2021 年 62 巻 5 号 p. 297-306
Morphological characters and multi-gene phylogenetic analyses were used to identify Retiboletus specimens collected in northern Thailand. Retiboletus brevibasidiatus is described as new to science, whereas R. fuscus and R. nigrogriseus are reported for the first time from Thailand. Retiboletus brevibasidiatus produces medium-sized basidiomes, with a dark blonde to clay pileus and densely reticulate stipe mostly on the upper part with pale yellow to chrome yellow basal mycelium. It is difficult to separate R. brevibasidiatus from other closely related species on the basis of macroscopic characters. However, the new species can be distinguished by microscopic characters, mostly the shorter basidia. The macro- and micro-morphology of the R. fuscus and R. nigrogriseus collections from Thailand fit well with the previous descriptions of materials from China and Japan. Detailed descriptions, molecular phylogeny, and illustrations of the three species are provided.
Retiboletus was described by Binder and Bresinsky (2002), typified by R. ornatipes (Peck) Manfr. Binder & Bresinsky, a species native to North America. Retiboletus belongs in the Boletaceae, Boletales (He et al., 2019) and differs from other Boletaceae genera by its usually reticulate stipe with a base usually darker than the apical region. No part of the basidiomata stain blue after bruising like many other boletes, but rather yellow-orange to rusty brown. Moreover, the mushrooms often stain the fingers yellow on handling. This yellow staining is due to retipolides, a unique class of metabolites so far only found in Retiboletus (Justus et al., 2007). DNA phylogenetic analyses and the presence of retipolides prompted Binder and Bresinsky (2002) to erect Retiboletus as a new genus of Boletaceae. Retiboletus ornatipes and R. retipes (Berk. & M.A. Curtis) Manfr. Binder & Bresinsky, originally described in Boletus, are closely related species that are very difficult to separate in morphology. However, DNA analyses and chemotaxonomical characters, i.e. the structure of the unique metabolites named retipolides, allow separating the two species clearly (Binder & Bresinsky, 2002). Retiboletus griseus (Frost) Manfr. Binder & Bresinsky from Northern America and R. nigerrimus (R. Heim) Manfr. Binder & Bresinsky from Australia were also transferred to Retiboletus based on morphological characters and sequence data (Binder & Bresinsky, 2002). However, the detection of retipolides in those two taxa remains to be established (Binder & Bresinsky, 2002; Gruber, Kerschensteiner, Marumoto, & Steglich, 2013). As Binder and Bresinsky (2002) proposed the new bolete genus Retiboletus to accommodate Boletus retipes Berk. & M.A. Curtis, B. ornatipes Peck, B. flavoniger Halling, G.M. Muell. & L.D. Gómez, B. griseus Frost, and B. nigerrimus R. Heim. The first three species are characterized by the occurrence of retipolides (Binder & Bresinsky, 2002; Justus et al., 2007). Later, Gruber et al. (2013) studied the chemotaxonomy of the latter two species, and they detected a new pulvinic acid (retiboletic acid), and two phenylalaninol derivatives (nigerrimin A and B), but no retipolides. However, retiboletic acid and phenylalaninol derivatives could be other compounds characteristic of Retiboletus, especially R. griseus, and R. nigerrimus, because so far, they have been found only in Retiboletus. Thus, although intraspecific variation in secondary metabolites may occur, retipolides and other compounds have some chemotaxonomical value (Hellwig, 1998; Binder & Bresinsky, 2002; Gruber et al., 2013).
Recently, Retiboletus collections from China were studied, and their molecular phylogenetic analyses confirmed the monophyly of Retiboletus (Wu et al., 2014; Zeng, Liang, Wu, Li, & Yang, 2016). To date, nine Retiboletus species have been reported from China (Wu et al., 2016; Zeng et al., 2016, 2018; Liu, Li, & Bau, 2020). Retiboletus ater Yan C. Li & T. Bau, R. brunneolus Y.C. Li & Zhu L. Yang, R. nigrogriseus N.K. Zeng, S. Jiang & Zhi Q. Liang, R. pseudogriseus N.K. Zeng & Zhu L. Yang, R. sinensis N.K. Zeng & Zhu L. Yang, R. sinogriseus Yan C. Li & T. Bau and R. zhangfeii N.K. Zeng & Zhu L. Yang were described as new to science while two new combinations R. fuscus (Hongo) N.K. Zeng & Zhu L. Yang and R. kauffmanii (Lohwag) N.K. Zeng & Zhu L. Yang were introduced.
