Exploring the Relationships between Four New Species of Boletoid Fungi from Northern China and Their Related Species

The family Boletaceae primarily represents ectomycorrhizal fungi, which play an essential ecological role in forest ecosystems. Although the Boletaceae family has been subject to a relatively global and comprehensive history of work, novel species and genera are continually described. During this investigation in northern China, many specimens of boletoid fungi were collected. Based on the study of their morphology and phylogeny, four new species, Butyriboletus pseudoroseoflavus, Butyriboletus subregius, Tengioboletus subglutinosus, and Suillellus lacrymibasidiatus, are introduced. Morphological evidence and phylogenetic analyses of the single or combined dataset (ITS or 28S, rpb1, rpb2, and tef1) confirmed these to be four new species. The evidence and analyses indicated the new species’ relationships with other species within their genera. Detailed descriptions, color photographs, and line drawings are provided. The species of Butyriboletus in China were compared in detail and the worldwide keys of Tengioboletus and Suillellus were given.

[1], a family with more than 70 genera, is one of the most prominent and diverse among the basidiomycetes [2]. It is mainly characterized by being tubulose with infrequent lamellate or loculate hymenophora, and by a fleshy context. Most Boletaceae species have value for humans and are essential for mutualistic symbiosis with trees [3][4][5][6]. Although the family Boletaceae was established nearly two centuries ago, the species diversity of the family increased significantly in the last few decades [7][8][9][10][11][12][13][14][15][16][17][18][19]. Because the morphology of Boletaceae has convergent characteristics, the classification did not correspond to the phylogeny of Boletaceae for a long time. With the development of molecular biology, the method of genealogical concordance phylogenetic species recognition (GCPSR) [20] was used to identify species of fungi, resolved some doubts about the status of taxa, and contributed to a better understanding of the relationships of the genera in this family [5,21,22]. In the past two decades, new genera and new species have rapidly increased, and the evolution of ectomycorrhizas of Boletales was gradually disclosed [23,24].

Taxonomy
Basidiospores (   Notes: Butyriboletus pseudoroseoflavus is characterized by a pink to greyish rose pileus, greenish yellow pores changing to greenish blue when bruised, a stipe surface almost wholly covered with yellow reticulation, a stipe of unchanging color when injured, and large and narrow basidiospores. Morphologically and phylogenetically, Bu. pseudoroseoflavus is similar to Bu. roseoflavus (Hai B. Li & Hai L. Wei) D. Arora & J.L. Frank, which was initially described in specimens from eastern China (Zhejiang province) and southwestern China (Yunnan province) by Li et al. [67]. However, Bu. pseudoroseoflavus differs from Bu. roseoflavus in its adnate to decurrent hymenophore and its relatively larger and narrower basidiospores, with a more considerable Q value and pleurocystidia larger than cheilocystidia [5]. In morphological features, Bu. pseudoroseoflavus is also similar to Bu. cepaeodoratus cepaeodoratus always has a duller color, its stipe stains blue when injured, and its basidiospores are broader than those of Bu. pseudoroseoflavus [68]. Both stipe and context of Bu. roseogriseus and Bu. primiregius turn blue when injured, and have broader basidiospores, Q = (1.95) 2.20-2.42 (2.57) and Q = 3.5, respectively [32,56]. The pores of Bu. regius are unchanging to blue when bruised; the stipe is usually ventricose when young, showing at the base rare faintly reddish or purplish spots, with basidiospores weakly dextrinoid [69]. Butyriboletus fuscoroseus is characterized by its brown-pink, reddish brown, or purplish brown pileus, decurrent hymenophore, stipe staining blue when bruised or cut, and the narrow basidiospores [56]. Phylogenetically, Bu. pseudoroseoflavus is similar to Bu. abieticola. However, Bu. abieticola