First Report of Buchwaldoboletus lignicola (Boletaceae), a Potentially Endangered Basidiomycete Species, in South Korea

Abstract

During the 2014 survey of the mushroom flora of Gwangneung forest in South Korea, we collected two specimens of boletoid mushroom growing on a felled tree of Pinus koraiensis. These specimens were characterized by a light brown to reddish-brown pileus with appressed tomentum, pore surface bluing instantly when bruised, golden-yellow mycelium at the base of stipe, and lignicolous habitat. Both specimens were identified as Buchwaldoboletus lignicola, a rare basidiomycete, based on morphological characteristics and sequences of internal transcribed spacer (ITS; fungal barcode). Here, we describe these specimens and provide the first report of this genus in South Korea.

Boletaceae Chevall. is a family from the order Boletales in Basidiomycota and is comprised of boletoid fruitbodies with pores. There are about 50 recognized genera and 800 species globally [1]. Most species in this family are known to form ectomycorrhizal associations with various trees. However, some of the basal genera of Boletaceae, such as Buchwaldoboletus Pilát, are saprophytic and lignicolous. Currently, the genus Buchwaldoboletus consists of 13 species and is divided into three groups, namely Lignicola, Sphaerocephalus, and Hemichrysus [2–5]. Buchwaldoboletus lignicola is the type species of Buchwaldoboletus and was first described by Kallenbach in 1929, as Boletus lignicola. Later, Pilát (1969) separated this species from Boletus and described a new genus Buchwaldoboletus, based on its decurrent and arcuate hymenophore, lack of veil, stipe with yellow mycelium, and saprophytic habit. This species is known to be distributed in South and East Asia, North America, Europe, and North Africa [3,6–10].

As part of the surveys of macrofungi in the unexploited areas of Korea, the project funded by the Korea National Arboretum, we encountered two rare boletoid mushrooms in Gwangneung Forest. They were identified as B. lignicola based on their morphological features and phylogenetic analysis of the internal transcribed spacer (ITS) region. It is the first report of this species in Korea. Here, we provide detailed morphological characteristics of B. lignicola and present new locality (South Korea) and a host plant (Pinus koraiensis Siebold & Zucc.) of the species.

Two Buchwaldoboletus fruiting bodies were collected at Gwangneung forest, Pocheon-si, Gyeonggi Province, South Korea in 2014. They were dried and deposited at the herbarium of the Korea National Arboretum. Macro-morphological features were determined based on field notes and color photos of fresh specimens. Micro-morphological features were observed from dried specimens after sectioning and mounting in 3% KOH solution. Basidia and cystidia were stained with a solution of 1% Congo Red under a light microscope (Olympus BX53, Tokyo, Japan). Measurements of microscopic characters were obtained using ProgRes Capture Pro v.2.8.8 (Jenoptik Co., Jena, Germany). For basidiospore descriptions, the measured numbers of basidiospore, the number of basidiomata, and the number of collection sites were respectively denoted with the abbreviation [n/m/p]. In describing basidiospore dimensions, we used the notation (a–) b–c (–d). The range b–c represents 95% of the measured values and “a” and “d” are lowest and highest measured values, followed by the mean spore length and width; Q is the range of the length/width ratio for all measured basidiospores; Qm is the average Q value ± sample standard deviation.

DNA was extracted from fruiting bodies using a modified CTAB procedure [11]. For the amplification of the ITS region, primer sets ITS5 and ITS4 were used in PCR mixture of 0.5 pM of each primer, 0.25 mM dNTPs, 1.5 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl, 2.5 U of Taq DNA polymerase, and 15 ng of template DNA [12]. PCR conditions for ITS were as follows: an initial denaturation step at 94 °C for 4 min, followed by 34 cycles of 94 °C for 40 s, 52 °C for 40 s, and 72 °C for 60 s; and a final elongation step at 72 °C for 8 min. PCR products were purified using an ExoSAP-IT PCR Product Cleanup Reagent (USB, Cleveland, OH). The PCR products were directly sequenced using a BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with the manufacturer’s instructions. The same primers used for PCR were employed for sequencing. Capillary electrophoresis and data collection were performed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). We edited the sequences using PHYDIT v. 3.2 [13] and deposited them to GenBank.

