Aureoboletus pseudorussellii sp. nov., A Newly Discovered Species in Genus Aureoboletus of Zhejiang Province, China

Abstract

A morphological and phylogenetic analysis has been conducted on a new species, Aureoboletus pseudorussellii, belonging to the genus Aureoboletus Pouzar (nrLSU + TEF1-α) and provided a detailed description. It grows on the ground under the mixed forest of Fagaceae and Pinaceae in Zhejiang Province, and is named after its similarity to A. russellii. The distinctive features of this species include a pileus uncracked in maturity, with a subtomentose surface, which darkens to reddish brown to blackish brown when injured. It is also marked by a yellowish white to white short veil at the margin. The stipe is adorned with prominent white to pale reddish brown carinate stripes, and its context ranges from yellowish white to orange, showing no discoloration when bruised. Basidiospores (14–)14.5–20.5(–21) × (7.5–)8–9(–9.5) µm. A detailed description of the novel species and its differences from similar species is provided below.

1. Introduction

Aureoboletus Pouzar, typified by A. gentilis (Quél.) Pouzar, was established in 1957 [1]. The genus is distinguished by its viscid and downy pileus, featuring a hymenophore adorned with yellow tubes and pores, and a surface that is slightly sunken at the top of the stipe [2,3]. The stipe is neither reticulate nor glandulose, with a cylindrical and a slightly swollen lower portion, and it often forms symbiotic mycorrhiza with plants from the Fagaceae and Pinaceae families [4].

The recognition of Aureoboletus as a distinct genus was a lengthy process. Initially, in 1942, it was categorized as a mere section within Xerocomus [5]. Subsequently, numerous species previously belonging to this genus were reassigned to Boletellus, Boletus, and Pulveroboletus [5–11]. In 1957, however, Pouzar [1] reestablished it as an independent genus, with A. gentilis designated as the type species. Yet, this reclassification was not promptly embraced by the mycological community for a considerable time [8–13]. Aureoboletus was subsequently equated with Sinoboletus by Zang [14] in 1992, before eventually being acknowledged as a synonym of Aureoboletus by Wu et al. [15]. It was only after molecular analysis was performed on Aureoboletus that the taxo­nomy of this genus was firmly established [16,17]. Klofac classified 13 species within Aureoboletus and presented an identification key for the genus in 2010 [4]. As of now, the Index Fungorum acknowledges 58 species belonging to Aureoboletus [1,4,18–25], among which 35 species have been identified in China [26–41].

Recently, our team has acquired several boletus specimens from Zhejiang Province. Following morphological and phylogenetic evaluations, some of these specimens are indeed part of Aureoboletus, and classified as a novel species. Detailed description is presented in this study.

2. Materials and methods

2.1. Specimen information

The specimens were contributed by Bin Chen (August 19 2024) and Xiaomin Yan (August 24 2024), were collected on the ground under the mixed forest of Fagaceae and Pinaceae in Meishu Village, Zuoxi Town, Qingyuan County, Lishui City, Zhejiang Province, and were dried at a temperature range of 41–56 °C under the guidance of Zhou [42]. Specimens were deposited in the Specimen Museum of Kunming Edible Fungi Research Institute (KEF).

2.2. Morphological studies

The macroscopic morphological characteristics were meticulously observed and recorded from field studies of basidiomata. Color references were selected from “Taschenlexikon der Farben, 3rd edn” [43]. For the micromorphology observation and description, we adopted established methodologies from previous studies [44–47]. The microstructures were examined from dry specimens using a LEICA DM5000 B microscope (Leica GmbH, Wetzlar, Germany) and were illustrated by hand.

The notation for measured basidiospores follows the format “n/m/p”, denoting that “n” basidiospores were assiduously measured from “m” specimens collected from “p” sampling sites. The size of the basidiospores is expressed as “(a) b–c (d)”, with “b–c” representing at least 90% of the observed values, while “a” and “d” denote the extreme values. Q signifies the length-to-width ratio of the spores. Q_m denotes the average Q value ± the sample standard deviation of the basidiospores [48].