For several years, we have collected Boletaceae in northern Thailand, where fungal diversity is high (Hyde et al., 2018). In this study, three Retiboletus species from northern Thailand are investigated based on both morphology and phylogenetic analyses. One new species is introduced, and two taxa are reported for the first time from Thailand.
The Retiboletus collections were made from northern Thailand. Each collection was photographed and described based on field notes and photos of fresh basidiomata. Macro-chemical reactions on fresh basidiomata, habitat, elevation, and localization of the collecting site were recorded. Color codes were given following Kornerup and Wanscher (1978). Finally, the specimens were dried using a hot air dryer at ca. 50 °C and deposited in the Mae Fah Luang University herbarium (MFLU) or the Chiang Mai University herbarium (CMUB). The dried specimens were used for microscopic observations. Micro-characters were studied and photographed using Eclipse Ni (Nikon, Tokyo) microscope. Tissues were observed in 5% (w/v) potassium hydroxide (KOH) or 28–30% (w/v) ammonium hydroxide (NH4OH) solutions or stained with 1% (w/v) ammoniacal congo red solution. The microscopic structures were observed at a magnification of 400× or 1,000×, and measured using Tarosoft (R) Image Frame Work program v. 0.9.7. At least 50 basidiospores, 20 basidia, pleurocystidia, cheilocystidia, terminal cells and hyphae of the pileipellis and stipitipellis were measured. The notation ‘[n/m/p]’ represents the number of basidiospores n measured from m basidiomata of p collections. The dimensions of microscopic structures are presented in the following format: (a–)b–c(–d), with the average, in which b represents the 5th percentile, c the 95th percentile and extreme values a and d are shown in parentheses. Q, the length/width ratio, is presented in the same format. All microscopic features were drawn by free hand, using a drawing tube. Faces of Fungi (Jayasiri et al., 2015) and MycoBank numbers (Westerdijk Fungal Biodiversity Institute) are provided for each species.
2.2. Molecular phylogeny 2.2.1. DNA extraction, amplification, sequencing and sequence alignmentGenomic DNA was extracted from CTAB-preserved tissues or dry specimen (OR0110) using a CTAB isolation procedure adapted from Doyle and Doyle (1990). Tissues were ground with a Retsch 300 mill with 0.2% ß-mercaptoethanol was added to the CTAB lysis buffer just prior to extraction; samples were lysed for one hour at 60 °C; proteins and polysaccharides were removed by two consecutive extractions with chloroform: isoamylalcohol (24: 1), after which DNA was precipitated by the addition of 0.8 volume isopropanol to the aqueous phase; the pellet was washed once in 600 μL 70% (v/v) ethanol, air-dried and suspended in 100 μL TE pH 8.0; RNA was then digested with RNase A. The PCR amplification of atp6, rpb2 and tef1 were performed following the procedures described in Raspé et al. (2016). For amplification of atp6, we used ATP6-1M40F and ATP6-2M (Raspé et al., 2016). The primers EF1-983F and EF1-2218R (Rehner & Buckley, 2005) were used to amplify tef1. The primers bRPB2-6F and bRPB2-7.1R (Matheny, 2005) were used to amplify rpb2. 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. (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 the two internal primers, EF1-1577F and EF1-1567R (Rehner & Buckley, 2005). Newly generated sequences were assembled in Geneious Prime ver. 2019.2.3 (Biomatters). The newly generated atp6, rpb2 and tef1 sequences , together with sequences of 17 specimens retrieved from GenBank were aligned using MAFFT version 7 (Katoh & Standley, 2013) on the server accessed at http://mafft.cbrc.jp/alignment/server/.
