Phylogenetic Framework, a New Species, and Two New Species Records for China in Rhizopogon

Abstract

Rhizopogon, a globally distributed genus of edible ectomycorrhizal fungi, is the focus of this study. This study describes a new species, Rhizopogon qujingensis sp. nov., along with two newly recorded species of the genus Rhizopogon from China. The new species is distinguished from other known Rhizopogon species by its distinctly phylogenetic position and unique morphological characteristics, the species is characterized by comparatively smaller basidiomata, a peridium that exhibits yellow tinting after injury and contains red-brown pigment, along with smaller, narrowly oblong to elongate spores. Furthermore, R. rubescens and R. evadens are recorded for the first time in China, with their taxonomic status confirmed through integrated morphological and molecular analysis, thereby extending their known geographical distribution. Phylogenetic analysis was conducted to elucidate the relationships between Chinese Rhizopogon species and other taxa within the genus. This research contributes essential data for advancing the understanding of global Rhizopogon species diversity and biogeographic patterns.

1. Introduction

The genus Rhizopogon Fr. apud Fr. & Nordholm (Basidiomycota, Agaricomycetes, Boletales, Rhizopogonaceae) is characterized by hypogeous or semi-hypogeous basidiomes [1,2]. It forms ectomycorrhizal associations predominantly with Pinaceae (Pinus, Pseudotsuga, and Tsuga), and is among the most species-rich sequestrate fungal genera [3,4,5,6,7]. Rhizopogon spp. possesses considerable economic value as edible fungi; basidiomata are commercially marketed as “shoro” in Japan [8,9]. Harvesting and sale constitute significant income sources for local communities in the Yunnan and Sichuan Provinces in China [10]. Notably, Japan has achieved breakthroughs in mycorrhizal synthesis and large-scale plantations of Rhizopogon, successfully realizing the production of mushrooms on a large scale, establishing a technological paradigm for sustainable resource utilization [11,12].

Taxonomic exploration of Rhizopogon commenced with its initial description from Europe by Elias Magnus Fries in 1817. Subsequent studies have revealed substantial species diversity and a broad global distribution encompassing North America [2], North Africa [13], Europe [14,15], and Asia [10,16,17,18].

Taxonomic frameworks have evolved significantly with the application of molecular tools, particularly sequence analysis of nuclear ribosomal regions (nuc-ssu, nuc-lsu, ITS) and mitochondrial genes (atp6, mt-lsu) [19,20,21,22,23,24,25,26]. Smith and Zeller [3] established two subgenera: Rhizopogonella and Rhizopogon, the latter comprising sections Amylopogon, Fulviglebae, Rhizopogon, and Villosuli. Trappe [27] subsequently transferred subgenus the species of the subgenus Rhizopogonella to the genus Alpova. Current classification recognized five subgenera: Versicolores, Rhizopogon, Roseoli, Villosuli, and Amylopogon [21,22].

Although Rhizopogon has high diversity and wide geographical distribution in western North America and Europe [28,29], its species richness in China is still relatively low [30]. Historically, only ten species have been reported in China (including R. cylindrisporus, R. piceus, R. nigrescens, R. rubescens, and R. luteolus, etc.) with distributions documented in Shanxi, Yunnan, Guangdong, Sichuan, and Inner Mongolia [10,30,31,32,33,34,35,36]. Among them, R. rubescens was previously considered a synonym of R. roseolus [34,37], a taxon later reclassified as R. jiyaozi for populations in southwestern China [30]. Currently, only four species (R. jiyaozi, R. songmaodan, R. flavidus, and R. sinoalbidus) are accepted with robust morphological and molecular evidence, all exclusively associated with Pinus in southwestern China. Moreover, research on Rhizopogon in China lags significantly behind studies on other hypogeous genera such as the genus Tuber [38]. This discrepancy is largely due to limited attention given to Rhizopogon and a lack of molecular evidence supporting some of the reported species. As a result, there is a considerable gap in our understanding of the true diversity and distribution of this genus in China.

