Two new Erythrophylloporus species (Boletaceae) from Thailand, with two new combinations of American species

Abstract Erythrophylloporus is a lamellate genus in the family Boletaceae that has been recently described from China based on E.cinnabarinus, the only known species. Typical characters of Erythrophylloporus are reddish-orange to yellowish-red basidiomata, including lamellae, bright yellow basal mycelium and smooth, broadly ellipsoid, ellipsoid to nearly ovoid basidiospores. During our survey on diversity of Boletaceae in Thailand, several yellowish-orange to reddish- or brownish-orange lamellate boletes were collected. Based on both morphological evidence and molecular analyses of a four-gene dataset (atp6, tef1, rpb2 and cox3), they were recognised as belonging in Erythrophylloporus and different from the already known species. Two new species, E.paucicarpus and E.suthepensis are therefore introduced from Thailand with detailed descriptions and illustrations. Moreover, two previously described Phylloporus species, P.aurantiacus and P.fagicola, were also revised and recombined in Erythrophylloporus. A key to all known Erythrophylloporus species is provided.


Introduction
Most fungi in the family Boletaceae are pileate-stipitate with poroid hymenophore, but some have a lamellate hymenophore. Lamellate Boletaceae are currently classified in four genera, Phylloporus Quél, which contains about 84 species worldwide, Phylloboletellus Singer from South America and Mexico, the two recently described genera Phylloporopsis Angelini et al., from the New World and Erythrophylloporus Ming Zhang & T.H. Li from Asia, each of which circumscribes only one species (http:// www.indexfungorum.org, Farid et al. 2018;Zhang and Li 2018).
The genus Erythrophylloporus was recently described from China, with E. cinnabarinus Ming Zhang & T.H. Li as the type species. According to Zhang & Li (2018), the typical characters of the genus are orange to reddish-orange basidiomes, reddish-orange to yellowishred lamellae turning greyish-green when bruised, bright yellow to orange yellow context staining blackish-blue to dark blue when exposed, bright yellow basal mycelium, smooth and broadly ellipsoid to nearly ovoid basidiospores and yellowish-brown pigmented cystidia. During our survey on the diversity of Boletaceae in Thailand, several collections of lamellate boletes were discovered. Some collections were recognised to belong to Erythrophylloporus by possessing yellowish-orange to deep orange to reddish-orange basidiomata with bright yellow basal mycelium and smooth basidiospores. We also found that two described Phylloporus species, P. aurantiacus Halling & G.M. Mueller from Costa Rica and P. fagicola Bandala from Mexico (Halling et al. 1999, Montoya andBandala 2011), share similar morphological characters with the genus Erythrophylloporus, but until now, have not been included in a molecular phylogeny. In this study, a combination of phylogenetic and morphological evidence indicated that our Thai collections were new species, that, together with the two aforementioned American Phylloporus species, belong in Erythrophylloporus. Therefore, we introduce two new species with detailed descriptions and illustrations and propose two new combinations. As some of the species we studied have some characters that do not fit with the protologue of the genus, we emend its description.

Specimen collecting
Specimens were obtained and photographed from community forests and Doi Suthep-Pui National Park, Chiang Mai Province, northern Thailand during the rainy season in 2015 to 2016. The specimens were wrapped in aluminium foil and taken to the laboratory. After description of macroscopic characters, all specimens were dried in an electric drier at 45-50 °C. Examined specimens were deposited in the herbaria CMUB, MFLU, BKF or BR (Index Herbariorum; Thiers, continuously updated).

Morphological studies
Macroscopic descriptions were made based on detailed field notes and photos of fresh basidiomata. Colour codes follow Kornerup and Wanscher (1978). Macrochemical reactions (colour reactions) of fresh basidiomata were determined using 10% potassium hydroxide (KOH) and 28-30% ammonium hydroxide (NH 4 OH) in water. Microscopic structures were observed from dried specimens mounted in 5% KOH, NH 4 OH, Melzer's reagent or 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 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-)bc-d (-e), in which c represents the average, b the 5 th percentile, d the 95 th percentile and a and e the minimum and maximum values, respectively. Q, the length/width ratio, is presented in the same format. A section of the pileus surface was radially and perpendicularly cut 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. 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 a SEM stub with double-sided tape. The sample was coated with gold, examined and photographed with a JEOL JSM-5910 LV SEM (JEOL, Japan).

