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Research Article
New species of Retiboletus (Boletales, Boletaceae) from China based on morphological and molecular data
expand article infoHai-Ying Liu, Yan-Chun Li§, Tolgor Bau
‡ Jilin Agricultural University, Changchun, China
§ Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
Open Access

Abstract

Species of the genus Retiboletus in China were investigated based on morphology and phylogenetic analysis of DNA sequences from the nuclear ribosomal large subunit (nrLSU) and the translation elongation factor 1-α gene (TEF1-α). Nine species were recovered from China, including two new and seven known species. The new species, namely Retiboletus ater and R. sinogriseus, from southwestern and northeastern China respectively, are documented and illustrated in this paper. Retiboletus ater is morphologically characterized by its black to grayish black pileus, white to grayish hymenophore, black to blackish stipe and white to grayish white context. Retiboletus sinogriseus is morphologically characterized by its brown to grayish-brown pileus, yellow to grayish-yellow hymenophore, pale yellow to brownish stipe and yellow to brownish-yellow context. Descriptions and line drawings of these two novel species and their comparisons with allied taxa are presented.

Keywords

Boletes, morphology, new taxa, phylogeny, taxonomy

Introduction

The genus Retiboletus Manfr. Binder & Bresinsky was erected to accommodate Boletus ornatipes Peck and allied species (Binder and Bresinsky 2002). The genus is morphologically characterized by the combination of the following characters: pileus convex to plane, dry, subtomentose, black, dark gray, mustard yellow or olive-brown; hymenophore pallid, grayish or yellow, unchanging or staining brown or orange-brown when bruised; hymenial cystidia present; stipe reticulate; context pallid, yellow or vivid yellow, unchanging or bruising orange-brown; clamp connections absent; spore deposit olive-brown to yellow-brown, basidiospores smooth, ellipsoid to subfusoid, inamyloid and partly dextrinoid (Binder and Bresinsky 2002; Zeng et al. 2016). The separation of Retiboletus from Boletus s. str. and its establishment at the generic rank is strongly supported (Wu et al. 2016; Zeng et al. 2016, 2018; Badou et al. 2018). So far thirteen species of this genus have been described from North/Central America and East Asia, seven out of which have been reported from China (Binder and Bresinsky 2002; Wu et al. 2016; Zeng et al. 2016, 2018).

During field investigation of Boletaceae across China, we encountered two impressive Retiboletus species from southwestern and northeastern China, respectively. These species can be easily recognized by their conspicuous colors in the field. Molecular phylogenetic analysis of this genus based on the nuclear ribosomal large subunit (nrLSU) and the translation elongation factor 1-α gene (TEF1-α) indicated that they represent two distinct species. Combined with morphological characters, Retiboletus ater and R. sinogriseus, are proposed and described herein. It is noteworthy that an additional collection from northeastern China, labeled R. aff. kauffmanii (HY56), was included in our molecular phylogenetic analysis. But its classification can’t be clarified due to its paucity of mature material. Further collections are needed to better estimate its taxonomic status.

Materials and methods

Morphological studies

Specimens were described and photographed in the field and deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN) and Herbarium of Jilin Agriculture University (HMJAU). In the descriptions, macroscopic characters were based on field notes and color slides of the specimens. Color codes are from Kornerup and Wanscher (1981). Microscopic characters were from the observations of the specimens through light microscopy. For microscopic study, dried materials were sectioned and mounted in 5% KOH solution. Sections of the pileipellis were cut tangentially and halfway between the center and margin of the pileus. All measurements were made in KOH mounts and observed under the light phase. All line drawings of microstructures were made from rehydrated material. Melzer’s reagent was used for testing color reactions of the tissue fragments to the solution. The notations “basidiospores (n/m/p)” indicate that the measurements were made on n basidiospores from m basidiomata of p collections. The expressions (a)b–c(d) stand for the dimensions of basidiospores; the range b–c contains a minimum of 90% of the measured values, a and d in the brackets stand for the extreme values. The following abbreviations are used: Q (length/width ration of basidiospores) and Qm (average Q ± standard deviation).