Systematic field surveys conducted over the past three years in northeast and southwest China have yielded numerous Rhizopogon specimens from the root zones of Pinus and Quercus. Preliminary integrated morphological and molecular analyses of these collections suggested the potential presence of a new species and two species previously unrecorded in China. However, these findings required formal description and rigorous taxonomic validation. This study aims to elucidate Rhizopogon diversity within China by (1) formally describing the novel species R. qujingensis sp. nov.; (2) confirming and documenting the two new records for China: R. rubescens and R. evadens. through integrated analysis; (3) clarifying the taxonomic status of these species through comparative analyses with closely related taxa; and (4) addressing critical knowledge gaps regarding the species richness and distribution of Chinese Rhizopogon. These findings are expected to provide a foundational framework for future systematic and evolutionary studies of this genus.

2. Materials and Methods

2.1. Morphological Characteristics Examination

Nine fresh basidiomata (specimens HKAS95544, HKAS95545, HKAS95546, HKAS95547, YNAU0608, YNAU0620, YNAU0630, YNAU1771, and YNAU2053) were collected from the Yunnan, Sichuan, and Heilongjiang Provinces. All voucher specimens are deposited in the Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences (HKAS) and Yunnan Agricultural University. Specimens were field-dried using silica gel for morphological analysis, molecular studies, and permanent voucher preservation. Macroscopic features were documented from rehydrated material using a Leica S8APO stereomicroscope. For microscopic characterization, freehand sections were prepared using a razor blade. Sections were placed in 5% (w/v) aqueous KOH, stained with Congo red reagent, and examined using a Leica DMi8 (Leica Microsystems, Wetzlar, Germany) inverted electron microscope. Morphological structures were described and measured according to standard taxonomic protocols [10,16].

2.2. DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA of basidiomata was extracted using the Aidlab™ (Aidlab Biotechnologies Co., Ltd., Beijing, China) Plant Genomic DNA extraction kit following the manufacturer’s protocol. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using the ITS1F and ITS4 [39,40]. The polymerase chain reaction (PCR) was performed in 25 μL volumes containing: 2.5 μL 10 × PCR buffer (with Mg²⁺), 1.5 μL dNTP mix (1 mM each), 1 μL bovine serum albumin (BSA; 0.1%), 1 μL of each primer (5 μM), 1.0 μL template DNA (diluted 1:25) extracts (obtained following the manufacturer’s protocol), 0.5 μL MgCl₂ (25 mM), and 0.15 μL Taq DNA polymerase (1.5 U; Takara Biotechnology, Dalian, China) and nuclease-free water to volume. Thermocycling conditions comprised an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. Three microliters of each PCR product were run on 1% (w/v) agarose gels and stained with ethidium bromide [10]. The PCR products were purified and subjected to bidirectional sanger sequencing using the amplification primers at TsingKe Biological Technology (Kunming, China) ITS1F and ITS4. Sequences were edited manually using Sequencher™ 4.1.4 (Gene Codes Corporation, Ann Arbor, MI, USA). Preliminary taxonomic identification was performed via BLASTn queries against the NCBI GenBank database. Sequences generated in this study were deposited in GenBank (accession numbers provided in

Table 1. Collection details of self-collected voucher specimens in this study and corresponding GenBank accession numbers for ribosomal ITS sequences.

Species Name	Voucher/Isolation	Origin	GenBank No. of ITS
R. qujingensis	YNAU2053	China	PV584203
R. evadens	YNAU0620	China	PV534069
R. evadens	YNAU0630	China	PV534070
R. evadens	HKAS95544	China	PV534016
R. evadens	HKAS95545	China	PV534017
R. evadens	YNAU0608	China	PV534071
R. evadens	HKAS95747	China	PV534027
R. evadens	HKAS95746	China	PV534028
R. rubescens	YNAU1771	China	PV534094
R. sinoalbidus	Ectomycorrhiza3117-5	China	PX368895

).