2.2.2. Phylogenetic analysesMaximum likelihood (ML) phylogenetic tree inference was performed using RAxML version 8.2.10 (Stamatakis, 2014) on the CIPRES web server (Miller, Pfeiffer, & Schwartz, 2010), using a mixed-model analysis, and the GTRCAT model of substitution with 25 categories. Four species in the sub-family Leccinoideae, namely Borofutus dhakanus Hosen & Zhu L. Yang, Ionosporus longipes (Massee) Khmeln., Davoodian, Raspé, S.M.L. Lee & Halling, Rhodactina rostratispora Vadthanarat, Raspé & Lumyong, and Spongiforma thailandica Desjardin, Manfr. Binder, Roekring & Flegel were used as outgroup taxa. The partitions were defined as: atp6, tef1 exons, rpb2 exons, and tef1 introns plus rpb2 intron. Node support was obtained by rapid bootstrapping with 1,000 replicates. Bayesian inference (BI) was performed using the Markov Chain Monte Carlo (MCMC) method implemented in MrBayes 3.2.6 (Ronquist et al., 2012) with a mixed-model partition (partitions identical to the ones defined in the ML analysis). The best-fit nucleotide substitution model was separately determined for each partition with jModeltest version 2.1.10 (Darriba, Taboada, Doallo, & Posada, 2012) on CIPRES, using the Akaike Information Criterion. GTR+G, SYM+G, SYM+G and GTR+G were selected as best-fit models for atp6, tef1 exons, rpb2 exons, and introns, respectively. Two MCMC runs with five chains each were run for 2,000,000 generations and sampled every 200 generations. At the end of the runs, the average deviation of split frequencies was 0.00242. The burn-in phase was estimated by checking the stationarity in the log-likelihood plot generated by the sump command.
A total of 22 new sequences were generated and deposited in GenBank (Table 1). The alignment contained 23 specimens and was 2642 characters long (TreeBase number S25985). The authors could not obtain tef1 and rpb2 sequences from the type OR0110 because its DNA was too degraded. However, the atp6 sequence obtained from this specimen was 100% identical to the other studied specimens of R. brevibasidiatus.
| Species | Voucher | Origin | atp6 | tef1 | rpb2 | References |
| Borofutus dhakanus | OR0345 | Thailand | MG212533 | MG212578 | MG212620 | Vadthanarat et al. (2018) |
| Ionosporus longipes | LEE1180 | Singapore | MT085461 | MT085471 | MH712031* | This study, *Khmelnitsky et al. (2019) |
| Retiboletus brunneolus | HKAS 52680 | China | – | KF112179 | KF112690 | Wu et al. (2014) |
| Retiboletus fuscus | HKAS63590 | China | – | KF112178 | KF112691 | Wu et al. (2016) |
| R. fuscus | HKAS63624 | China | – | KT990829 | KT990466 | Wu et al. (2016) |
| R. fuscus | HKAS74756 | China | – | KT990830 | KT990467 | Wu et al. (2016) |
| R. fuscus | OR0231 | China | MG212556 | MG212600 | MG212642 | Vadthanarat et al. (2018) |
| R. fuscus | OR0738 | Thailand | MT085462 | MT085472 | MT085477 | This study |
| Retiboletus griseus | MB03-079 | U.S.A. | KT823964 | KT824030 | KT823997 | Raspé et al. (2016) |
| Retiboletus kauffmanii | G.Wu352 | China | – | KP739301 | KP739299 | Zeng et al. (2016) |
| R. kauffmanii | HKAS63584 | China | – | KT990828 | KT990465 | Wu et al. (2016) |
| R. kauffmanii | OR0278 | China | MG212557 | MG212601 | MG212643 | Vadthanarat et al. (2018) |
| R. nigrogriseus | BC0178 | Thailand | MT085463 | MT085473 | – | This study |
| R. nigrogriseus | BC0179 | Thailand | MT085464 | MT085474 | MT085478 | This study |
| R. nigrogriseus | OR0049 | Thailand | KT823967 | KT824033 | KT824000 | Raspé et al. (2016) |
| R. nigrogriseus | OR0108 | Thailand | MT085465 | – | – | This study |
| R. nigrogriseus | OR0127 | Thailand | MT085466 | MT085475 | – | This study |
| R. nigrogriseus | SV0218 | Thailand | MT085467 | – | – | This study |
| R. nigrogriseus | FHMU 2800 | China | – | MH367488 | – | Zeng et al. (2018) |
| Retiboletus ornatipes | MBsn | U.S.A. | MT219514 | MT219516 | MT219515 | This study |
| Retiboletus sinensis | HKAS59832 | China | – | KT990827 | KT990464 | Wu et al. (2016) |
| Retiboletus brevibasidiatus | OR0110 | Thailand | MT085468 | – | – | This study |
| R. brevibasidiatus | OR0570 | Thailand | MT085469 | MT085476 | MT085479 | This study |
| R. brevibasidiatus | OR0708 | Thailand | MT085470 | – | – | This study |
| Retiboletus zhangfeii | HKAS53418 | China | – | KT990824 | KT990462 | Wu et al. (2016) |
| Rhodactina rostratispora | SV170 | Thailand | MG212560 | MG212605 | MG212645 | Vadthanarat et al. (2018) |
| Spongiforma thailandica | DED7873 | Thailand | MG212563 | KF030436* | MG212648 | Vadthanarat et al. (2018), *Nuhn et al. (2013) |
In the three-gene phylogeny, the new species was sister to R. sinensis with high statistical support (BS = 100%, PP = 1) (Fig. 1). Sequences from the other Retibletus specimens collected in Thailand grouped with either R. fuscus or R. nigrogriseus sequences with high support (BS = 100%, PP = 1).