DNA extraction, PCR and DNA sequencing

Protocols for DNA extraction, PCR, sequencing and sequence alignment followed those in Vadthanarat et al. (2019), Gelardi et al. (2019), Zhang et al. (2019) and references therein. The primer pair used for amplifying the nrLSU region was LROR and LR5 (Vilgalys and Hester 1990). DNA sequences were compiled with SeqMan (DNASTAR Lasergene 9). Sequences were aligned with MUSCLE 3.6 (Edgar 2004) and manually adjusted where necessary. Edited sequences were deposited in GenBank (Table 1).

Phylogenetic analysis

19 sequences (10 of nrLSU and 9 of TEF1-α) from 10 collections were newly generated in this study and aligned with selected sequences from GenBank and previous studies (Binder and Bresinsky 2002; Ortiz-Santana et al. 2007; Zeng et al. 2016, 2018) (Table 1). Boletus edulis Bull. and Boletus reticuloceps (M. Zang, M.S. Yuan & M.Q. Gong) Q.B. Wang & Y.J. Yao were chosen as outgroup. The combined nuclear dataset (nrLSU + TEF1-α) was analyzed with maximum likelihood (ML). Maximum-likelihood tree generation and bootstrap analysis were performed with the program RAxML 7.2.6 (Stamatakis 2006) running 1000 bootstrap replicates combined with a ML search.

Table 1.

Specimens used in molecular phylogenetic study and their GenBank accession numbers.

Species Voucher Locality Accession Reference
nrLSU TEF1-α
Retiboletus ater Li1215 SW China MT010611 MT010621 This study
R. ater Li1224 SW China MT010612 MT010622 This study
R. brunneolus LC_LJW237 SW China MT010615 MT010625 This study
R. brunneolus Li993 SE China KF112424 KF112179 Wu et al. 2016
R. flavoniger RH7247 Costa Rica AF456828 Binder and Bresinsky 2002
R. flavoniger RH7189 Costa Rica AF456829 Binder and Bresinsky 2002
R. fuscus Wu445 SW China KT990636 KT990830 Wu et al. 2016
R. fuscus Cui47 SW China MT010614 MT010624 This study
R. griseus BD210 USA HQ161858 Binder and Bresinsky 2002
R. griseus snBoth USA KF030308 KF030414 Binder and Bresinsky 2002
R. griseus Halling10162 USA MT010608 MT010618 This study
R. kauffmanii Wu317 SW China KP739282 KP739301 Zeng et al. 2016
R. nigerrimus Tyni1 Japan AF456832 Binder and Bresinsky 2002
R. nigrogriseus FHMU2045 Southern China MH367475 MH367487 Zeng et al. 2018
R. nigrogriseus FHMU2800 Southern China MH367476 MH367488 Zeng et al. 2018
R. ornatipes 201/97 USA AF456815 Binder and Bresinsky 2002
R. ornatipes Halling10163 USA MT010617 MT010626 This study
R. ornatipes 161/97 USA AF456817 Binder and Bresinsky 2002
R. pseudogriseus Zeng647 Southern China MT010613 MT010623 This study
R. pseudogriseus FHMU375 Southern China MH367477 MH367489 Zeng et al. 2018
R. pseudogriseus Zeng668 SE China KP739285 Zeng et al. 2016
R. retipes 96/97 USA AF456830 Binder and Bresinsky 2002
R. retipes 22/97 USA AF456831 Binder and Bresinsky 2002
R. retipes 116/96 USA AF456823 Binder and Bresinsky 2002
R. retipes 57/97 USA AF456811 Binder and Bresinsky 2002
R. sinensis Zeng1299 SE China KP739291 KP739303 Zeng et al. 2016
R. sinensis Zeng1278 SE China KP739289 KP739302 Zeng et al. 2016
R. sinensis Zeng569 Southern China KP739286 Zeng et al. 2016
R. sinogriseus LJ258 NE China MT010610 MT010620 This study
R. sinogriseus LJ260 NE China MT010609 MT010619 This study
R. sp. CAL_F_1397 India KY290586 GenBank
R. aff. kauffmanii HY56 NE China MT010616 This study
R. vinaceipes CFMR:DR-1035 Dominican Republic MN250180 Ortiz-Santana et al. 2007
R. vinaceipes CFMR:BZ-2386 Belize MN250190 Ortiz-Santana et al. 2007
R. zhangfeii Li1951 SE China JQ928627 JQ928582 Wu et al. 2016
R. zhangfeii Li1073 SE China KT990630 KT990824 Wu et al. 2016
Boletus edulis HMJAU4637 NE China KF112455 KF112202 Wu et al. 2016
B. reticuloceps Liang521 SW China KT990537 KT990739 Wu et al. 2016
Pseudoaustroboletus valens LF690 Southern China KM274870 KM274878 Li et al. 2014
P. valens Li915 SE China KM274869 KM274877 Li et al. 2014