2.3. Phylogenetic Analyses

The ITS sequence from the studied specimen was compiled, together with sequences from references taxa curated in GenBank (http://www.ncbi.nlm.nih.gov/) (accessed on 12 August 2025) (Detailed information provided in

Table A1. Collection details of voucher specimens and corresponding GenBank accession numbers for ribosomal ITS sequences analyzed in this study.

Species Name	Voucher/Isolation	Origin	GenBank No. of ITS	References
R. evadens	GO-2009-164	Mexico	KJ595006	Unpublished
Uncultured Rhizopogon	ECM	Russia	KJ769300	[48]
R. superiorensis	GO-2008-25	Mexico	KJ595010	Unpublished
Uncultured R. roseolus	ECM	Portugal	KF007248	Unpublished
Uncultured Rhizopogon	ECM	Portugal	KF007241	Unpublished
R. roseolus	KPM-NC0018075	Japan	KF990475	[49]
Uncultured Rhizopogon	ECM	Portugal	KF007261	Unpublished
R. roseolus	MA-Fungi 47716	Spain	AJ810062	[26]
R. roseolus	MA-Fungi 47716	Spain	AJ810063	[26]
Uncultured Rhizopogon	ECM	Spain	FJ013089	Unpublished
Uncultured Rhizopogon	ECM	Canada	FJ554251	[50]
R. roseolus	ECM	Poland	EU379678	Unpublished
R. abietis	Trappe 7603	US	EU837243	Unpublished
R. evadens	OSC 63507	USA	EU837230	Unpublished
R. roseolus	ECM	Sweden	HM036649	[51]
R. pseudoroseolus	K98C31T213	New Zealand	GQ267483	[52]
R. luteorubescens	K91T310b	New Zealand	GQ267482	[52]
R. abietis	OSC: 41455	USA	KC346843	Unpublished
R. fallax	GO-2009-222	Mexico	KC152186	Unpublished
R. fallax	GO-2009-230	Mexico	KC152199	Unpublished
R. fallax	GO-2009-218	Mexico	KC152195	Unpublished
R. evadens	GO-2010-122	Mexico	KC152181	Unpublished
Uncultured fungus	ECM	USA	KC424535	[53]
R. luteorubescens	NJDRS16aYSWY	USA	KX833229	[54]
R. jiyaozi	YAAS-L2399	China	KP893836	[30]
R. sinoalbidus	YAAS-L2947	China	KP893818	[30]
Uncultured Rhizopogon	85	China	LC013778	[55]
R. vulgaris	ECM	USA	JN858081	[56]
R. bacillisporus	SOC1503	USA	JN022518	Unpublished
Uncultured Rhizopogon	ECM	Mexico	JN704815	Unpublished
R. salebrosus	ECM	USA	HQ914333	[57]
R. salebrosus	ECM	USA	HQ914332	[57]
Uncultured Rhizopogon	ECM	Japan	AB839388	[58]
Uncultured fungus	ECM	New Zealand	KM596883	Unpublished
R. roseolus	DG68	UK	JQ888193	[59]
Uncultured Rhizopogon	ECM	China	JQ991779	Unpublished
R. flavidus	YAAS: L2957	China	NR_158904	[30]
R. sinoalbidus	YAAS: L2949 (Holotype)	China	NR_158905	[30]
R. jiyaozi	YAASL: 2929	China	NR_158906	[30]
R. succosus	OSC JMT19321	USA	AF062933	[22]
R. luteolus	OSC JMT22516	Sweden	AF062936	[22]
R. verii	RHILUT_211013C	Germany	LN875266	[29]
R. songmaodan	HKAS-106768	China	MN655984	[10]
R. songmaodan	RW_2019a	China	MN846303	[10]
R. nigrescens	NAMA 2015-326	USA	MH910566	Unpublished
R. pseudoroseolus	223	USA	MK841867	Unpublished
R. albidus	347	USA	MK841991	Unpublished
R. pseudoroseolus	224	USA	MK841868	Unpublished
Rhizopogon sp.	Trappe 28417; SC 110931	USA	DQ680181	Unpublished
R. subsalmonius	1SSA-1653	Spain	AM085530	Unpublished
R. subsalmonius	OSC JMT17218	USA	AF062938	[22]
R. albidus	AHS 69642	USA	AM085519	Unpublished