DNA sequences of other reported Boletaceae species were obtained from GenBank for phylogenetic analyses (Table 1). They were aligned with DNA sequences generated for this study using ClustalX v. 1.81 [14]. Ambiguously aligned positions were adjusted manually using PHYDIT. The constructed datasets were analyzed with the algorithms of maximum parsimony (MP) using PAUP* v. 4.0 b10 and Bayesian inference using MrBayes v. 3.1.2 [15,16]. Parsimony analysis was performed with a heuristic search of 1000 random addition replicates and tree bisection-reconnection branch-swapping. MP bootstrap support values (MPBS) were assessed to evaluate the supports for internal nodes from 1000 replicates of the MP analysis. Posterior probabilities (PPs) were calculated using the Metropolis-coupled Markov Chain Monte Carlo method. Two parallel runs were performed with one cold and three heated chains for 3 million generations, starting with a random tree with sampling every 100th generation. We assessed the convergence of two independent runs to remove the trees which were not in convergence with a criteria of the average standard deviation of the split frequencies being below 0.01 using the burn-in command. The remaining trees which were converged used in calculating a 50% majority consensus tree and estimating PP. The distance matrix was calculated using PHYDIT with Kimura-2-Parameter distance method.

Table 1.

Information of ITS sequences used in this study.

Species	Voucher	Locality	GenBank accession
Aureoboletus tenuis	GDGM:32601	China	KF265358
Aureoboletus venustus	HKAS:77700	China	KU321702a
Boletus hiratsukae	TMI 18352	Japan	NR_119672a
Boletus nobilissimus	BUF Both4244	USA	NR_119671a
Boletus rubriceps	SFSU Arora11340	USA	NR_137806a
Borofutus dhakanus	HKAS 73785	Bangladesh	NR_120117a
Borofutus dhakanus	CMU-ST58-001	Thailand	KU168045
Buchwaldoboletus lignicola	KM157323	England	GQ981493
Buchwaldoboletus lignicola	–	Italy	HM003619
Buchwaldoboletus lignicola	–	Sweden	HM003618
Buchwaldoboletus lignicola	–	Scotland	HM003617
Buchwaldoboletus lignicola	3533	Canada	KM248950
Buchwaldoboletus lignicola	KA14-0711	South Korea	MH170896
Buchwaldoboletus lignicola	KA14-0907	South Korea	MH170897
Butyriboletus roseogriseus	PRM:923483	Czech	NR_151842a
Butyriboletus yicibus	SFSU Arora9727	China	NR_137796a
Chalciporus piperatus	K80S25b	New Zealand	GQ267470
Chalciporus piperatus	2591	Canada	KM248949
Chalciporus radiatus	GDGM50080	China	KP871806
Chalciporus radiatus	GDGM43285	China	KP871804a
Chalciporus rubinellus	2626	Canada	KM248951
Chalciporus rubinellus	191/81	USA	EU685111
Rubinoboletus rubinus	18508	UK	JF908793
Rubinoboletus rubinus	KW 50674F	Ukraine	KJ562360
Chalciporus trinitensis	18465	Guatemala	JF908790
Gyrodon lividus	17191	Italy	JF908786
Gyrodon lividus	REG Gl1	Germany	DQ534568
Harrya chromapes	–	Canada	KM248941
Harrya chromapes	ITS199	Japan	KC552019
Porphyrellus porphyrosporus	DJM1332	USA	JN021085
Pseudoboletus parasiticus	18898	Italy	JF908801
Pseudoboletus parasiticus	2164-QFB-25840	Canada	KM248932
Pulveroboletus flaviscabrosus	HKAS83190	China	KX453802a
Pulveroboletus rubroscabrosus	HKAS75537	China	KX453816a
Rubroboletus rhodoxanthus	MA-Fungi 47703	Portugal	AJ419189
Rubroboletus satanas	Bs2	Germany	DQ534567
Strobilomyces confusus	BRNM 766848	South Korea	KT121567
Strobilomyces pteroreticulosporus	BRNM 718716	South Korea	KT121565a
Strobilomyces strobilaceus	LE253886	Russia	JQ318985
Tylopilus microsporus	HMAS 84730	China	NR_137924a
Tylopilus neofelleus	YT20090720	Japan	KM975489
Tylopilus porphyrosporus	GO-2009-237	Mexico	KC152268
Xanthoconium affine	3735	USA	KM248938
Paxillus ammoniavirescens	IK-00554	Poland	KX610700
Paxillus rubicundulus	Orton:2905	United Kingdom	NR_147640a