Result

Molecular analysis

The combined nuclear dataset (nrLSU + TEF1-α) consists of 64 sequences and is 1526 bp long. The alignment was submitted to TreeBASE (S25798). Phylograms with branch lengths inferred with RAxML, including the support values, are illustrated (Fig. 1). The monophyly of Retiboletus was moderately supported (bootstrap = 56%) in our analysis (Fig. 1). Two new species were recovered within Retiboletus, including two collections of R. ater from southwestern China and two collections of R. sinogriseus from northeastern China. Phylogenetically, sequences of R. ater form a unique lineage with 100% bootstrap support, while R. sinogriseus is closely related to R. griseus with high bootstrap support (99%). The collection labeled R. aff. kauffmanii (HY56) from northeastern China, clusters together with R. kauffmanii and another Indian species labeled Retiboletus sp. (CAL_F_1397).

Figure 1. 

Maximum likelihood phylogenetic tree of Retiboletus inferred from the combined nuclear dataset (nrLSU + TEF1-α). Bootstrap frequencies (> 50%) are shown above supported branches. Newly sequenced collections are boldfaced in black. Species vouchers are provided after the species name.

Taxonomy

Retiboletus ater Yan C. Li & T. Bau, sp. nov.

MycoBank No: 834293
Figures 2a–c, 3

Etymology

ater referring to the color of the basidiomata.

Type

China. Yunnan Province: Jingdong County, Ailaoshan National Nature Reserve, alt. 2500 m, 14 July 2008, Y.C. Li 1215 (holotype: KUN-HKAS 56069!).

Figure 2. 

Habitat of the new Retiboletus species. a–c R. ater (from KUN-HKAS 56069) d–f R. sinogriseus (d from KUN-HKAS 91288 e–f from KUN-HKAS 91286).

Description

Basidiomata small to medium-sized. Pileus 3–5 cm in diameter, hemispherical to applanate, surface dry, densely subtomentose, black (4F3) to blackish (4E2) in the center and gray (3D1) or yellowish-gray (3C2-3) towards margin, context 2.5 cm thick in the center of the pileus, pallid gray (2D1) to cream (2C3-4), unchanging when bruised. Hymenophore adnate or slightly depressed around apex of stipe; pores angular, tubes up to 11 mm long, 0.3–1 mm wide, white (2B1) when young and yellowish (2A2) in age, becoming brownish-yellow (5C7-8) when injured. Stipe 4–6 × 0.8–1.2 cm, clavate to flexuous, solid; surface dry, blackish to gray, prominently and coarsely reticulate over the upper 1/3; context white (2A1) in the upper part and yellowish to cream yellow downwards, unchanging when injured; basal mycelium white (2A1). Taste and odor indistinct.