R. evadens	MICH AHS 65484 (Holotype)	USA	AF062927	[22]
R. odoratus	AHS 71319	USA	AM085526	Unpublished
R. rocabrunae	17067	Spain	JF908761	[60]
R. occidentalis	JMT 17564, OSC	USA	AF058305	[22]
R. ochraceorubens	MICH AHS 59643 (Holotype)	USA	AF062928	[22]
R. flavidus	YAAS-L2957 (Holotype)	China	KP893813	[30]
R. flavidus	YAAS-L2956	China	KP893814	[30]
R. subpurpurascens	MICH AHS 65669 (Paratype)	USA	AF062929	[22]
R. ellenae	MICH AHS 66137 (Holotype)	USA	AH011350	[22]
R. sinoalbidus	YAAS-L2944	China	KP893816	[30]
R. sinoalbidus	YAAS-L2949 (Holotype)	China	KP893820	[30]
R. colossus	MICH AHS 49480 (Holotype)	USA	AH011348	[22]
R. hawkerae	MICH AHS 68417 (Paratype)	USA	AH011351	[22]
R. ochraceisporus	MICH AHS 65963 (Paratype)	USA	AF071439	[22]
R. jiyaozi	YAAS-L2335	China	KP893823	[30]
R. jiyaozi	YAASL-2929 (Holotype)	China	KP893830	[30]
R. roseolus	JMT 8227, OSC	USA	AF058315	[22]
R. burlinghamii	JMT 17882, OSC	USA	AF058303	[22]
R. buenoi	MA-Fungi 47676 (Holotype)	Spain	AJ297263	[61]
R. boninensis	KPM-NC 26928	Japan	MK395372	Unpublished
R. songmaodan	HKAS-106767 (Holotype)	China	MN655983	[10]
R. songmaodan	HKAS-106766	China	MN655982	[10]
R. mohelnensis	IK-00184	Poland	KX610701	[62]
R. rubescens	NS182	Sweden	DQ068965	[63]
R. rubescens	T1PK2	Sweden	JX907816	[64]
R. mohelnensis	4599	Spain	ON072209	[65]
R. albidus	AHS 69642	USA	AM085519	Unpublished
R. albidus	MICH: 425	USA	NR_198824	Unpublished
R. evadens	MICH: 5448 (Holotype)	USA	NR_198822	Unpublished
R. avellaneitectus	MICH: 5434	USA	NR_198826	Unpublished
R. subsalmonius	MICH: 423	USA	NR_198806	Unpublished
R. odoratus	Mycorrhizal root	USA	MK841906	Unpublished
R. odoratus	Mycorrhizal root	USA	MK841887	Unpublished
R. bacillisporus	OSC: 134671	USA	KC346846	Unpublished
Rhizopogon sp.	TNL1988	USA	MH878765	Unpublished
R. confusus	PRM945153	Czech	HG999784	[2]
R. confusus	PRM879700	Czech	HG999780	[2]
R. songmaodan	rsc-315	China	MT821479	[10]
R. songmaodan	Synthesized ECM of Pinus armandii	China	MN846304	[10]
R. songmaodan	Synthesized ECM of P. armandii	China	MN846305	[10]
R. songmaodan	HKAS-106770	China	MN655985	[10]
R. songmaodan	HKAS-106769	China	MN655986	[10]
R. songmaodan	HKAS-106765	China	MN655981	[10]
R. evadens	HAY-F-007602	USA	PQ125058	Unpublished
R. evadens	OSC 62146	USA	KT968587	Unpublished
R. evadens	OULU: E.Ohenoja 7505-380	Spain	ON072206	[65]
R. evadens	OULU: O.Kaattari 7131-347	Spain	ON072211	[65]
R. evadens	TB-2010-MEX 26	Mexico	KC152180	Unpublished
R. evadens	GO-2009-323	Mexico	KC152182	Unpublished
R. evadens	HAY-F-007605	USA	PQ288476	Unpublished
R. rubescens	MICH: 607 (Holotype)	USA	NR_119472	[26]
R. mohelnensis	PRM 154616 (Holotype)	Czech	AJ810039	[26]
R. roseolus	ECM	Sweden	DQ068964	[63]
R. luteorubescens	MICH: 5462	USA	NR_119471	[26]
T. pseudocolumella	HKAS131259	China	KP090064	Unpublished
T. pseudocolumella	HKAS131259	China	KP090063	Unpublished
T. pseudocolumella	HKAS95533	China	OR631922	Unpublished