is characterized by a light rose-colored pileus, with tan-colored spots interspersed, a white context, a hymenium dextrinoid, and hyaline spiral incrustations on most hyphae [70]. Reference Table 2 provides the critical characteristics distinguishing Bu. pseudoroseoflavus from other species in China. Basidiomata middle to large. Pileus 7.0-13.0 cm in diameter, hemispherical or broadly hemispherical at maturity, with distinctly appendiculate margin initially, surface dry, covered with weak or distinct tomentum, pastel pink (11A4-5), context yellowish green (30A6), turning blue when cut. Hymenophore weakly decurrent, covered with a layer of whitish mycelium (1A1) when young, surface yellowish green (29A6), bluing when bruised, pores angular to nearly round, ca. 4-5/mm; tubes concolorous with hymenophore surface, about 1.1 cm long, turning blue when cut. Stipe 11.0-14.5 × 4.4-5.0 cm, subcylindrical or enlarged downwards, yellowish green (29A6) at maturity, covered with pastel red stains when young, upper 2/3 portion covered with yellowish green (29A6) reticulation, staining blue when bruised, context yellowish green (30A6), changing weakly to blue when cut.  Basidiomata middle to large. Pileus 7.0-13.0 cm in diameter, hemispherical or broadly hemispherical at maturity, with distinctly appendiculate margin initially, surface dry, covered with weak or distinct tomentum, pastel pink (11A4-5), context yellowish green (30A6), turning blue when cut. Hymenophore weakly decurrent, covered with a layer of whitish mycelium (1A1) when young, surface yellowish green (29A6), bluing when bruised, pores angular to nearly round, ca. 4-5/mm; tubes concolorous with hymenophore surface, about 1.1 cm long, turning blue when cut. Stipe 11.0-14.5 × 4.4-5.0 cm, subcylindrical or enlarged downwards, yellowish green (29A6) at maturity, covered with pastel red stains when young, upper 2/3 portion covered with yellowish green (29A6) reticulation, staining blue when bruised, context yellowish green (30A6), changing weakly to blue when cut.
Basidiospores (  Notes: Butyriboletus subregius is characterized by a pastel pink pileus, a yellowish green stipe covered with reticulation of the same color and staining blue when the hymenophore and stipe are bruised. Morphologically and phylogenetically, Bu. subregius resembles Bu. autumniregius, Bu. primiregius, Bu. querciregius, Bu. regius and Bu. fuscoroseus. However, Bu. autumniregius is distinguished by its autumn fruiting season, a stipe that commonly has dark red stains toward the base, and longer spores with a larger Q value [33]; Bu. primiregius is characterized by its late spring season, and a pileus tending to dingy olive-brown as it ages or exposed in sunlight [33]; Bu. querciregius differs from Bu. subregius in its mycorrhizal host, the dull color of a pileus, relatively longer spores with larger Q value [33]; Bu. regius is different from Bu. subregius in its pileus covered with distinct scales with aging, a context usually not bluing when cut, and spores longer with larger Q value [69]. Butyriboletus fuscoroseus is different from Bu. subregius in its brown-pink, reddish brown, or purplishbrown pileus, fine reticulation covered only on the upper half of stipe and context of stipe strongly bluing when cut [56]. Reference Table 2   Basidiomata medium to large. Pileus 6.5-9.0 cm in diameter, hemispherical to applanate, surface brownish yellow (5C8) to yellowish brown (5D8), glabrous, viscid when wet, context deep yellow (4A8), 0.6-1.5 cm thick, color unchanging when cut; hymenophore sinuate to decurrent; tubes up to 1.3 cm long, vivid yellow (3A8), changing to indistinct blue erratically or unchanging color when cut; hymenophore surface concolorous with tubes or olive yellow (3C8), staining blue when bruised; pores angular, ca. 2-3/mm. Stipe 7.2-16.0 × 1.4-2.2 cm, central, clavate to subcylindrical, solid, sometimes tapered downwards, surface concolorous with pileus surface, covered with fine reticulation at apex, context deep yellow (4A8), color unchanging when cut; basal mycelium yellow (3B8).