2.3. DNA extraction, amplification, and sequencing

The desiccated samples (KEF12827, KEF12828) were employed to extract whole genome DNA, adhering to the methodology outlined by Feng et al. [49]. The large subunit ribosomal region (nrLSU) genes and the translation elongation factor 1-a region (TEF1-a) genes were amplified using the primer pairs LR0R/LR5 [50,51] and TEF1-983F/TEF1-1567R [52], respectively.

The PCR amplification procedure is pre-denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 53 °C (for nrLSU and TEF1-a) for 40 s, and extension at 72 °C for 80 s, culminating in a final extension at 72 °C for 8 min [15].

The quality of DNA was detected in NanoDrop 2000 (Thermo Scientific, Waltham, MA) before being sequenced by TSINGKE Biological Technology (Kunming, China).

2.4. Phylogenetic analyses

The sequencing results were processed in MEGA X [53], and submitted to NCBI for sequence homology searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on August 30 2024) to ascertain the taxonomic placement of the specimens. All accessible sequences were retrieved from NCBI and integrated into phylogenetic analyses to elucidate the genetic relationship between our samples and other species within the genus. The robustness of the TEF1-α intron region was assessed by comparing the outcomes of single-gene and multi-gene analyses [15,42,54]. Based on the studies of Zhang [30,34,39], Fang [35], and others [19,38], Phylloporus imbricatus and Xerocomus aff. subtomentosus were chosen as outgroups. All the data used are listed in Table 1.

Table 1.

Data on Aureoboletus species used in phylogenetic analyses.

