) when exposed to the air, virgate to marmorate with brownish-grey (8F2), less so at the stipe base, at places scattered with brownish-black fine encrustations, unchanged to slowly slightly reddening when cut. Hymenophore tubulate, adnate, subventricose, slightly depressed around the stipe. Tubes (2)4-6 mm long half-way to the margin, brown to greyish-brown (8F3), not separable from the pileus context. Pores 0.4-1.5 mm wide at mid-radius, regularly arranged, mostly roundish to elliptical at first, becoming slightly angular with age, slightly elongated around the stipe, colour distribution even, dark brown to chocolate brown (9F4 to 10F3) at first, becoming chocolate brown to brown (10F4 to 7-8F4-5) with age. Odour rubbery. Taste slightly bitter at first, then mild. Spore print dark brown (8F4/5) in mass.
Macrochemical reactions. KOH, orange brown on cap, yellowish-black on stipe, yellowish-black on the pileus context and stipe context, brownish-black on hymenium; NH 4 OH, yellowish-brown on cap, yellowish-orange on stipe, orangey yellow to yellowish-orange on the pileus context, stipe context and hymenium.
Habitat and Distribution. solitary to gregarious up to 4 basidiomata, on soil in hill evergreen forest dominated by Fagaceae trees, with a few Dipterocarpus spp. and Shorea spp. or in Dipterocarp forest dominated by Dipterocarpus spp. and Shorea spp. with a few Lithocarpus sp., Castanopsis sp. and Quercus sp. Currently known only from Chiang Mai Province, Northern Thailand.
Cacaoporus pallidicarneus differs from C. tenebrosus by its basidiomata context colour which is paler, especially at the stipe base. A combination of the following characters are also distinctive: spore shape which is amygdaliform or elongated amygdaliform or sometimes ovoid with subacute apex in side view and ovoid in front view, while C. tenebrosus has ovoid spores, shorter basidia and differently shaped hymenophoral cystidia (see note under C. tenebrosus). Cacaoporus pallidicarneus has a stipitipellis which is a disrupted hymeniderm composed of caulocystidia and clavate cells, while the other species has a loose trichoderm or tangled trichoderm. Interestingly, one collection (SV0402) had a slightly paler context than C. tenebrosus but not as pale as C. pallidicarneus. The phylogenetic analyses indicated that this collection might be a species different from C. pallidicarneus and C. tenebrosus. However, the specimen was immature and, therefore, more collections are needed before the species can be formally recognised. Vadthanarat, Raspé & Lumyong, sp. nov. MycoBank: MB829656 Figs. 2b-d, 3b-c, 4b and 6 Etymology. Refers to the overall darkness of basidiomata, including the context.
Diagnosis. Cacaoporus tenebrosus is characterised by having a darker context than the other species, longer basidia and cylindrical to narrowly subclavate hymenophoral cystidia.
Macrochemical reactions. KOH, yellowish then brown to black on cap, stipe, pileus context, stipe context and hymenium; NH 4 OH, yellowish then orange to brown on cap, stipe, pileus context, stipe context and hymenium.
Habitat and distribution. Gregarious (up to 9 basidiomata) to fasciculate or solitary, on soil in hill evergreen forest dominated by Fagaceae trees, with a few Dipterocarpus spp. and Shorea spp. or in Dipterocarp forest dominated by Dipterocarpus spp., Shorea spp. with a few Lithocarpus sp., Castanopsis sp. and Quercus sp. Currently known only from Chiang Mai Province, Northern Thailand.
Microscopically, Cacaoporus tenebrosus differs from C. pallidicarneus by having a darker context, longer basidia (33.6-47 µm vs. 25.3-33.8 µm, respectively), longer and larger hymenophoral cystidia, which also differ in shape (cylindrical to narrowly subclavate in C. tenebrosus but fusiform to narrowly fusiform in C. pallidicarneus). Phylogenetically, all Cacaoporus collections with a dark context formed a clade sister to C. pallidicarneus (BS = 85% and PP = 0.88), but some (SV0224 and SV0422) were genetically somewhat distant from the other collections. However, we could not find any difference in morphology. Consequently, we consider them as the same species (C. tenebrosus). Study of more collections is needed to confirm or infirm that they belong to the same species.

Discussion
Morphologically, Cacaoporus is most similar to Sutorius, with which it shares the overall brown colour of basidiomata and encrustations in the flesh. However, the genus Cacaoporus has darker basidiomata, especially the hymenophore and pore surface and is more chocolate brown, not reddish-brown or purplish-brown like Sutorius, tubes that are not separable from the pileus context whereas they are easily separable in Sutorius, white to off-white basal mycelium which becomes reddish when bruised, whereas in Sutorius, the basal mycelium is more or less white and unchanging. Cacaoporus also produces dark brown spore deposits whereas in Sutorius, spore deposits are reddishbrown (Halling et al. 2012). Microscopically, the two genera differ in the shape of basidiospores, which is amygdaliform to ovoid or ovoid with subacute apex in side view in Cacaoporus, whereas Sutorius produces narrowly ellipsoid to ellipsoid or subfusoid to fusoid basidiospores. Phylogenetically, Cacaoporus and Sutorius are not closely relatedthe two genera belong in two different clades of the Pulveroboletus group.