DNA isolation, PCR amplification and DNA sequencing
Genomic DNA was extracted from fresh tissue preserved in CTAB or about 10-15 mg of dried specimens using a CTAB isolation procedure adapted from Doyle and Doyle (1990). Portions of the genes atp6, tef1, rpb2 and cox3 were amplified by the polymerase chain reaction (PCR) technique. The tailed primers ATP6-1M40F and ATP6-2M (Raspé et al. 2016) and the primer pairs EF1-983F/EF1-2218R (Rehner and Buckley 2005) and bRPB2-6F/bRPB2-7.1R (Matheny 2005) were used to amplify atp6, tef1 and rpb2, respectively. PCR conditions were the same as in Raspé et al. (2016). Part of the mitochondrial gene cox3 was amplified with the primers COX3M1-F and COX3M1-R , using KAPA2G™ Robust HotStart polymerase (Kapa Biosystems, Wilmington, MA, USA) and the following PCR programme: 2 min 30 s at 95 °C; 35 cycles of 25 s at 95 °C, 30 s at 48 °C, 30 s at 72 °C; 3 min at 72 °C. PCR products were purified by adding 1 U of Exonuclease I and 0.5 U FastAP Alkaline Phosphatase (Thermo Scientific, St. Leon-Rot, Germany) and incubated at 37 °C for 1 h, followed by inactivation at 80 °C for 15 min. Sequencing was performed by Macrogen Inc. (Korea and The Netherlands) with PCR primers, except for atp6, for which universal primers M13F-pUC(-40) and M13F(-20) were used; for tef1, additional sequencing was performed with two internal primers, EF1-1577F and EF1-1567R (Rehner and Buckley 2005).

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 version 7 (Katoh and Standley 2013) on the server accessed at http:// mafft.cbrE.jp/alignment/server/.
Maximum Likelihood (ML) phylogenetic tree inference was performed using RAxML-HPC2 version 8.2.10 (Stamatakis 2006) on the CIPRES web portal (Miller et al. 2009). The phylogenetic tree was inferred from a four-partitions combined dataset, using the GTRCAT model with 25 categories. Two Buchwaldoboletus and nine Chalciporus species from subfamily Chalciporoideae were used as the outgroup. 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 GTR+I+G for atp6 and cox3, SYM+I+G for tef1 and K80+I+G for rpb2. 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 1,000 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 0.007058 and the Potential Scale Reduction Factor (PSRF) values of all parameters were close to 1. The burn-in phase (25%) was estimated by checking the stationarity in the plot generated by the sump command.

Phylogenetic analysis
Twenty-five sequences were newly generated and deposited in GenBank (Table 1). The sequences from three specimens, OR0689, OR1135 (E. paucicarpus) and OR0615B (E. suthepensis), were not included in our phylogenic analyses because they were identical to the sequences of the type specimens of E. paucicarpus and E. suthepensis. The alignment contained 906 sequences (179 for atp6, 313 for tef1, 279 for rpb2, 135 for cox3) from 315 voucher specimens and was 2946 characters long (TreeBase number 24078). ML and BI trees showed similar topologies without any supported conflict (Bootstrap Support values, BS ≥ 70% and posterior probabilities, PP ≥ 0.90; Fig. 1). The four-gene phylogram indicated that the included taxa formed seven major clades, representing the Austroboletoideae, Boletoideae, Chalciporoideae, Leccinoideae, Xerocomoideae, Zangioideae and the Pulveroboletus group. Erythrophylloporus cinnabarinus (typus generis) grouped with the two new Erythrophylloporus species, E. paucicarpus and E. suthepensis, in a highly supported clade (BS = 100% and PP = 1). The two New World Phylloporus species (P. aurantiacus voucher REH7271 and P. fagicola voucher Garay215) Description. Basidiomata stipitate-pileate with lamellate hymenophore, small to medium-sized; Pileus subhemispheric to convex when young becoming convex to planoconvex to plano-subdepressed when old, dry, pruinose or velutinous, subtomentose to tomentose, yellowish-orange to red; pileus context vivid yellow to yellowish-orange. Hymenophore lamellae, slightly thick, decurrent, deeply yellowish-orange to deep orange or reddish-orange to orange red or brownish-orange to red. Stipe central to slightly excentric, cylindrical or clavate, yellowish-to reddish-orange to yellowish red, with scattered yellowish-to reddish-orange to red scales on surface, with bright yellow basal mycelium; stipe context solid, yellow to reddish-yellow or yellow with olivaceous Figure 1. Phylogenetic tree inferred from the four-gene dataset (atp6, rpb2, tef1 and cox3), including Erythrophylloporus species and selected Boletaceae using Maximum Likelihood and Bayesian Inference methods (ML tree is presented). The two Buchwaldoboletus and nine Chalciporus species in subfamily Chalciporoideae were used as outgroup. Most of the taxa not belonging to the Pulveroboletus group were collapsed into subfamilies. All generic clades, including one undescribed generic clade in Pulveroboletus group that were highly supported, were also collapsed. Bootstrap support values (BS ≥ 70%) and posterior probabilities (PP ≥ 0.90) are shown above the supported branches.