Basidia 26–38 × 6–10 μm, clavate, thin-walled, 4-spored, hyaline to yellowish in KOH. Basidiospores [60/3/2] (7)8–10.5(11) × 3–4.5(5) μm [Q = (1.89) 2–3.33 (3.67), Qm = 2.52 ± 0.42], subfusiform and inequilateral in side view with shallow suprahilar depression, elongate fusoid or narrowly oblong in ventral view, slightly thick-walled (up to 0.5 μm), brownish to yellowish-brown in KOH, olive-brown to brown in Melzer’s reagent, smooth. Hymenophoral trama boletoid; hyphae cylindrical, 3.5–9 μm wide, hyaline to yellowish in KOH, yellowish to brownish-yellow in Melzer’s reagent. Cheilo- and pleurocystidia 26–55 × 6–10 μm, abundant, subfusiform to fusiform, thin-walled, with yellowish-brown contents, surface without encrustations. Caulocystidia forming the reticulum over the stipe surface, similar to cheilo- and pleurocystidia. Pileipellis a trichoderm about 280 μm thick, composed of more or less vertically arranged, slightly interwoven, brown to dark brown hyphae, 5–15 μm wide; terminal cells 45–111 × 9–15 μm, narrowly clavate to subcylindrical or subfusiform, sometimes narrowly mucronate, rostrate, slightly thick-walled (up to 0.5 μm), hyaline to yellowish in KOH, yellowish to brownish-yellow in Melzer’s reagent. Pileal trama composed of thin- to slightly thick-walled (up to 0.5 μm) hyphae, 5–11 μm wide, hyaline to yellowish in KOH, yellowish to brownish-yellow in Melzer’s reagent. Clamp connections absent in all tissues.

Figure 3. 

Microscopic features of R. ater (KUN-HKAS 56069). a Basidiospores b basidia and pleurocystidium c cheilo- and pleurocystidia d pileipellis. Scale bars: 10 μm.

Habitat, ecology and distribution

Solitary on the ground in forests dominated by plants in the family Fagaceae; currently known from southwestern China.

Additional specimens examined

China. Yunnan Province: Jingdong County, Ailaoshan National Nature Reserve, alt. 2500 m, 14 July 2008, Y.C. Li 1224 (KUN-HKAS 56078).

Discussion

Retiboletus ater is characterized by the black to blackish or gray to yellowish-gray pileus, the white to yellowish hymenophore, the gray to brownish-gray stipe, the prominent and coarse reticulum over the upper 1/3 of the stipe and the trichoderm pileipellis with hyphae 9–15 μm wide. It generally shares the same colored pileus and hymenophore with R. fuscus (Hongo) N.K. Zeng & Zhu L. Yang, R. griseus (Frost) Manfr. Binder & Bresinsky, R. nigrogriseus N.K. Zeng, S. Jiang & Zhi Q. Liang, and R. pseudogriseus N.K. Zeng & Zhu L. Yang. However, R. fuscus is characterized by an overall reticulate stipe, slight longer basidiospores (9–12 × 3.5–4.5 μm) and narrower pileipellis hyphae (4–8 μm wide) (Zeng et al. 2016). Retiboletus griseus has a reticulum over the upper 2/3 of the stipe, a cream or grayish-brown stipe often with orange-yellow stains when hurt, and a distribution in North/Central America (Smith and Thiers 1971; Ortiz-Santana et al. 2007). Retiboletus nigrogriseus is characterized by the white to olivaceous contex in the stipe, the entirely reticulate stipe and the cutis pileipellis with hyphae 4–10 μm wide. Retiboletus pseudogriseus has a grayish white pileus which is covered with brown to blackish brown squamules, white context becoming brown when injured, and a slender and completely reticulate stipe.