Some species in Porphyrellus E.-J. Gilbert also have brown to dark brown to umber basidiomata similar to Cacaoporus. However, Porphyrellus differs from the new genus in having white to greyish-white hymenophore when young, becoming greyish-pink to blackish-pink with age, white to pallid context in pileus and stipe variably staining blue and/or reddish when cut and white basal mycelium that does not turn red when bruised (Wolfe 1979;Wu et al. 2016). Some species in Strobilomyces Berk also share some characters with Cacaoporus, including dark brown basidiomata, white to off-white basal mycelium that turns red when bruised and the context turning red when cut. However, Strobilomyces species clearly differ from Cacaoporus, especially in the pileus surface, which is coarsely fibrillose or shows conical to patch-like scales, in the hymenophore, which is whitish-cream or greyish-brown or vinaceous drab and stains reddish then blackish when bruised and also basidiospores, which are subglobose to obtusely ellipsoid with reticulation or longitudinally striate (Gelardi et al. 2012;Antonín et al. 2015;Wu et al. 2016). Moreover, Porphyrellus and Strobilomyces were phylogenetically inferred to belong in subfamily Boletoideae (Wu et al. , 2016Vadthanarat et al. 2018) distinct from Cacaoporus.
Phylogenetically, Cacaoporus was monophyletic and clustered in a well-supported clade with the genera Cyanoboletus and Cupreoboletus and one undescribed taxon, Boletus p.p. sp. (specimen voucher JD0693), belonging to the Pulveroboletus group of Wu et al. (2014Wu et al. ( , 2016. Cyanoboletus and Cupreoboletus, however, differ from Cacaoporus in important morphological characters. The former two genera have a yellow hymenophore and yellowish context and tissues instantly discolouring dark blue when injured, and olive-brown spore deposits (Gelardi et al. 2014(Gelardi et al. , 2015Wu et al. 2016). The undescribed taxon represented by the voucher specimen JD0693, which clustered within the same clade as Cacaoporus, Cyanoboletus and Cupreoboletus, is also morphologically very different from Cacaoporus, in having yellow tubes, reddish pores, pale yellow to off-white context and reddish-brown pileus and stipe.
Our survey on the diversity of Boletes in the north of Thailand has been conducted since 2012 and no Cacaoporus has been found in the forests at elevations lower than 850 m. Cacaoporus was found only between 850 m and 1460 m elevation. However, more collections are needed to confirm that the distribution of the genus is restricted to mid-to high-elevation forests and does not occur in the lower elevation, drier forests. Most collections were made from Fagaceae-dominated, evergreen hill forests. The dominant trees in these forests belong to the Fagaceae, including Lithocarpus, Castanopsis and Quercus, but some Dipterocarpaceae may also occur. At the lower end of its elevation range, however, Cacaoporus was also found in Dipterocarpaceaedominated forests (in which Fagaceae, especially Quercus spp., also occurs). The Dipterocarpaceae trees include Dipterocarpus, namely D. tuberculatus, D. obtusifolius and Shorea, namely S. obtusa and S. siamensis. The listed trees have previously been reported as ectomycorrhizal hosts of Boletaceae (Moser et al. 2009;Desjardin et al. 2009Desjardin et al. , 2011Hosen et al. 2013;Arora and Frank 2014;Halling et al. 2014;Wu et al. 2018) and presumably are also the hosts for Cacaoporus.
Interestingly, our phylogeny indicated that the genera Neoboletus and Sutorius formed two different clades, both with high support (BS = 85% and PP = 0.95 for Neoboletus; BS = 100% and PP = 1 for Sutorius). Recently, Wu et al. (2016) synonymised Neoboletus with Sutorius because, in their phylogeny based on a four-gene dataset (28S+tef1+rpb1+rpb2), Boletus obscureumbrinus, a species morphologically more similar to Neoboletus than to Sutorius, seemed to cluster with Sutorius rather than with the Neoboletus species, although with neither ML nor BI support. Moreover, the Neoboletus clade was not supported either. Later, Chai et al. (2019) treated the two genera as different genetic lineages based on morphology and phylogeny (28S+ITS+tef1+rpb2), in which B. obscureumbrinus clustered with the other Neoboletus species in a wellsupported clade. Our phylogenetic analyses, based on a different set of genes (atp6+ tef1+rpb2+cox3), confirm the separation of the two genera Neoboletus and Sutorius. The differences in gene trees obtained could be explained by a long-branch attraction artefact in datasets with different taxon and gene samplings and/or problems in the dataset (e.g. suboptimal alignment). Neoboletus obscureumbrinus is quite atypical amongst Neoboletus species and its phylogenetic affinities within this genus remain unclear (Fig. 7).
Cacaoporus is the second novel bolete genus described from Thailand, the first one being Spongiforma Desjardin, Manfr. Binder, Roekring & Flegel, described in 2009 (Desjardin et al.). However, fungal diversity in Thailand is high and still poorly known (Hyde et al. 2018), with a large number of species and possibly genera that remain to be described.