brown. Staining none or slightly reddening or greening or gradually bluing or dark violet, greyish to blackish-blue when bruised on the basidiomata or context or lamellae. Spore print olivaceous brown. Basidiospores ovoid or ellipsoid to broadly ellipsoid to subovoid, thin-walled, with non-bacillate surface. Basidia clavate to narrowly clavate. Cheilocystidia and pleurocystidia present, subcylindrical or narrowly conical to narrowly fusiform to ventricose with slightly or obtuse apex, thin-walled, sometimes thickwalled, originating more or less deeply in the sub hymenium or from hymenophoral trama, hyaline or sometimes containing yellowish-brown pigments. Pileipellis a subcutis to cutis to trichoderm to palisadoderm, composed of thin to slightly thick-walled hyphae. Remarks. Erythrophylloporus is easily distinguished from other lamellate Boletaceae genera by a combination of the following characters: the intense orange to red colour of the pileus and lamellae; bright yellow basal mycelium; ovoid or ellipsoid to broadly ellipsoid to subovoid basidiospores with non-bacillate surface; pleurocystidia originating more or less deeply in the subhymenium or from hymenophoral trama. Etymology. from Latin "pauci-" meaning few and "carpus" meaning fruits or what is harvested, refers to the low number of basidiomata produced.
Macrochemical reactions. KOH on pileus and stipe surface deep red at first, then red-brown to brown, with pale orange aura on the pileus; brown on pileus context, dark red-brown on stipe context; brownish-orange on hymenophore. NH 4 OH on pileus first red, then orange; on pileus context bluing at first then with a greenish tinge; on stipe surface and context briefly bluing; no reaction on hymenophore.
Habit and habitat. On soil, mostly solitary in dipterocarp forest dominated by Dipterocarpus tuberculatus, D. obtusifolius, Shorea obtusa, S. siamensis, Quercus spp. and Lithocarpus spp. Remarks. E. paucicarpus is characterised by the following combination of features: orange to brownish-to orange-red basidiomata, yellowish-orange lamellae that turn slightly red when bruised; pileus context yellow to yellowish-orange that slowly reddens when exposed and mostly occurring as solitary basidiomata.
In the inferred molecular phylogeny, E. paucicarpus clustered close to E. suthepensis and E. cinnabarinus (65% BS and 1 PP), but the two species are different from   E. paucicarpus in that they have darker lamellae which are orange to orange red or brownish-orange. Moreover, spores of E. paucicarpus are wider and longer (5.9-8 × 4.1-6 µm) than those of E. suthepensis (4.6-5.9 × 3.5-4.5 µm) and, on average, longer than those of E. cinnabarinus (5.5-7 × 4.5-5.5 µm) (Zhang and Li 2018). Erythrophylloporus paucicarpus also differs from both species by the slight reddening of the context and lamellae when exposed or bruised, whereas E. suthepensis context seems unchanging when exposed and lamellae turn blue when bruised. In E. cinnabarinus, the context slowly turns dark violet, blackish-blue to dark blue when exposed and lamellae turn greyish-blue, or greyish-green when bruised (Zhang and Li 2018).