Retiboletus brevibasidiatus Raspé & Chuankid, sp. nov. Figs. 2, 3.
MycoBank no.: MB 835032, Faces of Fungi FoF 07863.
Holotype: THAILAND. Chiang Mai Province, Mae Taeng District, near Mushroom Research Center, 19°06’34’’N, 98°44’27’’E, elev. 1,090 m, 9 Jul 2010, O. Raspé, OR0110 (MFLU10-0942).
Etymology: the epithet brevibasidiatus refers to the shorter basidia as compared to the most closely related and resembling species R. kauffmanii and R. sinensis.
Macromorphology: Basidiomata medium-sized. Pileus 43–88 mm diam, convex when young, subhemispherical to convex when aged, densely subtomentose; surface dry, dark blonde to clay (5D4–5) or hair brown to bronze (5E4–5) with brownish grey (6F8) patches; margin inflexed when young, then straight to slightly reflexed, abruptly paler than the rest of the pileus, at least in immature basidiomes; context 9–11 mm thick at center, 3–6 mm thick half-way between center and margin, pale yellow to light yellow (3A3–5), staining deeper chrome yellow (3A7) when bruised. Hymenophore poroid, sinuate-decurrent to decurrent; tubes 2–9 mm long, pale yellow to light yellow (3A3–5), deeper chrome yellow when bruised; pores 0.3–1.5 mm in diam, angular, irregular surface, pale yellow to light yellow (3A3–5), staining pale orange (between 4A6 and 5A6) with time when bruised. Stipe 47–75 mm long, 9–19 mm wide, central, solid, equal or slightly enlarged at base, usually curved, densely reticulate over the upper 2/3; surface dry, background color yellow (1A5) on upper part, lower part golden blonde (4B4), staining vivid yellow (2A8) at first and changed to light brown (6D8) later when injured; reticulum light yellow to yellow (3A4–6) entirely, or only above, and then brownish-grey below (between 4B3 and 5C3); context white to yellow, staining deeper chrome yellow when cut, at least when young. Spore print not obtained. Basal mycelium pale yellow to chrome yellow, with some fine rhizoids. Odor fungoid, weak. Taste bitter.
Macrochemical reactions: Pileipellis reddish with violet tinge around the spot in KOH solution, pale orange in NH4OH solution; pileus context pinkish white with KOH, yellow by reversal of the oxidation with an additional greenish tinge with NH4OH solution.
Micromorphology: [55/1/1] (8–)9–11(–11.5) × (3.6–)3.7–4.3(–4.4) µm, 10.3 × 4.0 µm on average, Q = (1.9–)2.4–2.9(–3), 2.59 on average, subfusiform to ellipsoid, greenish grey to greyish green (1C2–3) in water and KOH, smooth. Basidia (17–)18–25(–26) × 6–10 µm, 21.1 × 8.3 µm on average, clavate, four-spored, hyaline in KOH; sterigmata 2–4 μm long. Hymenophoral trama bilateral of the boletus-type, composed of hyphae 3–5 μm wide, hyaline in KOH. Cheilocystidia 34–46 × 7–13 µm, 40.7 × 9.9 µm on average, abundant, ventricose or subfusiform, usually with yellowish-brown (5E8) pigmented contents in water, but without cell wall encrustations. Pleurocystidia 24–40 × 7–11 µm, 32.1 × 9 µm on average, clavate or subfusiform, with yellowish-brown (5E8) pigmented contents in water, without cell wall encrustations. Pileipellis a trichoderm about 70–100 μm thick, composed of slightly interwoven hyphae 5–10 μm wide, brown (6E6) to blackish-brown in water and KOH; terminal cells 20–50 × 7–10 μm, narrowly clavate or subcylindrical. Pileal trama made of hyphae 6–10 μm, hyaline in KOH. Stipitipellis a disrupted hymeniderm about 60–90 μm thick, composed of emergent hyphae, hyaline in potassium, with 20–40 × 7–9 μm clavate or subfusiform terminal cells. Stipe trama composed of parallel hyphae, 5–8 μm wide, cylindrical, hyaline in KOH. Clamp connections not seen in any tissue.