^aType specimen.

Taxonomic description

Buchwaldoboletus lignicola (Kallenb.) Pilát, Friesia 9(1–2): 217 (1969) (Figure 1).

Figure 1.

Morphological characters of Buchwaldoboletus lignicola KA14-0711 and KA14-0907. (A–D) Fruiting bodies of B. lignicola; (E) golden-yellow mycelia at the base of stipe; (F, G) Pleurocystidia (stained with 1% Congo red solution; (H) Basidia and basidioles (stained with 1% Congo red solution); (I) Basidiospores in 3% KOH. (Scales bars: B–E = 5 cm, F–I = 10 μm).

Pileus 4–10 cm in diameter, convex with in rolled margin and covered with soft appressed tomentum, light brown to reddish-brown. Hymenophore tubulose, tubes detersible, decurrent, yellow to golden at first, then olivaceous yelllow, turning greenish-blue above tubes when cut or bruised; pores circular to angular or irregular, 1–3 per mm. Stipe 3–7 cm long, 0.6–1.7 cm thick, central or somewhat eccentric, cylindrical, sometimes base somewhat thickened, rust-yellow to reddish-brown with a golden-yellow mycelia at the base, context yellow. Partial veil absent.

Basidiospores [100/2/2] (7.0–) 7.2–8.8 (–11.2)×(3–) 3.1– 4(–5.4) µm, Q=(1.9–) 2–2.5 (–2.6), Qm = 2.22 ± 0.15, elliptic, smooth, thick-walled, pale yellow. Basidia [n = 40] 20.5–34 × 6–9 µm, clavate or broadly clavate, 4-spored, sterigmata 2–4 µm long. Pleuroystidia 28–51 × 6.3–10.2 µm, fusiform, ventricose-lageniform, often with a long rosrtrum, hyaline to yellowish in 3% KOH, thin-walled, smooth. Caulocystidia 31.4–42.5 × 5.5–8.5 µm, fusiform, fusiform to clavate, ventricose-lageniform, hyaline to yellowish in 3% KOH, thin-walled, smooth. Caulobasidia [n = 30] 22–30 × 5–9 µm, clavate, 4-spored, sterigmata 2.5–3.6 µm long. Clamp connections absent in all tissues.

Habitat: Solitary or scattered on dead conifer stump (Pinus koraiensis Siebold & Zucc, Pinaceae).

Edibility: unknown

Examined specimens: KA14-0711, Gwangneung Forest, Pocheon-si, Gyeonggi Province, Korea, July 28 2014, coll. Jo et al., KA14-0907 same place, August 13 2014, coll. Jo et al.

Remarks: Morphologically, B. lignicola is similar to B. hemichrysus, B. pseudolignicola, B. pontevedrensis, and B. xylophilus. However, B. hemichrysus has a golden-yellow pileus, red-brown to reddish-brown pore, and a ventricose stipe, and B. lignicola does not share those features [4,17,18]. Similarly, B. pseudolignicola, a member of Sphaerocephalus group, has a yellow to cinnamon-brown pileus, unlike B. lignicola, and has a smaller basidiospore than B. lignicola [4]. B. pontevedrensis is a recently described member of a group of Lignicola; it differs from B. lignicola by having a bigger pileus, shorter stipe, and slightly longer basidiospores [3]. Buchwaldoboletus xylophilus differs from B. lignicola by having shorter basidiospores [19].