Species	Voucher	Locality	LSU	TEF1-α	Reference
Aureoboletus abruptibulbus	4588	USA	KF030302	KF030401	[21]
A. albipes	FHMU2228*	China	OP630876	OP676221	[38]
A. albipes	FHMU2237	China	OP630877	OP676222	[38]
A. auriflammeus	CFMR BOS-699	USA	MK601706	MK721060	[18]
A. auriporus	BDCR0431	UK	HQ161871	–	[55]
A. auriporus	MAN020	Brazil	JQ003659	–	[56]
A. ayuukii	30448*	Mexico	OR421569	–	[19]
A. betula	MICH: KUO-10030904	USA	MK601736	–	[18]
A. betula	134/96	Germany	AF050642	–	[57]
A. catenarius	HKAS 54467*	China	NG_057093	KT990711	[31]
A. catenarius	HKAS54463	China	KT990509	KT990710	[31]
A. citriniporus	REH8719	USA	KF030298	–	[21]
A. citriniporus	JLF6091	USA	MH271618	–	[58]
A. clavatus	HKAS59802*	China	KR052044	KR052053	[28]
A. clavatus	GDGM42963	China	KR052046	KR052054	[28]
A. conicus	FHMU4730*	China	OP630878	–	[38]
A. duplicatoporus	HKAS:50498	China	KF112361	KF112230	[15]
A. duplicatoporus	GDGM52898	China	MN410708	–	[34]
A. elvirae	MEXU HO_29006*	Mexico	NG_243306	–	[19]
A. erythraeus	FHMU 3144*	China	NG_075380	MT650114	[36]
A. erythraeus	FHMU1053	China	MT650074	MT650112	[36]
A. formosus	GDGM44444	China	KT291750	MK165833	[30]
A. formosus	GDGM44441*	China	KT291749	KT291744	[30]
A. garciae	MEXU 29006*	Mexico	NG_075245	–	[20]
A. gentilis	ADK4865	Thailand	–	KT824027	[59]
A. gentilis	MG372a	China	KF112344	KF134014	[15]
A. gentilis	Pug1	USA	DQ534635	KF030399	[17]
A. glutinosus	GDGM 44477*	China	NG_068273	MH700205	[34]
A. glutinosus	GDGM44479	China	MH670256	MH700204	[34]
A. griseorufescens	ZM131	China	MH670279	–	[34]
A. griseorufescens	GDGM28490*	China	MH670278	–	[34]
A. guangdongensis	FHMU3797*	China	–	OM321040	[37]
A. innixus	MB03-104	USA	KF030239	KF030400	[21]
A. innixus	CFMR: BOS-544	USA	MK601707	MK721061	[18]
A. liquidus	TNS F-39710*	Japan	NG_056938	–	[22]
A. longicollis	HKAS:53398	China	KF112376	KF112238	[15]
A. longicollis	HKAS80127	China	KT990515	KT990719	[31]
A. marroninus	GDGM43288*	China	NG_057040	KT291746	[29]
A. miniatoaurantiacus	KD 22-004*	India	OR349659	–	[60]
A. miniatoaurantiacus	GDGM42855	China	MH670259	MH700202	[34]
A. miniatoaurantiacus	N.K. Zeng3077	China	MT829114	OP680540	[61]
A. minimus	GDGM 44400*	China	NG_243178	–	[62]
A. minimus	GDGM44401	China	OP901641	–	[63]
A. mirabilis	HKAS:57776	China	KF112360	KF112229	[15]
A. mirabilis	REH9765	USA	KP327661	KP327709	[23]
A. moravicus	MG374a	China	KF112421	KF112232	[15]
A. moravicus	PARMA 1544/11	Italy	KJ676958	KJ676959	[64]
A. nanlingensis	GDGM44759*	China	MH670276	MH700206	[34]
A. nephrosporus	HKAS 74929*	China	NG_057094	KT990721	[31]
A. nephrosporus	HKAS67931	China	KT990516	KT990720	[31]
A. ornatipes	FHMU1981*	China	OP630879	OP676223	[38]
A. projectellus	MICH: KUO-09111014	USA	MK601708	MK721062	[18]
A. projectellus	NYBG13392	USA	KP327622	KP327675	[23]
A. projectellus	AFTOL-ID 713	USA	AY684158	AY879116	[17]
A. pseudorussellii	KEF12827 *	China	PQ346298	PQ352347	This study
A. pseudorussellii	KEF12828	China	PQ346299	PQ352348	This study
A. pseudoauriporus	JAB_80	USA	MW662588	MW737490	[24]
A. pseudoauriporus	JAB_320	USA	MW662585	MW737489	[24]
A. quercus-spinosae	GDGM 43755*	China	NG_057121	MK165836	[33]
A. quercus-spinosae	GDGM43758	China	KY039968	MK165837	[33]
A. raphanaceus	GDGM 45911*	China	NG_068304	–	[34]
A. raphanaceus	GDGM44832	China	MH670268	MH700194	[34]
A. raphanaceus	GDGM52590	China	MH670272	MH700193	[34]
A. readii	MEXU 30443*	Mexico	OR421566	–	[19]
A. readii	MEXU 30440	Mexico	OR421567	–	[19]
A. roxanae	CFMR: BOS-698	USA	MK601709	MK721063	[18]
A. roxanae	DS626-07	USA	KF030311	KF030402	[21]
A. rubellus	HKAS105265*	China	MN304782	–	[35]
A. rubellus	FHMU4453	China	–	OP676227	[38]
A. rubellus	FHMU4877	China	–	OP676226	[38]
A. russellii	BD391	UK	HQ161874	–	[55]
A. russellii	CFMR: BOS-716	USA	MK601710	MK721064	[18]
A. shichianus	HKAS:76852	China	KF112419	KF112237	[15]
A. shichianus	HKAS43373	China	AY647211	DQ408145	[65]
A. singeri	CFMR BZ-2395	USA	MK601711	MK721065	[18]
A. sinobadius	GDGM 71932*	China	NG_068305	–	[34]
A. sinobadius	GDGM70666	China	MN204547	–	[34]
A. solus	FHMU4377	China	–	OP680547	[38]
A. solus	GDGM49600	China	MN410707	–	[34]
A. tenuis	FHMU2225	China	OP615201	–	[38]
A. tenuis	GDGM42601*	China	KF534789	KT291745	[30]
A. thibetanus	HKAS:76655	China	KF112420	KF112236	[15]
A. thibetanus	HKAS89494	China	KT990525	KT990729	[31]
A. thibetanus	AFTOL-ID 450	USA	AY700189	DQ029199	[17]
A. tomentosus	HKAS59694	China	KT990513	KT990714	[31]
A. velutipes	GDGM 44713*	China	NG_068272	MH700191	[34]
A. velutipes	GDGM42608	China	MH670251	MH700190	[34]
A. venustus	FHMU4776	China	OP615204	OP680548	[38]
A. venustus	HKAS 77700*	China	NG_057099	–	[32]
A. viscidipes	HKAS77103	China	KT990519	KT990723	[31]
A. viscidipes	FHMU5526	China	OP615206	–	[38]
A. viscosus	OR0361	Thailand	–	MH614703	[66]
A. yunnanensis	HKAS75050	China	KT990520	KT990724	[31]
A. yunnanensis	GDGM26359	China	MN204560	MN549670	[34]
A. yunnanensis	HKAS 57581*	China	KF112422	KF112233	[15]
A. zangii	HKAS74766	China	KT990522	KT990726	[31]
A. zangii	HKAS63217*	China	KT990526	–	[31]
A. zangii	HKAS74751	China	KT990521	KT990725	[31]
Phylloporus imbricatus	HKAS 54859	China	JQ967203	JQ967160	[67]
Xerocomus aff. subtomentosus	HKAS 58865	China	KF112389	KF112294	[15]