Erythrophylloporus paucicarpus is different from the two New World species by the reddening of the context, whereas in E. fagicola, it turns blue and, in E. aurantiacus, the colour remains unchanged when exposed. Moreover, E. fagicola has somewhat thickwalled (0.8-3.5 µm) pleurocystidia (Montoya and Bandala 2011), which are not found in E. paucicarpus. Although the basidiospores of E. paucicarpus and E. aurantiacus are similar in size (E. aurantiacus = 6.0-7.5 × 4-5.5 µm), they differ in shape, being more ovoid in E. aurantiacus than in E. paucicarpus. Erythrophylloporus paucicarpus also differs from E. aurantiacus by macro-chemical reactions. In the latter, the pileus surface and pileus context are unchanging with NH 4 OH , while in E. paucicarpus, the pileus becomes orange to red and the pileus context initially turns blue then with a greenish tinge. Holotype. THAILAND, Chiang Mai Province, Muang District, Doi Suthep-Pui National Park, 18°48'47"N, 98°55'56"E, elev. 645 m, 25 August 2015, S. Vadthanarat, SV0236, (holotype CMUB, isotype BKF, BR).
Macrochemical reactions. KOH orange-brown on pileus and stipe surface; yellowish-brown on pileus and stipe context and hymenophore. NH 4 OH yellowish-brown on pileus and stipe surface and hymenophore; yellowish on pileus and stipe context.
Morphologically, E. suthepensis is quite similar to E. cinnabarinus in that they have similar colours in pileus and lamellae; the lamellae in both species also turn more or less blue to dark blue when bruised. Erythrophylloporus suthepensis and E. cinnabarinus are also similar, based on some pleurocystidia containing yellowish-brown to dark brown pigments, but those features are not found in E. paucicarpus and in the two New World Erythrophylloporus species (Halling et al. 1999, Montoya andBandala 2011). However, the pleurocystidia containing brown pigments seem to be more frequent in E. cinnabarinus, which also has, on average, larger basidiospores than E. suthepensis (Zhang and Li 2018).
The pinkish-red hymenophoral trama of E. suthepensis was not found in either E. paucicarpus or in the two American Erythrophylloporus species. In our observation of the two American specimens (E. aurantiacus voucher REH7271 and E. fagicola voucher Garay215), we found that the hymenophoral trama was yellowish hyaline when observed in water. The original description of E. cinnabarinus does not mention the colour of the hymenophoral trama and we could not obtain a specimen to observe this character. However, other morphological characters and phylogenetic evidence are enough to differentiate E. suthepensis from E. cinnabarinus.
Our phylogenetic analyses of a four-gene dataset revealed that Phylloporus aurantiacus from Costa Rica and P. fagicola from Mexico clustered in the Erythrophylloporus clade with high support (BS = 100% and PP = 1). Both species possess the distinctive morphological characters of Erythrophylloporus, which include yellowish-orange to reddish-orange basidiomata, orange to orange brown lamellae, bright yellow basal mycelium, ovoid or ellipsoid to broadly ellipsoid basidiospores with smooth surface and subcylindrical to subfusoid to ventricose cheilocystidia and pleurocystidia (Halling et al. 1999, Montoya andBandala 2011). Therefore, the following two new combinations are proposed: close to E. cinnabarinus (typus generis). Morphologically, they are characterised by having yellowish-orange to reddish-to brownish-orange basidiomata with bright yellow basal mycelium and smooth, ellipsoid, broadly ellipsoid to subglobose basidiospores. The other lamellate Boletaceae in Phylloporus, Phylloboletellus and Phylloporopsis are solely similar to the new species by having a lamellate hymenophore instead of a poroid hymenophore. However, Phylloporus differs from Erythrophylloporus species by having whitish-to yellowish-pale brown basidiomata with yellow to golden-yellow lamellae, with off-white to whitish to yellow basal mycelium and most species in the genus have basidiospores with more or less bacillate ornamentation under SEM (Neves & Halling 2010, Neves et al. 2012, Zeng et al. 2013). The single Phylloboletellus species, Ph. chloephorus Singer differs from Erythrophylloporus by having longitudinally ridged basidiospores (Bandala et al. 2004). The sole species of Phylloporopsis, Phy. boletinoides, differs by having beige to olive-cream or olive buff lamellate to subporoid hymenophore, with anastomosing and interveined gills and basal mycelium whitish to yellowish (Farid et al. 2018). Moreover, those genera are phylogenetically distant from Erythrophylloporus. (Bandala et al. 2004, Neves & Halling 2010, Neves et al. 2012, Zeng et al. 2013, Farid et al. 2018. Interestingly, Phylloporus coccineus Corner, described from Singapore (Corner 1970), is similar to Erythrophylloporus species, in that it produces crimson to scarlet, lamellate basidiomata with orange to orange-red lamellae and yellow basal mycelium, broadly ellipsoid to subglobose and smooth basidiospores. It probably should also be transferred to Erythrophylloporus, but we refrain from doing so until specimens become available for molecular study. According to the protologue of P. coccineus, it differs from the newly described Asian species of Erythrophylloporus by having larger basidiospores (7.5-10 × 6.5-8 µm), larger cheilocystidia (70-120 × 10-18 µm) and larger caulocystidia (up to 200 × 10-16 µm) (Corner 1970).