Habitat: Solitary or in small groups on the leaves litter in forests dominated by Fagaceae (Quercus, Lithocarpus, Castanopsis) and/or Dipterocarpaceae (Shorea, Dipterocarpus).
Known distribution: Currently only known from northern Thailand.
Other materials examined: THAILAND. Lampang Province, Mueang Pan District, along Road 1252, 18°55’50’’N, 99°23’30’’E, elev. 1,420 m, Olivier Raspé & Komsit Wisitrassameewong, OR0570; Chiang Mai Province, Mae On District, Behind Tharnthong Lodges, 18°52’06’’N, 99°18’06’’E, elev. 760 m, 8 Aug 2013, O. Raspé & Benjarong Thongbai, OR0708 (MFLU13-0610).
Note: Retiboletus brevibasidiatus is macro-morphologically characterized by its medium-sized basidiomes, dark blonde to clay or hair brown to bronze pileus, with brownish grey spots, context staining deeper yellow and pores changing to pale orange after bruising, stipe densely reticulate mostly on the upper part with pale yellow to chrome yellow basal mycelium. When compared to other Retiboletus species, R. brevibasidiatus is mostly similar to the two Chinese species R. kauffmanii and R. sinensis in pileus, hymenophore, and stipe colors. When cut, the context of R. kauffmanii does not change color, but it does in R. brevibasidiatus and R. sinensis. The most important discriminative character of R. brevibasidiatus is the short basidia (18–26 µm long). The most closely related and resembling species R. kauffmanii and R. sinensis produce longer basidia (23–32 µm and 27–39 µm, respectively). Also, the cheilocystidia and pleurocystidia of R. brevibasidiatus are broader (7–12 µm) than those of R. sinensis (4.5–7 µm), and tend to be broader than those of R. kauffmanii (6–10 µm). The new species also resembles R. ornatipes and R. retipes by the yellow tinge of the pileus and the strongly pigmented (yellowish brown) cystidia in KOH (Smith & Thiers, 1971; Binder & Bresinsky, 2002). However, upon closer examination, the pileus color of R. brevibasidiatus is more blonde than yellow. Based on the descriptions of R. ornatipes by Ortiz-Santana, Lodge, Baroni, & Both (2007), the chemical reactions on pileus and pileus context are the most remarkable characters that could be used to difference R. brevibasidiatus from R. ornatipes. The pileus of R. ornatipes turns brown with KOH and shows no reaction to NH4OH solution, whereas the pileus of R. brevibasidiatus reddens with a violet tinge around the spot in KOH and turns pale orange with NH4OH solution. The fresh pileus context of R. brevibasidiatus turns pinkish white with KOH and changes to yellow with a greenish tinge with NH4OH solution, but those characters were not found in pileus context of R. ornatipes (Ortiz-Santana et al., 2007). Retiboletus retipes mainly differs from R. brevibasidiatus in its yellowish pulverulent pileus and stipe (Berkeley, 1872). Phylogenetically, R. brevibasidiatus groups with R. sinensis (BS=100% and PP=1), which together are sister to R. kauffmanii (BS=95% and PP=0.99).
Retiboletus fuscus (Hongo) N.K. Zeng & Zhu L. Yang Figs. 4, 5.
MycoBank no.: MB 811563, Faces of Fungi FoF 08036.
≡ Boletus griseus var. fuscus Hongo, J Jap Bot 4:301, 1974 (basionym)
Macromorphology: Basidiomata medium-sized. Pileus about 50 mm diam, broadly convex; surface dry, densely subtomentose, greyish brown (8F3) to black; margin straight; context 6–10 mm thick, dull yellowish white (4A2) or paler, slowly staining pale yellowish-grey (paler than 4B2) when cut. Hymenophore poroid, adnexed; tubes 2.5–6 mm long, dull yellowish white (4A2), inconsistently staining pale orange-grey (paler than 5B2) when bruised; pores 0.3–0.8 mm diam, angular, irregular surface, dull yellowish white (4A2). Stipe 80 mm long, 10–15 mm wide, central, cylindrical and tapering downward, solid, almost entirely reticulate; surface dry, background color cream (4A3) in the upper part, darker to the base; reticulum concolorous with the pileus; context fibrous, upper part yellowish white (4A2) unchanging or slightly yellowing when bruised, lower part yellow (3A6). Basal mycelium off-white to pale beige. Spore print not obtained. Odor rubbery. Taste acidulous.