Discussion
In this study, four new species, Butyriboletus pseudoroseoflavus, Butyriboletus subregius, Tengioboletus subglutinosus, and Suillellus lacrymibasidiatus, were discovered in northern China based on morphological studies and phylogenetic analyses.
Seven species of Butyriboletus were previously reported in China, and all of them were collected from tropical and subtropical regions of China. The two new species of Butyriboletus we proposed here are the first reports of this genus in northern China. Moreover, according to Arora et al. [33], the species diversity of the genus should be more abundant in temperate climes than tropical, subtropical, or boreal ones. Based on this, we presume that northern China may be a species diversity hotspot of Butyriboletus waiting to be explored further.
Butyriboletus subregius is easily confused with Bu. autumniregius, Bu. primiregius, Bu. querciregius, and Bu. regius, morphologically. The primary distinguishing characteristics are the fruiting time and different ecological niches. According to Queiroz [78], these differences mean that the features formerly treated as secondary species criteria are relevant to species delimitation, to the extent that they provide evidence of a lineage separation. Although one ITS sequence of Bu. loyo (Phillippi) Mikšík, was uploaded to the GeneBank [79], the authors did not give a detailed morphological description to prove identification accuracy, so it was excluded from the current phylogeny. However, Bu. loyo is unique within this genus, given its combined morphological characteristics of being equilateral in profile and having red-brown basidiospores and a viscid pileus.
Due to the different color of the hymenophore surface and tubes and the usually vivid red color of basidiomata, Farid et al. [19], Bozok et al. [80], and Biketova et al. [37] all recommended Exsudoporus as a genus separate from Butyriboletus, including B. floridanus  [19] showed that the B. subsplendidus (W.F. Chiu) Kuan Zhao, G. Wu, & Zhu L. Yang clade has affinity with other Butyriboletus. Our phylogram accords with the findings of Chai [28] and Biketova et al. [37] that B. subsplendidus is a sister to the Exsudoporus clade. We agree with Biketova et al. [37] that Exsudoporus should be elevated to genus status, and B. subsplendidus and B. hainanensis N.K. Zeng, Zhi Q. Liang, & S. Jiang should separate from Butyriboletus and represent two distinct genera, as their apparently different characteristics from other species of Butyriboletus.
Tengioboletus is a small genus, with only three species previously reported in southern China. Tengioboletus reticulatus was the first species of the genus collected at Liaoning province in northeastern China [81]. In our study, one new species, T. subglutinosus, was also collected at Jilin province in northeastern China. This means a geographical extension of Tengioboletus into temperate zones, which may also indicate a potentially wide distribution, given that their previously known main distribution was subtropical and tropical China. Our study showed that sequences of Tengioboletus formed an independent clade, which corresponded to the findings of Wu et al. [5] and supported Tengioboletus as a separate genus. As found by Wu et al. [5], Porphyrellus E.-J. Gilbert is a polyphyletic genus in the phylogram ( Figure 1); it formed two clades; one clade named "Porphyrellus?" is a sister to Strobilomyces Berk., as was implied by Han et al. [82]. Clarification of the relationships among the genera will require additional specimens and future studies.
Rubroboletus Kuan Zhao & Zhu L. Yang, Neoboletus, Sutorius, and Lanmaoa G. Wu & Zhu L. Yang shares some characteristics with Suillellus, such as the orange-red surface of the hymenophore and the bluing color change. Nevertheless, none of them has the amyloid hyphae of the context [5,25,[83][84][85]. In contrast, Rubroboletus species have a vivid or dark red pileus with rose-to-red reticulation, and the stipes of species of Neoboletus usually have fine dots but never reticulation. The basidiomata of Sutorius always have a dull color and a reddish color change [28,86]; the hymenophore of Lanmaoa is thin, with a thickness about 1/3-1/5 times that of the pileal context at the position halfway to the pileus center.