The newly generated sequences are marked in bold. (–) means the data is missing.

^*Holotype.

Single gene dataset was sequentially submitted to MAFFT (http://mafft.cbrc.jp/alignment/server/, an online website, login on September 15 2024) for precise alignment, adhering to the methodology outlined by Katoh and Standley [68]. Subsequently, the aligned sequences of the two genes were merged within PhyloSuite [69]. Unavailable gene sequences were considered as missing data. In congruence with the findings from both single-gene and multi-gene phylogenetic assessments, the intron genes were found to be conserved [70]. ML and BI analyses were conducted on the integrated datasets using RAxML version 8 [71] and MrBayes v3.2 [72], respectively. For the ML analysis, the GTRGAMMAI model [73], was employed, with statistical robustness verified through 1000 iterations of rapid non-parametric bootstrapping [74]. In the BI analysis, the partition replacement model for the combined datasets was evaluated according to the Akaike information criterion (AIC) in PartitionFinder 2 [75], which was then applied to the phylogenetic analysis. The results of phylogenetic analysis are visualized and beautified by FigTree v1.4.4 [76], with only bootstrap frequencies of 50% or higher and posterior probabilities of 0.95 or above displayed on the branches.

3. Results

3.1. Molecular phylogenetic results

Four novel sequences, comprising two nrLSU and two TEF1-α, were derived from two distinct samples.

The nuclear large subunit dataset was composed of 94 taxonomic units and spanned 961 characters, while the TEF1-α dataset has 70 entries and 721 bases. The integrated dataset, which merged the nrLSU and TEF1-α, included 100 taxa and featured 1682 nucleotide positions. TrN + I + G and TIMeF + I + G have been identified as the best alternative models for the nrLSU and TEF1-α partitions, respectively. BI analyses were performed with two MCMC runs, each with four chains, and the number of generations was set to 2,500,000. After completion, the average deviation of the splitting frequency is 0.007020. The ML tree and BI tree exhibited congruent topologies, with only the ML trees that possessed both BS and PP values being presented (Figure 1).

Figure 1.

The ML phylogenetic tree of Aureoboletus, showcasing BS ≥50% and PP ≥0.95. The novel species is marked in red, with the type specimen denoted in bold.