In the phylogenetic analysis (Fig. 1), R. ater forms an independent lineage within Retiboletus, future studies would require more molecular sequence data to help fully resolve its evolutionary relationships to the other species.

Retiboletus sinogriseus Yan C. Li & T. Bau, sp. nov.

MycoBank No: 834294
Figures 2d–f, 4

Etymology

sino (Latin) = China, reflecting that the basidiomata were collected in China + griseus for similarity of the basidiomata of this species to Retiboletus griseus.

Type

China. Liaoning Province: Anshan City, Qianshan, alt. 400 m, 25 Aug 2015, J. Li 260 (holotype: KUN-HKAS 91288!).

Figure 4. 

Microscopic features of R. sinogriseus (KUN-HKAS 912889). a Basidiospores b basidia and pleurocystidium c pleurocystidia d cheilocystidia e pileipellis. Scale bars: 10 μm.

Description

Basidiomata medium-sized. Pileus 6–7.2 cm in diameter, subhemispherical to applanate, sometimes convex; surface tomentose, grayish-brown (5D2-3) to brown (5E4), rimose when dry, always cracked into small squamules on grayish (4B1) to whitish (2A1) background; context 1–2 cm thick in the center of pileus, white (1A1), unchanging when injured. Hymenophore adnate, sometimes slightly depressed around apex of stipe; pores angular, 0.3–1 mm wide, tubes up to 14 mm long, yellow (4C3-4) to grayish-yellow (4B2-3), unchanging when injured. Stipe 6–8 × 1.1–1.5 cm, subcylindrical, solid; surface dry, pale yellow at apex, blackish-yellow towards the base, entirely covered with moderately developed reticulum; context white to cream in the upper part and yellowish to yellow downwards, unchanging when injured; basal mycelium yellow. Taste and odor indistinct.

Basidia 21–27 × 9–11 μm, clavate, thin-walled, four-spored; sterigmata 4–5 μm long. Basidiospores [40/2/2] (9) 10.0–13.5 (–14.0) × (3) 4.0–5.0 (–5.5) μm, Q = (2.25–) 2.5–3.25 (–3.42), Qm = 2.88 ± 0.32, subfusiform to ellipsoid, slightly thick-walled (up to 0.5 μm), hyaline to yellowish in KOH, olive-brown to yellowish-brown in Melzer’s reagent, smooth. Hymenophoral trama boletoid. Cheilo- and pleurocystidia 35–56 × 7–12 μm, abundant, subfusiform to fusiform, thin-walled, with yellowish-brown to brown contents, without encrustations. Pileipellis a subcutis, 100–120 μm thick, composed of thin-walled filamentous hyphae 4–7 μm wide, with subcylindrical to clavate terminal cells 33–72 × 4–6 μm, sometimes with subacute apex, colorless to pale yellowish-brown in KOH, yellow-brown to brownish in Melzer’s reagent. Pileal trama composed of thin-walled hyphae 4–9 μm wide, colorless to pale yellowish-brown in KOH, yellow-brown to brownish in Melzer’s reagent. Clamp connections absent in all tissues.

Habitat, ecology and distribution

Solitary on the ground in mixed forests dominated by plants in the families Fagaceae and Pinaceae; currently known from northeastern China.

Additional specimens examined

China. Liaoning Province: Anshan City, Qianshan, alt. 400 m, 25 Aug 2015, J. Li 258 (KUN-HKAS 91286).