Macrochemical reactions: Hymenophore orange in KOH solution; stipitipellis, pileus context and stipe context pink in KOH solution; pileipellis reaction subnull in KOH solution.
Micromorphology: Basidiospores [55/1/1] 9.5–12 × 4.0–5 µm, 10.69 × 4.48 µm on average, Q = (1.9–)2.1–2.7(–3), 2.39 on average, subfusiform to ellipsoid, greenish grey in water (1C2) and olive grey (1D2) in KOH, smooth. Basidia (25–)26–36(–37) × (7.5–)8–10 μm, 30.9 × 8.9 μm on average, clavate, four-spored, hyaline in KOH; sterigmata 2–4 μm long. Hymenophoral trama bilateral, of the boletus type; hyphae 5–7 µm diam. Cheilocystidia 31–43 × (5–)6–8 µm, 36.7 × 7 µm on average, subfusiform or narrowly fusiform, hyaline with some brown to yellowish-brown contents (5E5–8) when observed in water, no encrustations. Pleurocystidia 28–42 × 6–10 µm, abundant, 32.5 × 7.4 µm on average, subfusiform or narrowly fusiform, hyaline with some brown to yellowish-brown contents (5E5–8) when observed in water, no encrustations. Pileipellis a trichodermium about 70–120 μm thick, composed of more or less vertically arranged, interwoven, dark brown (7F8) to black hyphae in water, 4–10 μm wide; terminal cells 30–60 × 6–10 μm, narrowly clavate or subcylindrical, with some obtuse apex. Pileal trama composed of smooth hyphae 5–7 μm diam. Stipitipellis hymeniform, composed of smooth, hyaline hyphae, or dark blonde to honey yellow (5D4–6) hyphae in the reticulum, with clavate, subcylindrical, or subfusiform terminal cells; 25–30 × 10–13 μm and occasionally with clavate, four-spored basidia. Stipe trama composed of cylindrical, smooth, parallel hyphae 4–6 μm diam. Clamp connections not seen in any tissue.
Habitat: Solitary on the forest ground, near Pinus kesiya Royle ex Gordon.
Known distribution: Japan (Hongo 1974), southwestern China, and northern Thailand.
Material examined: THAILAND Chiang Rai Province, Mae Fah Luang District, 17 Jul 2014, Benjarong Thongbai, OR0738 (MFLU14-0460).
Notes: Retiboletus fuscus can be recognized by its greyish brown or dark grey pileus, dull yellowish white context sometimes slightly yellowing when bruised, concolorous stipe with darker reticulum on surface, and off-white mycelium at base. Zeng et al. (2016), elevated Boletus griseus var. fuscus Hongo to species rank based on a multi-gene (LSU, ITS, tef1) phylogeny. The macro- and micro-morphological characters of the specimen from Thailand were similar to R. fuscus from China and Japan. The combined phylogenetic analyses indicated the sequences from the Thai specimen clustered with those of R. fuscus from China with BS = 100%, PP = 1.0. Zeng et al. (2016) reported the species from Fagaceae forests. In Thailand, the specimen was collected in the vicinity of P. kesiya trees.
Retiboletus nigrogriseus N.K. Zeng, S. Jiang & Zhi Q. Liang Figs. 6, 7.
MycoBank no.: MB 825542, Faces of Fungi FoF 08037.