). The combined sequences were aligned using MAFFT v.7.0 with the online server implementation. The resulting multiple sequence alignment was refined using Gblocks 0.91b to eliminate ambiguously aligned regions under default parameters, followed by manual adjustment in Bioedit v.7.2.5 [41]. The optimal nucleotide substitution model was determined using Modeltest-NG v.2.0 under the Akaike information criterion (AIC) [42]. Maximum likelihood (ML) analysis was conducted in RAxML v.7.2.8 [43] using the selected model. Branch support was assessed via 1000 non-parametric bootstrap replicates. Bayesian inference (BI) was performed using MrBayes v.3.1.2 [44] with two parallel runs of four Markov Chain Monte Carlo (MCMC) chains each, sampling every 100 generations for 1 million generations. Stationarity was confirmed when the average standard deviation of split frequencies fell below 0.01, with the initial 25% of samples discarded as burn-in. Posterior probabilities (PPs) were calculated from the remaining trees.

Figure 1. Phylogenetic reconstruction of based on ITS sequence data, inferred using RAxML maximum likelihood analysis. Branch support values are indicated adjacent to corresponding nodes: bootstrap support (BS) values ≥ 70% derived from maximum likelihood (ML) analysis and posterior probabilities (PP) ≥ 0.90 obtained from Bayesian inference. Red labels denote the newly described species; blue labels indicate species newly recorded in China.

Figure 1. Phylogenetic reconstruction of based on ITS sequence data, inferred using RAxML maximum likelihood analysis. Branch support values are indicated adjacent to corresponding nodes: bootstrap support (BS) values ≥ 70% derived from maximum likelihood (ML) analysis and posterior probabilities (PP) ≥ 0.90 obtained from Bayesian inference. Red labels denote the newly described species; blue labels indicate species newly recorded in China.

3. Results

3.1. Molecular Phylogenetics

A total of 117 ITS nrDNA sequences were analyzed, with three sequences of Truncocolumella pseudocolumella designated as the outgroup. This dataset included nine newly generated sequences from Chinese collections. Following alignment and exclusion of ambiguously aligned regions, the final matrix comprised 899 bp. Both maximum likelihood (ML) and Bayesian inference (BI) analyses resulted in similar phylogenetic tree topologies. The ML phylogram (Figure 1) is presented, with nodes exhibiting strong statistical bootstrap support from ML and posterior probability values from BI, corroborating the robustness of the inferred phylogeny.

3.2. Taxonomy

Rhizopogon qujingensis S. P. Wan, H. W. Zhang & F. Q. Yu sp. nov. (Figure 2).

MycoBank: 858797

Etymology: The epithet qujingensis refers to Qujing, the locality of the type collection.

Type: CHINA, Yunnan Province, Qujing city, in humus soil beneath litter under a monospecific forest of P. armandii Franch., 25.763234° N, 104.060502° E, elevation 2178 m, 2 December 2023, YNAU2053 (holotype).