The ITS dataset included 45 taxa and 795 characters, of which 364 were parsimony-informative. The MP tree was 1329 steps long with a consistency index of 0.5813, retention index of 0.6765, and homoplasy index of 0.4698. The Bayesian analyses were conducted with a model of GTR + I + G and the first 9000 trees were discarded as burn-in (burninfrac = 0.30). The phylogenetic trees of the ITS dataset in this study showed a similar main branch topology as presented in previous studies [20–24].

Boletaceae was monophyletic (Figure 2). Among the Boletaceae clade, subfamily Chalciporoideae Wu & Yang (including Buchwaldoboletus and Chalciporus) formed a basal group. This group appears to be parasitic or saprophytic. Although Chalciporoideae was only supported by MPBS (87%), the clade grouping Buchwaldoboletus and Chalciporus were strongly supported in both MPBS and PP (Figure 2). The ITS sequences of our two B. lignicola specimens (KA14-0711 and KA14-0907) formed a group with five previously reported European and Canadian B. lignicola specimens (HM003617, HM003618, HM003619, GQ981493 and KM248950) with strong support values (MPBS = 99%, PP = 0.99) (Figure 2).

Figure 2.

One of 104 most parsimonious trees from a heuristic analysis of ITS sequences. Broad black branches indicate maximum parsimony bootstrap value (MPBS) >60% and Bayesian posterior probabilities >0.95. Only MPBS values >50% are shown above or below branches. The symbol “T” indicated the type materials.

Buchwaldoboletus lignicola is a basidiomycete fungus that is distributed mainly in Europe and North America [4,25]. However, this species is rarely observed in Asia. For this reason, only a few studies and sequences have been published for B. lignicola. In Japan, three species of Buchwaldoboletus (B. xylophilus, B. pseudolignicola, and B. sphaerocephalus) have been recorded [26] excluding B. lignicola. However, there has not been a report of genus Buchwaldoboletus species in Korea. B. lignicola has been found on conifer stumps, including Larix decidua Mill., Picea abies (L.) H. Karst., Pinus sylvestris L., Pinus strobus L., and rarely on Prunus avium L. [4,7,27]. In addition to the report of the species in Korea, this study presents a new host for B. lignicola, Korean pine (Pinus koraiensis).

Some considered B. lignicola to be saprotrophic based on its lignicolous habitat [28]. Other studies, however, present that the species is often found with Phaeolus schweinitzii (Fr.) Pat., a brown-rot tree pathogen [8,29]. Additionally, Nuhn et al. [21] suggest that B. lignicola has a mycoparasitic nutritional mode, based on the confrontation assay between the hyphae of B. lignicola and P. schweinitzii. Although P. schweinitzii was not observed at the habitat of B. lignicola in this study, previous research has shown that B. lignicola can occur where P. schweinitzii occurs.

Although B. lignicola is not recorded in the IUCN Red List, the species is listed as a critically endangered species in Bulgaria [30], endangered species in the Czech Republic [31], and vulnerable in Great Britain [32]. In South Korea, nine species of macrofungi (Albatrellus dispansus, Amanita hemibapha subsp. javanica, Ganoderma neojaponicum, Grifola frondosa, Hericium coralloides, Inonotus obliquus, Lyophyllum fumosum, Oudemansiella brunneomarginata, and Phellinus linteus) are registered as protected forest species by the Korea Forest Service. Considering its rarity, B. lignicola may need to be considered as a protected forest species of South Korea in the future.

Funding Statement

This work was supported by research grants of the Korea National Arboretum [project no. KNA 1-1-25, 19-2] and the National Institute of Horticultural and Herbal Science [project no. PJ01262501].

Disclosure statement

No potential conflict of interest was reported by the authors.