Macromorphology: Basidiomata medium to large-sized. Pileus 60–115 mm diam, convex when young, applanate to slightly plano-concave in age; surface dry, finely tomentose, brownish grey (5C2) to black with patches of light brown (6D7) or white; margin straight to slightly reflexed; context 10–20 mm thick at center, 5–7 mm thick half-way between center and margin, white to yellowish white, bruising greyish (1B1) then slowly violaceous grey (15D2), with a yellowish to greenish tinge above stipe. Hymenophore poroid, adnate at first, then sinuate to sinuate and narrowly decurrent; tubes 3–7 mm long, white to yellowish white, bruising greyish (1B1) then slowly violaceous grey (15D2); pores 0.1–0.4 mm diam, angular, irregular surface, white, bruising dark grey (1F1). Stipe 65–85 mm long, 10–29 mm wide, central, solid, equal or tapering upwards to clavate or sub-bulbous, sometimes curved, densely reticulate over the upper 2/3; surface dry, background color cream (4A3) or white around the apex, darker towards the base, with more or less pronounced yellow to pale orange stains; reticulum pale grey (1B2), dark brown (6F7) or black; context fleshy fibrous, marbled white to yellowish white bruising greyish white (1B1) or olive green (3D7) tinged with silver white to greyish yellow (2B3–5), with some yellow or orange-yellow stains on the sides and base of stipe. Basal mycelium white. Sporeprint not obtained. Odor fungoid-sclerodermatoid. Taste slightly bitter.
Macrochemical reactions. pileipellis and stipitipellis yellow to orange-yellow in KOH solution, unchanging to yellowish in NH4OH solution; pileus context and stipe context pale yellow in KOH solution, unchanging to yellowish in NH4OH solution.
Micromorphology: Basidiospores [285/5/5] (9–)10–12(–13.5) × 4–5 µm, 10.95 × 4.52 µm on average, Q = (1.9–)2.1–2.7(–3), 2.42 on average, elliptical to subfusiform, greenish grey (1C2) in water and KOH, smooth. Basidia 26–40(–41) × (8–)9–11(–12) µm, 33.2 × 10 µm on average, clavate, four-spored, hyaline in KOH; sterigmata 3–5 μm long. Hymenophoral trama bilateral of the boletus type, composed of hyphae 4–10 μm in wide, hyaline with some rust-brown (6E8) hyphae in KOH. Cheilocystidia (35–)36–63 × (7–)8–13 µm, 50.9 × 11.4 µm on average, abundant, ventricose or subfusiform, usually with reddish brown to dark brown (7E–F8) intracellular pigments, without encrustations. Pleurocystidia same as cheilocystidia. Pileipellis a trichodermium about 100–150 μm thick, composed of hyphae 6–10 μm wide, greyish yellow (4C5) to reddish brown (8E8) in KOH; terminal cells 17–50 × 5–10 μm, subfusiform, clavate, or subcylindrical, with obtuse apex. Pileal trama made up of hyphae 5–9 μm wide, hyaline in KOH. Stipitipellis hymeniform, about 70–140 μm thick, composed of emergent hyphae, golden (4C6) to raw umber (5F8), or blackish in water and KOH, with 25–60 × 7–17 μm, clavate, ventricose, or subfusiform terminal cells. Stipe trama composed of longitudinally arranged, parallel hyphae, 4–10 μm wide, cylindrical, almost hyaline in KOH, brown to mustard brown (5E5–6) in water and ammonium solution. Clamp connections not seen in any tissue.
Habitat: Solitary to gregarious on the ground in forests with Lithocarpus spp., Castanopsis spp., Shorea spp., and Dipterocarpus spp.
Known distribution: Southern China and Thailand.
Material examined: THAILAND. Chiang Mai Province, Mae Taeng District, 30 Jun 2010, O. Raspé & S.C. Karunarathna, OR0049 (MFLU10-0881); —Pa Pae, 9 Jul 2010, O. Raspé & S.C. Karunarathna, OR0108 (MFLU10-0940); —, 12 Jul 2010, O. Raspé & S.C. Karunarathna, OR0127 (MFLU10-0959); —, 22 Jun 2018, B. Chuankid, BC0178 (MFLU18-1772) and BC0179 (MFLU18-1773); —, Mueang District, Doi Suthep-Pui National Park, 18°47’16’’N, 98°55’25’’E, elev. 860 m, 14 Aug 2015, S. Vadthanarat, SV0218 (BKF, SDBR-CMU).
Note: Macro- and micro-morphology of R. nigrogriseus specimens from Thailand match the description of the specimens from China (holotype, FHMU 2800), except for the pileipellis structure, which was reported to be a subcutis by Zeng et al. (2018), whereas we observed a trichodermium. This discrepancy may be due to observations being made on basidiomes at different stages of development. It is indeed common in boletes to have a trichodermium in young basidiomes which more or less collapses and may look like a subcutis with age. In addition, R. nigrogriseus is characterized by its pileus and stipe context changing brownish to fuliginous when injured, stipe with grey to black reticulum and white basal mycelium. When observed in KOH, greyish yellow to reddish brown pigments were found in terminal cells of the pileipellis. The hymeniform stipitipellis is composed of emergent hyphae, golden to raw umber, or blackish pigments in water and KOH.