In phylogenetic tree, Aureoboletus pseudorussellii (KEF12827, KEF12828) formed an independent branch and clustered into a clade with A. mirabilis, A. projectellus, and A. russellii, and has the closest phylogenetic relationship with A. russellii.

3.2. Taxonomy

Aureoboletus pseudorussellii D.F. Sun, R. Hua, F. Zhou & J.B. Zhang, sp. nov. (Figures 2 and 3).

Figure 2.

Habitat of Aureoboletus pseudorussellii. (a, b) KEF12827 (holotype), photos by Bin Chen; (c) KEF12828, photos by Xiaomin Yan. Scale bars = 10 mm.

Figure 3.

Aureoboletus pseudorussellii (KEF12827, Holotype). (a) Basidiospores; (b) Basidia; (c) Cheilocystidia; (d) Pleurocystidia; (e) Pileipellis. The scale is 10 µm. Drawn by F. Zhou.

Mycobank number: MB857541, Fungal Names No.: FN 571200.

Etymology: The epithet “pseudorussellii” signifies the new species resemblance to A. russellii.

Holotype: China. Zhejiang Province: Lishui City, Qingyuan County, Zuoxi Town, Meishu Village, elev. 913 m, August 19 2024, ZF032 (KEF12827).

Description: Basidiomata are small to medium-sized. Pileus 3–8 cm in diameter, hemispherical to flattened, occasionally with a central depression, featuring a dry surface that remains uncracked with age, white (4A1) to yellow-brown (6A5) subtomentose turning reddish brown (7C7) to blackish brown (7D3) after bruised when young, with an ochre (6A3) to brown (6B4) center, and fringed with a yellow-white (4A2) to white (4A1) short veil at the margin. Context 0.3–0.9 cm thick at the pileus center, whitish (1A1) to pale yellow white (1A2), remains unchanged when injured. Hymenophore is adnate and depressed around the top of stipe; pores are circular, 1–3 per mm, exhibiting a pale-yellow (2A2) in their youth, transitioning to a deeper yellow (2A4) or a yellow brown (4B2) as they mature; tubes extending up to 10 mm in length, yellow (2A2) to pale brown (2B2), no color change even when damaged. Stipes 8.4–15 × 0.6–2.1 cm, central and solid, clavate, curved in the lower part, the upper part 0.6–1.1 cm diameter, the lower part up to 2.1 cm, reddish brown (7C5), surface viscid when wet, uneven, with obvious white (7A1) to pale reddish brown (7B2) carinate stripes; context is yellowish-white (5A2) to orange (5A5), no reaction when bruised; the base of the stipe is covered by dense yellowish-white mycelium. Odor indistinct.

Basidia 38–53 × 14–19 µm, thin-walled, clavate, with four sterigmata, 3.5–8 µm long. Basidiospores [40/2/2] (14–)14.5–20.5(–21) × (7.5–)8–9(–9.5) µm, Q = (1.57–)1.60–2.40(–2.44), Qm = 2.04 ± 0.28, amygdaloid to fusiform, with thin walls and a surface marked by distinct carinate stripes, light yellow to golden in KOH. Hymenophoral trama boletoid, composed of slender hyphae, 3–7 µm wide, hyaline to pale grayish in KOH. Cheilocystidia, commonly found, are 57–68 × 11–13 µm, fusiform to subclavate, thin walls and hyaline to pale gray in KOH. Pleurocystidia are 56–70 × 15–18 µm, frequently occurring, stout clavate to cystidioid. Pileipellis is composed of interlaced hyphae, appearing colorless to light gray in KOH, with thin walls, a diameter of 5–11 µm, subcylindrical to subclavate terminal cells (28–61 × 6–11.5 µm). Pileal trama is composed of hyphae 4–8 µm in diameter, thin walls, and hyaline in KOH. Clamp connections are absent across all tissues.

Habitat: Under the mixed forests of Fagaceae (Quercus) and Pinaceae (Pinus).

Distribution: Zhejiang Province, China.