Diagnosis: R. qujingensis is distinguished by a white peridium exhibiting rose-pink discoloration upon cutting or bruising and becoming yellow-brown upon drying; peridium thin (40.0–220.0 μm), composed of pigmented, interwoven hyphae. Gleba yellowish brown at maturity, with small, labyrinthine chambers. Basidiospores narrowly oblong to elongate, smooth, hyaline, 4.3–9.5 × 2.3–4.4 μm.

Description: Basidiomata subglobose to irregular, 1.3 × 1.1 cm, compliant to slightly soft to rubbery. Fresh coloration, white when young, maturing to yellow-brown; surfaces discolor rose-pink when cut or bruised (Figure 2a); dried specimens yellow-brown, shrunken, often exhibiting depressed areas (Figure 2b). Growth solitary, occurring in humus-rich soils. Peridial surface white to yellow (Figure 2c), bearing rhizomorphs and sparse hyphae, white to yellow (Figure 2d). Odor indistinct, faintly fungal. Peridium readily separable, thin (40.0–220.0 μm thick), comprising interwoven, septate hyphae, 4.0–8.0 μm in diam, hyaline with red-brown pigmentation, becoming rose-pink when cut. Gleba initially white when, maturing yellowish to yellowish-brown; chambers small, numerous, labyrinthine; xtrama composed of interwoven, hyaline and gelatinized hyphae, 4.4–6.1 μm diam, thin-walled (Figure 2e). Hymenium lining chamber surfaces, composed of basidia and brachybasidioles embedded in a pith of interwoven, hyaline, gelatinized hyphae (Figure 2f). Basidia predominantly four-spored, smooth, clavate to cylindrically clavate, thin-walled (Figure 2g). Brachybasidioles ellipsoid to clavate, 11.8–22.0 × 3.7–7.4 μm (Figure 2h). Basidiospores narrowly oblong to elongate, smooth, 4.3–9.5 × 2.3–4.4 μm, mean 6.90 × 3.35 μm (SD: 1.30 [length], 0.53 [width]), Q = 1.0–4.1 (Figure 2i).

Distribution: The species is currently known only from a single collection site in Qujing City, Yunnan Province, China.

Notes: Phylogenetic analysis places R. qujingensis within subgenes Versicolores, forming a distinct lineage sister to R. songmaodan. While sharing same peridia discoloration patterns and glebal configuration with R. songmaodan, R. qujingensis has a markedly thinner peridium and more slender basidiospores.

Rhizopogon evadens [3] (Figure 3).

Description: Basidiomata subglobose to irregular, 1.2–3.0 × 1.1–2.5 cm. Surface smooth, bearing a shallow depression; coloration white to brownish-yellow when fresh (Figure 3a), becoming rugose and tan upon drying (Figure 3b). Odor inconspicuous. Peridium constituting the outermost layer of the basidiomata, readily abraded; pigmentation tan; in some species staining pink to red where bruised or cut; thickness 38.0–200.0 μm thick (Figure 3c). Surface bearing adherent hyaline hyphae, 5.0–8.0 μm wide, septate, unbranched. Gleba initially white or off-white in immature specimens, maturing to light yellow to dark yellow; labyrinthine, exhibiting numerous small pores; composed of interwoven, hyaline hyphae, becoming pasty at maturity (Figure 3d). Hymenium lining the cavities. Central issue (pith) consisting of interwoven, hyaline, gelatinized hyphae (Figure 3e). Hymenium composed of basidia and brachybasidioles (Figure 3f,g). Basidia 4–8-spored, smooth-walled, cylindrical to clavate, 5.8–8.3 × 2.8–3.5 μm. Brachybasidioles ellipsoid to clavate, 10.0–19.6 × 4.8–6.9 μm (Figure 3g). Basidiospores ellipsoid to ovoid, smooth-walled, 11.8–15.0 (–19.0) × 4.3–6.1 (–8.7) μm, mean 13.40 × 5.20 μm (SD: 0.80 [length], 0.45 [width]), Q = 1.9–3.5 (Figure 3h). Trama composed of laxly interwoven, hyaline, gelatinized hyphae, 4.8–6.9 μm in diameter, predominantly unbranched.