In the phylogenetic analyses of Zeng et al. (2018), a collection from Thailand (OR049) was included, which seemed to be related to R. nigrogriseus, but they did not study the morphology of this collection. The sequences of OR049 were first published in Raspé et al. (2016), who also did not give a morphological description of the material. Here, we provide both three-gene phylogenetic evidence and morphological evidence to support the identification of the original material from Thailand as R. nigrogriseus.
The combined sequences data supported R. brevibasidiatus as distinct from other Retiboletus species included in the tree (Fig. 1). Our molecular data also allowed identification of other collections from Thailand as R. fuscus and R. nigrogriseus, which were previously described from Japan and China, respectively. The collection labelled as “OR049_R. nigerrimus” in Raspé et al. (2016) from Thailand is here confirmed as R. nigrogriseus. Retiboletus brevibasidiatus, R. kauffmanii, and R. sinensis formed a highly supported clade (BS = 95%, PP = 0.99).
Zeng et al. (2016) stated that the species with dark grey to black pileus and white basal mycelium were grouped in the same clade (R. nigerrimus and R. zhangfeii, clade I), whereas the species with brown pileus and white basal mycelium were grouped in another clade (R. griseus, R. fuscus and R. pseudogriseus, clade II). The species with yellow basal mycelium, including the type species of the genus, were in the third clade (R. kauffmanii, R. ornatipes, R. retipes, and R. sinensis, clade III). In our phylogenetic tree, however, R. griseus was part of a well-supported clade (BS = 96%, PP = 0.99) with R. nigrogriseus and R. zhangfeii. The claim by Zeng et al. (2016) that clades I and II are characterized by ‘black’ and brown pileus, respectively, seems artificial to us. Indeed, R. fuscus R. griseus, and R. pseudogriseus, which belonged to clade II, were said to have greyish (R. griseus) or greyish brown to greyish black (R. pseudogriseus and R. fuscus) pileus in the key (Zeng et al., 2016). Therefore, all the species in that clade may have grey or dark grey (blackish) pileus. Also, because clade II of Zeng et al. (2016) was relatively weakly supported (BS = 84%, PP < 0.9) and most nodes within it also not or weakly supported, we prefer to lump clade I and II of Zeng et al. (2016) in a single clade (clade 1 in Fig. 1) characterized by white basal mycelium. Our clade 2, which includes the type species R. ornatipes, has the following morphological synapomorphies: yellow basal mycelium and deep yellow context, at least in the stipe. The color of basal mycelium, stipe context, and to a lesser extent of the pileus, are therefore useful for separating Retiboletus species.
Retiboletus ornatipes/retipes were described from Central and North America (Binder & Bresinsky, 2002; Ortiz-Santana et al., 2007; Zeng et al., 2016) while R. kauffmanii and R. sinensis were both described from China, and R. brevibasidiatus from Thailand. As previously found in a number of ectomycorrhizal mushroom genera, including bolete genera, specimens from different continents that were previously thought to be conspecific were frequently shown to belong to different species (e.g., Feng et al., 2012; Badou et al., 2018; Davoodian et al., 2018; Delgat et al., 2019). Biogeographical evidence could, therefore, also be useful to separate species when morphological differences are limited. Ecological evidence such as elevation could also be used to separate species, since R. kauffmanii was found at high elevations (over 2,000 m), whereas R. brevibasidiatus was found between 700 and 1,500 m. However, more data are needed to confirm the specificity of this ecological character and its usefulness for species identification.
The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the countries where they were performed.
The authors are grateful to Wim Baert and Myriam de Haan for their help in the molecular laboratory. Thanks to Dr. Naritsada Thongklang for her help and encouragements. Our thanks are due to the Research and Researchers for Industries grant (PHD57I0015) for financial support to BC and OR. We acknowledge the National Parks authorities of Thailand for granting the permit number 0907.4/4769 for collecting in Doi Suthep-Pui National Park to SV, OR, and SL. Thanks to Shaun Pennycook for checking the novel species epithet. Finally, we would like to thank Manfred Binder for allowing us to study his collections of R. ornatipes and R. griseus.