Erythrophylloporus species formed two clades, an Asian species clade (BS = 65% and PP = 1) and a New World species clade (BS = 100% and PP = 1) (Fig. 1). The Asian one contains three species, E. cinnabarinus, E. paucicarpus and E. suthepensis, while the American clade contains the remaining two species E. aurantiacus and E. fagicola. Erythrophylloporus aurantiacus and E. fagicola seem to be genetically very close to each other, much closer than the species in the Asian clade. Only morphological differences between the two species were used to separate them from each other. Erythrophylloporus fagicola produces larger basidiospores than E. aurantiacus and pleurocystidia are somewhat thick-walled (0.8-3.5 µm thick) in E. fagicola, whereas they are thin-walled in E. aurantiacus and the latter has non-staining context, whereas the former has a cyanescent context. However, the descriptions were based on a limited number of collections and more samples are desirable to verify whether the morphological traits observed are good characters differentiating the two species or merely extremes of a continuum in morphological variation within a single species.
Regarding the phylogenetic affinities of Erythrophylloporus, Zhang and Li (2018) reported that it was likely close to the genus Rugiboletus G. Wu & Zhu L. Yang and Lanmaoa G. Wu & Zhu L. Yang, based on a multilocus dataset of nrLSU, tef1, rpb1 and rpb2, although this relationship was not supported in their phylogram. In our phylogeny, based on a multilocus dataset of atp6, tef1, rpb2 and cox3, with wider taxon sampling, Erythrophylloporus also clustered within the Pulveroboletus group, but was sister to Singerocomus with high bootstrap support (96%) but relatively weak posterior probability support (0.86). Singerocomus contains three species, S. atlanticus A.C. Magnago, S. inundabilis (Singer) T.W. Henkel and S. rubriflavus T.W. Henkel & Husbands that have some similar morphological characters to Erythrophylloporus, including red-orange to red pileus and light yellow basal mycelium. The three existing Singerocomus species are clearly different from all known Erythrophylloporus species by having a poroid, non-cyanescent hymenophore (Henkel et. al. 2016, Magnago et al. 2018). However, the hymenophore structure (lamellate vs. poroid) is not sufficient to separate genera in Boletaceae. Phylloporus currently contains both lamellate and poroid species, although some poroid species have already been transferred to another genus, Hourangia (Zhu et al. 2015). Phylogenetic analyses, including the remaining poroid Phylloporus species, are needed to verify their taxonomic position.
Erythrophylloporus putatively forms ectomycorrhizal associations with trees in family Fagaceae, including the genera Fagus, Lithocarpus and Quercus (Neves and Halling 2010, Montoya and Bandala 2011, Zhang and Li 2018. The two Thai Erythrophylloporus species were found in forests dominated by Dipterocarpaceae trees, mainly Dipterocarpus, including D. tuberculatus, D. obtusifolius and Shorea, including S. obtusa and S. siamensis. However, some Quercus and Lithocarpus trees (Fagaceae) were also observed in the vicinity and could also be the ectomycorrhizal partners. Further study is needed to confirm the ectomycorrhizal relationships of Erythrophylloporus.