Ecology and Distribution: Hypogeous to subhypogeous occurring in symbiotic association with P. densata Mast. Distribution encompasses Europe, America, and Asia. Within China, primarily recorded from the Yunnan and Sichuan Provinces.

Additional specimen examined:

China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, in forests of P. densata with associated Q. pannosa, 29.15695851° N, 99.92065430° E, elevation 3387 m, 4 October 2021, wsp1492 (YNAU0620); China, Yunnan Province, Lijiang City, symbiotic host P. yunnanensis Franch. Surrounded by Rhododendron sp. and Quercus sp., 27.00.03.35° N, 100.10.51.06° E, elevation 3260 m, 13 July 2015, wsp400 (HKAS95544); China, Sichuan Province, Ganzi Tibetan Autonomous Prefecture, forest of Q. guyavifolia H. Lév., 29.03163016° N, 99.70884830° E, elevation 3557 m, 4 October 2021, wsp1480 (YNAU0608); China, Yunnan Province, Chuxiong Prefecture, under Quercus sp. in mixed forests with P. yunnanensis, 25.18.58.8° N, 101.22.50.4° E, alt. 2519 m, 29 August 2015, wsp673 (HKAS95747); China, Yunnan Province, Lijiang City, Jade Dragon Snow Mountain Ecological Station, 13 July 2015, wsp401 (HKAS95545); China, Yunnan Province, Chuxiong Prefecture, under Quercus sp. in mixed forests with P. yunnanensis, 25.18.58.8° N, 101.22.50.4° E, elevation 2519 m, 29 August 2015, wsp674 (HKAS95746).

Rhizopogon rubescens [8] (Figure 4).

Description: Basidiomata subglobose to irregular, 0.4 × 0.5 cm, surface smooth; coloration pale pink to light brown when fresh (Figure 4a), becoming rugose and reddish brown upon drying, exhibiting subtle color gradations (Figure 4b). Odor inconspicuous. Peridium, the outermost layer of the basidiomata, readily abraded; pigmentation reddish brown; 85.0–460.0 μm thick (Figure 4c). Surface sparsely adorned with adherent hyaline hyphae, 1.0–6.0 μm wide, septate, unbranched. Gleba in immature specimens white to off-white when immature, becoming light yellow to earthy yellow at maturity; structure labyrinthine, bearing numerous small pores; composed of interwoven hyaline hyphae, becoming pasty at maturity (Figure 4d). Hymenium lining cavity surfaces. Central tissue (pith) of interwoven, hyaline, gelatinized hyphae (Figure 4e). Hymenium comprising basidia and brachybasidioles (Figure 4f,g). Basidia predominantly two-spored, smooth-walled, cylindrical to clavate, 12.2 × 4.7 μm. Brachybasidioles ellipsoid to clavate, 1.9–2.1 × 0.9–1.1 μm (Figure 4g). Trama laxly interwoven, hyaline, gelatinized hyphae, 1.8–5.2 μm in diameter, septate, branched.

Ecology and Distribution: Hypogeous, occurring in soils of mixed forests dominated by P. sylvestris var. mongolica and forests of Acer truncatum Bunge in Heilongjiang, China. Previous reports document occurrence in Europe and North America.

Voucher Specimen examined: China, Heilongjiang Province, mixed forests of P. sylvestris var. mongolica and A. truncatum, 46.760187° N, 130.365395° E, elevation 108.7085 m, 1 September 2023, PV534094 (YNAU1771).