Discussion

Retiboletus sinogriseus has a grayish-brown to brown pileus, a pale yellow to blackish-yellow stipe. Such traits are very similar to those of R. griseus. Interestingly, R. sinogriseus clusters with R. griseus with strong statistical support (Fig. 1). However, R. griseus, originally described from North America but not found in China yet, has a distinctly pallid hymenohore and broad pileipellis hyphae which are up to 17 μm wide (Singer 1947; Smith and Thiers 1971; Ortiz-Santana et al. 2007). Additionally, the R. sinogriseus/R. griseus clade is clustered with R. zhangfeii N.K. Zeng & Zhu L. Yang, R. nigrogriseus and R. nigerrimus (R. Heim) Manfr. Binder & Bresinsky (however without bootstrap support). In this assemblage, R. zhangfeii differs significantly from R. sinogriseus by its differently colored pileus, hymenophore, stipe and context (Zeng et al. 2016). Retiboletus nigrogriseus has a black to gray pileus, white to grayish white hymenophore, white to olivaceous contex in the stipe and much smaller basidiospores 8–10.5 × 3.5–4.5 μm. Retiboletus nigerrimus, originally described from Papua New Guinea, has a pileus with a distinctive blue tinge, a context lemon yellow in pileus and orange in the base of stipe and longer and narrower basidiospores 11.5–14.5 × 3.6–4.6 μm (Heim 1963).

Nine species of Retiboletus were recorded from China, including two new species described herein. For the convenience of identification, a key to the species in China is given below.

Key to Retiboletus species in China

1 Hymenophore bright yellow to brownish-yellow, stipe yellow to orange-yellow, mycelium on the base of stipe yellow to brownish-yellow 2
Hymenophore whitish to grayish white, stipe black to blackish or grayish-black, mycelium on the base of stipe whitish to grayish-white 4
2 Pileus grayish-brown to brown without olivaceous tinge, context in pileus white to grayish-white R. sinogriseus
Pileus yellow-brown to olive-brown, context in pileus yellow to pale yellow 3
3 Basidiomata medium-sized to large, pileus up to 15 cm in diameter, basidiospores 9–13 × 4–5 μm, cheilo- and pleurocystidia 30–60 × 6–10 μm R. kauffmanii
Basidiomata small to medium-sized, pileus up to 8 cm in diameter, basidiospores 8–11 × 3.5–4 μm, cheilo- and pleurocystidia 20–46 × 4.5–7 μm R. sinensis
4 Context in the stipe white to grayish-white with olivaceous tinge 5
Context in the stipe white to grayish-white with grayish-yellow tinge 6
5 Hymenophore white when young, lilac to purplish when old, basidiospores 9–11 × 4–5 μm R. zhangfeii
Hymenophore white to grayish-white without lilac to purplish tinge, basidiospores relatively small 8–10.5 × 3.5–4 μm R. nigrogriseus
6 Stipe entirely reticulate 7
Stipe without reticulum or with reticulum restricted to the upper part 8
7 Pileus brown to blackish-brown, basidiospores 9.5–11 × 4–4.5 μm, pileipellis hyphae up to 8 μm wid R. pseudogriseus
Pileus grayish-brown to grayish-black, basidiospores slightly narrower 9–12 × 3.5–4 μm, pileipellis hyphae broad up to 13 μm wide R. fuscus
8 Pileus pale brown to grayish-brown, stipe without reticulum, basidiospores 10–12.5 × 4.5–5 μm, pleurocystidia 50–80 × 9–14.5 μm R. brunneolus
Pileus black to blackish, stipe covered with reticulum over the upper 1/3, basidiospores much smaller 8–10.5 × 3–4.5 μm, pleurocystidia relatively small 26–55 × 6–10 μm R. ater

Acknowledgements

The authors are indebted to Dr. Jing Li (Kunming Institute of Botany, Chinese Academy of Sciences) for her kind help in collecting the specimens. They are grateful to Prof. Roy E. Halling (the New York Botanical Garden) for providing American specimens. The anonymous reviewers are acknowledged for their valuable comments and suggestions. This study was supported by the Funds of the National Natural Science Foundation of China (31570025, 31750001, 31872618), the Youth Innovation Promotion Association, CAS (2016348), the Key Research Program of Frontier Sciences of CAS (QYZDY-SSW-SMC029-5) and the Ten Thousand Talents Program of Yunnan (YNWR-QNBJ-2018-125).

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