Additional specimens examined: China. Zhejiang Province, Lishui City, Qingyuan County, Zuoxi Town, Meishu Village, elev. 918 m, August 24 2024, ZF039 (KEF12828).

Note: Aureoboletus pseudorussellii features a hemispherical to applanate pileus, uncracked with age, exhibiting a pale yellow to yellow-brown subtomentose surface that darkens to reddish brown to blackish brown upon bruising in youth, and adorned with a yellow-white to white, short veil at the margin; a surface is marked by distinct white to pale reddish brown carinate stripes stipes, a yellowish-white to orange context that does not react to bruising; basidiospores (14–)14.5–20.5(–21) × (7.5–)8–9(–9.5) µm, with pronounced carinate stripes. Morphologically and phylogenetically, A. pseudorussellii is closest to A. russellii. However, A. russellii differ from A. pseudorussellii in its color of pileus and stipe, and the reaction in color of context when injured, and the size of basidia, pleurocystidia, and cheilocystidia.

4. Discussion

Aureoboletus pseudorussellii exhibits similar characteristics in its stipes to those of A. mirabilis (Murrill) Halling, A. projectellus (Murrill) Halling, and A. russellii (Frost) G. Wu & Zhu L. Yang. However, A. mirabilis, initially described from North America, can be distinguished by its longer and smooth basidiospores ((20) 22–27 (28) × 9–13 µm), larger pleurocystidia (72–85 × 14–21 µm), and a pileus that transitions from grayish ruby to cherry to ruby with pale-colored spots, featuring distinctly coarse, pale yellow to dull yellow reticulations on the stipes [23]. Aureoboletus projectellus has larger and smooth basidiospores ((27.3) 30.1 ± 2.3 (36.4) × (7.8) 10.8 ± 1.2 (13.3) µm), and smaller basidia ((36.4) 41 ± 3.9 (44.2) × (12.7) 14.9 ± 1.1 (17.9) µm), and larger pleurocystidia ((82.9) 84.2 ± 11.5 (85.8) × (15.6) 18.8 ± 2.5 (23.4)), and the color of stipe is yellowish to brownish yellow to reddish [23]. Aureoboletus russellii also has pronounced carinate stripes basidiospores and stipes, but can be differentiated by its reddish brown with a yellowish background pileus, and its context turns pink to pale red when bruised, the context of the stipe turns reddish brown when injured, along with smaller basidia (32–40 × 10.4–15.2 μm), larger pleurocystidia (66.5–76.8 × 15.2–24.7 μm), and shorter cheilocystidia (30–52 × 11.2–13.6 μm) [31].

In phylogenetic analyses, Aureoboletus pseudorussellii and A. mirabilis, A. projectellus, A. russellii are clustered into a clade, and have the closest phylogenetic relationship with A. russellii, and combined with morphological structure, we named the new species as A. pseudorussellii.

It is documented that Aureoboletus mirabilis, A. projectellus and A. russellii are fit for consumption [19,77]. Consequently, the newly discovered species we have acquired might also be edible; however, this hypothesis necessitates substantiation through subsequent research endeavors.

Funding Statement

This study received support from Yunnan Province Key Laboratory of Edible Fungi Germplasm Innovation and Functional Components (202402AN360003) and the Yunnan Province Science and Technology Talent and Platform Plan Project (202305AD160051, 202205AD160042).

Author contributions

Conceptualization was carried out by F.Z., J.-B.Z., R.H., and D.-F.S. Methodology was devised by F.Z., J.-B.Z., Q.-M.L., S.-X.L., X.L., X.-K.L., C.-L.L., and L.W. The experiment was conducted by F.Z. Formal analysis was performed by F.Z. and J.-B.Z. Resources were provided by F.Z. and J.-B.Z. The original draft was written by F.Z., with review and editing assistance from R.H. and D.-F.S. Supervision was provided by R.H. and D.-F.S., who also administered the project and acquired funding.

Disclosure statement

No potential conflict of interest was reported by the author(s).