4. Discussion

The genus Rhizopogon exhibits substantial species diversity, with a well- documented research history in Europe and North America extending to the early 18th century [30]. In Asia, confirmed taxa included Japanese records: R. boninensis, R. roseolus, R. alpinus, R. nitidus, and R. yakushimensis sp. nov. and Chinese reports enumerate ten taxa: R. rubescens var. ochraceus, R. rubescens var. pallidimaculatus, R. rubescens var. rileyi, R. nigrescens, R. piceus, R. shanxiensis, R. jiyaozi, R. songmaodan, R. flavidus, and R. sinoalbidus. Molecular analyses support the recognition of four species: R. jiyaozi, R. songmaodan, R. flavidus, and R. sinoalbidus within established subgenera Roesoli, Versicolores, Rhizopogon, and Amylopongon, respectively [10,30].

In the present study, integrated morphological and molecular evidence supports the recognition of a new species: R. qujingensis. Phylogenetic analysis places this taxon within the subgenus Versicolores (Figure 1), demonstrating a sister relationship to R. songmaodan. Despite their relatively close phylogenetic relationship, the species diverge into distinct, strongly supported clades (ML/BS = 95%; BI/PP = 0.98), corroborating their independent evolutionary trajectories.

Phylogenetically, the subgenus Versicolores is closely related to the subgenus Rhizopogon yet exhibits striking differences in peridial structure; morphologically, it more closely resembles the subgenus Roseoli but is distinguished by the absence of the latter’s yellow pigmentation [22]. Within Versicolores subgenus, R. qujingensis is morphologically distinguished from related species by a unique combination of macro- and micromorphological features. Unlike R. subsalmonius [22] which shows no staining reaction and R. evadens [22] which stains red but possesses a white, non-yellow peridium unaffected by KOH, R. qujingensis exhibits immediate rose-pink discoloration when bruised or cut. R. qujingensis has a thinner peridium (40.0–220.0 μm) compared to R. rocabrunae (300.0–600.0 μm) and lacks the characteristic orange pigments and prominent squamules found in the latter [45]. Moreover, R. rocabrunae develops brown to black squamules and produces larger, elongated ellipsoid spores with higher Q_m values (2.59–2.87 μm), R. qujingensis has smaller, narrowly oblong to elongate spores (4.3–9.5 × 2.3–4.4 μm). Compared to R. songmaodan [10], which shares similar rubbery gleba and staining reaction, R. qujingensis has narrower spores and distinct red-brown pigmentation in its peridial hyphae. Additionally, R. qujingensis differs from R. albidus in its lack of peach to salmon coloration and from R. odoratus [46] by lacking an aromatic odor. It differs ecologically from the conifer-associated R. subsalmonius (with Abies) and R. albidus (with Abies) [47] by growing solitary in humus-rich soils. These diagnostic characteristics—including its rose-pink staining reaction, thin peridium with red-brown pigmentation, narrow spore morphology, and humus-rich habitat—collectively differentiate R. qujingensis from all compared species.

Phylogenetic analyses confirmed that the seven specimens collected in this study (YNAU0608, YNAU0620, YNAU0630, HKAS95544, HKAS95545, HKAS95746, and HKAS95747) represent R. evadens, showing strong support (BS = 96%; PP = 1.0) for clustering with European and North American taxa. The ectomycorrhizal fungal genus Rhizopogon exhibits a global distribution and demonstrates a strong preference for, and high specificity towards, Pinaceae hosts [22]. Within China, its diversity is centered in the Sichuan and Yunnan provinces. Specimens examined in this study were collected under P. densata, P. yunnanensis, and Quercus spp. The latter observation diverges from established host patterns. This observation suggests that Rhizopogon has greater ecological flexibility and a wider range of host species than previously thought. Our recent research further supports this idea, as we have successfully identified and confirmed, through ITS molecular evidence, the formation of ectomycorrhizae between R. sinoalbidus and Q. franchetii (GenBank accession no.: PX368895). This discovery marks a departure from the strict Pinaceae specificity seen in nearly all previously studied Rhizopogon species from North America and Europe, indicating a unique ecological adaptation within Chinese taxa that deserves further exploration.

This finding emphasizes the necessity to reevaluate the host-range limitations of Rhizopogon and serves as an important reference for future research into the ecological adaptability of this genus.